| 1. |
Allard ST,
Cleland WW,
Holden HM,
( 2004 ) High resolution X-ray structure of dTDP-glucose 4,6-dehydratase from Streptomyces venezuelae. PMID : 14570895 : DOI : 10.1074/jbc.M310134200 Abstract >>
Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found in some macrolide antibiotics. In Streptomyces venezuelae, there are seven genes required for the biosynthesis of this unusual sugar. One of the genes, desIV, codes for a dTDP-glucose 4,6-dehydratase, which is referred to as DesIV. The reaction mechanisms for these types of dehydratases are quite complicated with proton abstraction from the sugar 4'-hydroxyl group and hydride transfer to NAD+, proton abstraction at C-5, and elimination of the hydroxyl group at C-6 of the sugar, and finally return of a proton to C-5 and a hydride from NADH to C-6. Here we describe the cloning, overexpression, and purification, and high resolution x-ray crystallographic analysis to 1.44 A of wild-type DesIV complexed with dTDP. Additionally, for this study, a double site-directed mutant protein (D128N/E129Q) was prepared, crystallized as a complex with NAD+ and the substrate dTDP-glucose and its structure determined to 1.35 A resolution. In DesIV, the phenolate group of Tyr(151) and O(gamma) of Thr(127) lie at 2.7 and 2.6 A, respectively from the 4'-hydroxyl group of the dTDP-glucose substrate. The side chain of Asp(128) is in the correct position to function as a general acid for proton donation to the 6'-hydroxyl group while the side chain of Glu(129) is ideally situated to serve as the general base for proton abstraction at C-5. This investigation provides further detailed information for understanding the exquisite chemistry that occurs in these remarkable enzymes.
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2. |
Yin Y,
Lu H,
Khosla C,
Cane DE,
( 2003 ) Expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin/methymycin polyketide synthase. PMID : 12733905 : DOI : 10.1021/ja034574q Abstract >>
Picromycin synthase (PICS) is a multifunctional, modular polyketide synthase (PKS) that catalyzes the conversion of methylmalonyl-CoA to narbonolide and 10-deoxymethynolide, the macrolide aglycone precursors of the antibiotics picromycin and methymycin, respectively. PICS modules 5 and 6 were each expressed in Escherichia coli with a thioesterase domain at the C-terminus to allow release of polyketide products. The substrate specificity of PICS modules 5+TE and 6+TE was investigated using N-acetylcysteamine thioesters of 2-methyl-3-hydroxy-pentanoic acid as diketide analogues of the natural polyketide chain elongation substrates. PICS module 5+TE could catalyze the chain elongation of only the syn diketide (2S,3R)-4, while PICS module 6+TE processed both syn diastereomers, (2S,3R)-4 and (2R,3S)-5, with a 2.5:1 preference in k(cat)/K(m) for 5 but did not turn over either of the two anti diketides. The observed substrate specificity patterns are in contrast to the 15-100:1 preference for 4 over 5 previously established for several modules of the closely related erythromycin PKS, 6-deoxyerythronolide B synthase (DEBS).
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3. |
Wang L,
Vining LC,
( 2003 ) Control of growth, secondary metabolism and sporulation in Streptomyces venezuelae ISP5230 by jadW(1), a member of the afsA family of gamma-butyrolactone regulatory genes. PMID : 12904539 : DOI : 10.1099/mic.0.26209-0 Abstract >>
Three new genes (jadW(1), jadW(2) and jadW(3)) were isolated from a region of the Streptomyces venezuelae ISP5230 chromosome at the left-hand end of the jad cluster for jadomycin B (JdB) biosynthesis. The deduced amino acid sequence of jadW(1) showed strong similarity to gene products associated in several streptomycetes with gamma-butyrolactone autoregulators controlling morphological differentiation and secondary metabolism. Examination of JadW(1) for conserved domains detected a repeat sequence characteristic of proteins in the AfsA regulatory family. Insertional inactivation of jadW(1) reduced the growth rate of S. venezuelae cultures in aerated liquid media containing complex nitrogen sources and altered growth morphology in minimal medium. It also affected sporulation on agar media. Cultures of jadW(1)-disrupted mutants grown under conditions supporting biosynthesis of JdB or chloramphenicol by the wild-type strain failed to produce either of the antibiotics. Complementing the disrupted strain by transformation with pJV435, containing a cloned copy of the gene, improved sporulation and restored antibiotic biosynthesis in transformants to titres close to those of the wild-type similarly transformed with pJV435 as a control. The results are consistent with a role for jadW(1) in regulating morphological and metabolic differentiation. Further sequence analysis of jadR(2), which functions with jadR(1) in stress-induced activation of JdB biosynthesis, indicated that this gene encodes a gamma-butyrolactone receptor homologue. The growth-rate-sensitive phenotype of the jadW(1)-disrupted mutant, and the proximity of jadW(1) to jadR(2) indicate that this region of the jad gene cluster contains a regulatory mechanism incorporating gamma-butyrolactone signalling and sensitivity to environmental stress.
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4. |
Tsai SC,
Lu H,
Cane DE,
Khosla C,
Stroud RM,
( 2002 ) Insights into channel architecture and substrate specificity from crystal structures of two macrocycle-forming thioesterases of modular polyketide synthases. PMID : 12379102 : DOI : 10.1021/bi0260177 Abstract >>
Modular polyketide synthases (PKSs) synthesize the polyketide cores of pharmacologically important natural products such as erythromycin and picromycin. Understanding PKSs at high resolution could present new opportunities for chemoenzymatic synthesis of complex molecules. The crystal structures of macrocycle-forming thioesterase (TE) domains from the picromycin synthase (PICS) and 6-deoxyerythronolide B synthase (DEBS) were determined to 1.8-3.0 A with an R(crys) of 19.2-24.4%, including three structures of PICS TE (crystallized at pH 7.6, 8.0, and 8.4) and a second crystal form of DEBS TE. As predicted by the previous work on DEBS TE [Tsai, S. C., et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 14808-14813], PICS TE contains an open substrate channel and a hydrophobic dimer interface. Notwithstanding their similarity, the dimer interfaces and substrate channels of DEBS TE and PICS TE reveal key differences. The structural basis for the divergent substrate specificities of DEBS TE and PICS TE is analyzed. The size of the substrate channel increases with increasing pH, presumably due to electrostatic repulsion in the channel at elevated pH. Together, these structures support previous predictions that macrocycle-forming thioesterases from PKSs share the same protein fold, an open substrate channel, a similar catalytic mechanism, and a hydrophobic dimer interface. They also provide a basis for the design of enzymes capable of catalyzing regioselective macrocyclization of natural or synthetic substrates. A series of high-resolution snapshots of a protein channel at different pHs is presented alongside analysis of channel residues, which could help in the redesign of the protein channel architecture.
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5. |
Lu H,
Tsai SC,
Khosla C,
Cane DE,
( 2002 ) Expression, site-directed mutagenesis, and steady state kinetic analysis of the terminal thioesterase domain of the methymycin/picromycin polyketide synthase. PMID : 12379101 : DOI : 10.1021/bi026006d Abstract >>
The thioesterase (TE) domain of the methymycin/picromycin synthase (PICS) was functionally expressed in Escherichia coli, and the optimal N-terminal boundary of the recombinant TE was determined. A series of diketide-N-acetylcysteamine (SNAC) thioesters were tested as substrates. PICS TE showed a strong preference for the 2-methyl-3-ketopentanoyl-SNAC substrate 5 over the stereoisomers of the reduced diketides 1-4, with an approximately 1.6:1 preference for the (2R,3S)-2-methyl-3-hydroxy diastereomer 2 over the (2S,3R)-diketide 1. The closely related DEBS TE, the thioesterase from the 6-deoxyerythronolide B synthase, showed a more marked 4.4:1 preference for 2 over 1, with only a slightly greater preference for the 3-ketoacyl-SNAC substrate 5. The roles of several active site residues in PICS TE were examined by site-directed mutagenesis. Serine 148, which is part of the apparent catalytic triad consisting of S148, H268, and D176, was found to be essential for thioesterase activity, while replacement of D176 with asparagine (D176N) gave a mutant thioesterase that retained substantial, albeit reduced, hydrolytic activity toward diketide-SNAC substrates. Mutation of E187 and R191, each of which is thought to play a role in substrate binding, had only minor effects on the relative specificity for diketide substrates 1, 2, and 5. Finally, when PICS TE was fused to the C-terminus of DEBS module 3, the resultant chimeric protein converted diketide 1 with methylmalonyl-CoA to triketide ketolactone 6 with improved catalytic efficiency compared to that of the previously developed DEBS module 3-(DEBS)TE construct.
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6. |
Kim BS,
Cropp TA,
Beck BJ,
Sherman DH,
Reynolds KA,
( 2002 ) Biochemical evidence for an editing role of thioesterase II in the biosynthesis of the polyketide pikromycin. PMID : 12368286 : DOI : 10.1074/jbc.M207770200 Abstract >>
The pikromycin biosynthetic gene cluster contains the pikAV gene encoding a type II thioesterase (TEII). TEII is not responsible for polyketide termination and cyclization, and its biosynthetic role has been unclear. During polyketide biosynthesis, extender units such as methylmalonyl acyl carrier protein (ACP) may prematurely decarboxylate to generate the corresponding acyl-ACP, which cannot be used as a substrate in the condensing reaction by the corresponding ketosynthase domain, rendering the polyketide synthase module inactive. It has been proposed that TEII may serve as an "editing" enzyme and reactivate these modules by removing acyl moieties attached to ACP domains. Using a purified recombinant TEII we have tested this hypothesis by using in vitro enzyme assays and a range of acyl-ACP, malonyl-ACP, and methylmalonyl-ACP substrates derived from either PikAIII or the loading didomain of DEBS1 (6-deoxyerythronolide B synthase; AT(L)-ACP(L)). The pikromycin TEII exhibited high K(m) values (>100 microm) with all substrates and no apparent ACP specificity, catalyzing cleavage of methylmalonyl-ACP from both AT(L)-ACP(L) (k(cat)/K(m) 3.3 +/- 1.1 m(-1) s(-1)) and PikAIII (k(cat)/K(m) 2.9 +/- 0.9 m(-1) s(-1)). The TEII exhibited some acyl-group specificity, catalyzing hydrolysis of propionyl (k(cat)/K(m) 15.8 +/- 1.8 m(-1) s(-1)) and butyryl (k(cat)/K(m) 17.5 +/- 2.1 m(-1) s(-1)) derivatives of AT(L)-ACP(L) faster than acetyl (k(cat)/K(m) 4.9 +/- 0.7 m(-1) s(-1)), malonyl (k(cat)/K(m) 3.9 +/- 0.5 m(-1) s(-1)), or methylmalonyl derivatives. PikAIV containing a TEI domain catalyzed cleavage of propionyl derivative of AT(L)-ACP(L) at a dramatically lower rate than TEII. These results provide the first unequivocal in vitro evidence that TEII can hydrolyze acyl-ACP thioesters and a model for the action of TEII in which the enzyme remains primarily dissociated from the polyketide synthase, preferentially removing aberrant acyl-ACP species with long half-lives. The lack of rigorous substrate specificity for TEII may explain the surprising observation that high level expression of the protein in Streptomyces venezuelae leads to significant (>50%) titer decreases.
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7. |
Chen H,
Yamase H,
Murakami K,
Chang CW,
Zhao L,
Zhao Z,
Liu HW,
( 2002 ) Expression, purification, and characterization of two N,N-dimethyltransferases, tylM1 and desVI, involved in the biosynthesis of mycaminose and desosamine. PMID : 12119032 : DOI : 10.1021/bi020245j Abstract >>
Methylation catalyzed by an S-adenosylmethionine- (AdoMet-) dependent methyltransferase is an effective means to alter the hydrophilicity and/or nucleophilicity of a molecule. While a large number of enzymes capable of catalyzing methylation at carbon, oxygen, sulfur, and nitrogen atoms are known, only a few are able to catalyze N,N-dimethylation. Mycaminose and desosamine are aminohexoses found in several macrolide antibiotics, such as tylosin and methymycin, respectively. Both sugars contain a C-3 N,N-dimethylamino group which has been shown to confer the biological activity of these unusual sugars. Recently, sequence analysis as well as genetic studies has led to the assignment of tylM1 in the tylosin biosynthetic gene cluster and desVI in the methymycin biosynthetic gene cluster as genes encoding the corresponding N,N-dimethyltransferases. To verify the proposed roles of the tylM1 and desVI genes, we have overexpressed and purified their encoded products, synthesized the predicted substrates, and characterized the catalytic function of these proteins. Our studies showed that TylM1 and DesVI are homodimeric proteins and have nearly identical biochemical properties. These enzymes do not have strong preference for binding either the unmethylated substrate or the monomethylated intermediate. It is the chemical reactivity of the nitrogen functional group that determines the relative rate of a particular methylation step. Thus, our results not only establish TylM1 and DesVI as new members of a small family of enzymes that are capable of catalyzing N,N-dimethylation of an amino group but also provide evidence indicating that the methylation catalyzed by AdoMet-dependent methyltransferases proceeds in a stepwise manner and is nucleophilic in nature.
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8. |
Chang Z,
Vining LC,
( 2002 ) Biosynthesis of sulfur-containing amino acids in Streptomyces venezuelae ISP5230: roles for cystathionine beta-synthase and transsulfuration. PMID : 12101301 : DOI : 10.1099/00221287-148-7-2135 Abstract >>
A 0.5 kb fragment of Streptomyces venezuelae ISP5230 genomic DNA was amplified by PCR using primers based on consensus sequences of cysteine synthase isozyme A from bacteria. The deduced amino acid sequence of the PCR product resembled not only cysteine synthase sequences from prokaryotes and eukaryotes but also eukaryotic cystathionine beta-synthase sequences. Probing an Str. venezuelae genomic library with the PCR product located a hybridizing colony from which pJV207 was isolated. Sequencing and analysis of the Str. venezuelae DNA insert in pJV207 detected two ORFs. The deduced amino acid sequence of ORF1 matched both cysteine synthase and cystathionine beta-synthase sequences in GenBank, but its size favoured assignment as a cystathionine beta-synthase. ORF2 in the pJV207 insert was unrelated in function to ORF1; in its sequence the deduced product resembled acetyl-CoA transferases, but disruption of the ORF did not cause a detectable phenotypic change. Disruption of ORF1 failed to elicit cysteine auxotrophy in wild-type Str. venezuelae, but in the cys-28 auxotroph VS263 it prevented restoration of prototrophy with homocysteine or methionine supplements. The change in phenotype implicated loss of the transsulfuration activity that in the wild-type converts these supplements to cysteine. This study concludes that disruption of ORF1 inactivates a cbs gene, the product of which participates in cysteine synthesis by transsulfuration. Enzyme assays of Str. venezuelae mycelial extracts confirmed the formation of cysteine by thiolation of O-acetylserine, providing the first unambiguous detection of this activity in a streptomycete. Enzyme assays also detected cystathionine gamma-synthase, cystathionine beta-lyase and cystathionine gamma-lyase activity in the extracts and showed that the substrate for cystathionine gamma-synthase was O-succinyl-homoserine. Based on assay results, the cys-28 mutation in Str. venezuelae VS263 does not inactivate the cysteine synthase gene but impairs expression in cultures grown in minimal medium.
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9. |
Yang K,
Han L,
He J,
Wang L,
Vining LC,
( 2001 ) A repressor-response regulator gene pair controlling jadomycin B production in Streptomyces venezuelae ISP5230. PMID : 11733141 : DOI : 10.1016/s0378-1119(01)00723-5 Abstract >>
A second regulatory gene (jadR(1)) is located immediately upstream of the putative repressor gene (jadR(2)) in the jad cluster for biosynthesis of the antibiotic jadomycin B in Streptomyces venezuelae ISP5230. It encodes a 234-amino acid polypeptide with a sequence resembling those of response regulator proteins in two-component control systems. Features in the conserved C-terminal domain of JadR(1) place the protein in the OmpR-PhoB subfamily of response regulators. In mutants where jadR(1) was deleted or disrupted, jadomycin B was not produced, implying that the gene has an essential role in biosynthesis of the antibiotic. Cloning jadR(1) from S. venezuelae in pJV73A, and introducing additional copies of the gene into the wild-type parent by plasmid transformation gave unstable strains with pJV73A integrated into the chromosome. The transformants initially showed increased production of jadomycin B but gave lower titers as excess copies of jadR(1) were lost; mature cultures stabilized with a wild-type level of antibiotic production. The mutant from which jadR(1) had been deleted could not be transformed with pJV73A. Altering the composition of jadR genes in the chromosome by integration of vectors carrying intact and disrupted copies of jadR(1) and jadR(2) provided evidence that the two genes form a regulatory pair different in function from previously reported two-component systems controlling antibiotic biosynthesis in streptomycetes.
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10. |
He J,
Magarvey N,
Piraee M,
Vining LC,
( 2001 ) The gene cluster for chloramphenicol biosynthesis in Streptomyces venezuelae ISP5230 includes novel shikimate pathway homologues and a monomodular non-ribosomal peptide synthetase gene. PMID : 11577160 : DOI : 10.1099/00221287-147-10-2817 Abstract >>
Regions of the Streptomyces venezuelae ISP5230 chromosome flanking pabAB, an amino-deoxychorismate synthase gene needed for chloramphenicol (Cm) production, were examined for involvement in biosynthesis of the antibiotic. Three of four ORFs in the sequence downstream of pabAB resembled genes involved in the shikimate pathway. BLASTX searches of GenBank showed that the deduced amino acid sequences of ORF3 and ORF4 were similar to proteins encoded by monofunctional genes for chorismate mutase and prephenate dehydrogenase, respectively, while the sequence of the ORF5 product resembled deoxy-arabino-heptulosonate-7-phosphate (DAHP) synthase, the enzyme that initiates the shikimate pathway. A relationship to Cm biosynthesis was indicated by sequence similarities between the ORF6 product and membrane proteins associated with Cm export. BLASTX searches of GenBank for matches with the translated sequence of ORF1 in chromosomal DNA immediately upstream of pabAB did not detect products relevant to Cm biosynthesis. However, the presence of Cm biosynthesis genes in a 7.5 kb segment of the chromosome beyond ORF1 was inferred when conjugal transfer of the DNA into a blocked S. venezuelae mutant restored Cm production. Deletions in the 7.5 kb segment of the wild-type chromosome eliminated Cm production, confirming the presence of Cm biosynthesis genes in this region. Sequencing and analysis located five ORFs, one of which (ORF8) was deduced from BLAST searches of GenBank, and from characteristic motifs detected in alignments of its deduced amino acid sequence, to be a monomodular nonribosomal peptide synthetase. GenBank searches did not identify ORF7, but matched the translated sequences of ORFs 9, 10 and 11 with short-chain ketoreductases, the ATP-binding cassettes of ABC transporters, and coenzyme A ligases, respectively. As has been shown for ORF2, disrupting ORF3, ORF7, ORF8 or ORF9 blocked Cm production.
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11. |
Magarvey N,
He J,
Aidoo KA,
Vining LC,
( 2001 ) The pdx genetic marker adjacent to the chloramphenicol biosynthesis gene cluster in Streptomyces venezuelae ISP5230: functional characterization. PMID : 11495988 : DOI : 10.1099/00221287-147-8-2103 Abstract >>
The pdx-4 mutation in Streptomyces venezuelae ISP5230 confers a growth requirement for pyridoxal (pdx) and is a marker for the genetically mapped cluster of genes associated with chloramphenicol biosynthesis. A gene regulating salvage synthesis of vitamin B6 cofactors in S. venezuelae was cloned by transforming a pdx-4 mutant host with the plasmid vector pDQ101 carrying a library of wild-type genomic DNA fragments, and by selecting for complementation of the host's pdx requirement. However, the corresponding replicative plasmid could not be isolated. Southern hybridizations and transduction analysis indicated that the complementing plasmid had integrated into the chromosome; after excision by a second crossover, the plasmid failed to propagate. To avoid loss of the recombinant vector, a pdx-dependent Streptomyces lividans mutant, KAA1, with a phenotype matching that of S. venezuelae pdx-4, was isolated for use as the cloning host. Introduction of pIJ702 carrying an S. venezuelae genomic library into S. lividans KAA1, and selection of prototrophic transformants, led to the isolation of a stable recombinant vector containing a 2.5 kb S. venezuelae DNA fragment that complemented requirements for pdx in both S. venezuelae and S. lividans mutants. Sequence analysis of the cloned DNA located an intact ORF with a deduced amino acid sequence that, in its central and C-terminal regions resembled type-I aminotransferases. The N-terminal region of the cloned DNA fragment aligned closely with distinctive helix-turn-helix motifs found near the N termini of GntR family transcriptional regulators. The overall deduced amino acid sequence of the cloned DNA showed 73% end-to-end identity to a putative GntR-type regulator cloned in cosmid 6D7 from the Streptomyces coelicolor A3(2) genome. This location is close to that of pdxA, the first pdx marker in S. coelicolor A3(2) identified and mapped genetically in Sir David Hopwood's laboratory. The S. venezuelae gene and S. coelicolor pdxA are postulated to be homologues regulating vitamin B6 coenzyme synthesis from pdx.
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12. |
Chang Z,
Sun Y,
He J,
Vining LC,
( 2001 ) p-Aminobenzoic acid and chloramphenicol biosynthesis in Streptomyces venezuelae: gene sets for a key enzyme, 4-amino-4-deoxychorismate synthase. PMID : 11495989 : DOI : 10.1099/00221287-147-8-2113 Abstract >>
Amplification of sequences from Streptomyces venezuelae ISP5230 genomic DNA using PCR with primers based on conserved prokaryotic pabB sequences gave two main products. One matched pabAB, a locus previously identified in S. venezuelae. The second closely resembled the conserved pabB sequence consensus and hybridized with a 3.8 kb NcoI fragment of S. venezuelae ISP5230 genomic DNA. Cloning and sequence analysis of the 3.8 kb fragment detected three ORFs, and their deduced amino acid sequences were used in BLAST searches of the GenBank database. The ORF1 product was similar to PabB in other bacteria and to the PabB domain encoded by S. venezuelae pabAB. The ORF2 product resembled PabA of other bacteria. ORF3 was incomplete; its deduced partial amino acid sequence placed it in the MocR group of GntR-type transcriptional regulators. Introducing vectors containing the 3.8 kb NcoI fragment of S. venezuelae DNA into pabA and pabB mutants of Escherichia coli, or into the Streptomyces lividans pab mutant JG10, enhanced sulfanilamide resistance in the host strains. The increased resistance was attributed to expression of the pair of discrete translationally coupled p-aminobenzoic acid biosynthesis genes (designated pabB/pabA) cloned in the 3.8 kb fragment. These represent a second set of genes encoding 4-amino-4-deoxychorismate synthase in S. venezuelae ISP5230. In contrast to the fused pabAB set previously isolated from this species, they do not participate in chloramphenicol biosynthesis, but like pabAB they can be disrupted without affecting growth on minimal medium. The gene disruption results suggest that S. venezuelae may have a third set of genes encoding PABA synthase.
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13. |
Wang L,
McVey J,
Vining LC,
( 2001 ) Cloning and functional analysis of a phosphopantetheinyl transferase superfamily gene associated with jadomycin biosynthesis in Streptomyces venezuelae ISP5230. PMID : 11390684 : DOI : 10.1099/00221287-147-6-1535 Abstract >>
Sequence analysis of a XhoI/SacI fragment of chromosomal DNA downstream of jadL in the Streptomyces venezuelae ISP5230 gene cluster for jadomycin biosynthesis detected a partial ORF similar in its deduced amino acid sequence to the hetI product involved in synthesizing a regulator of heterocyst spacing in ANABAENA: By probing a phage library of S. venezuelae DNA with the XhoI/SacI fragment, the authors identified and isolated a hybridizing clone. The nucleotide sequence of its DNA contained three complete ORFs (jadM, N and X) and one incomplete ORF (jadO). The jadM ORF lay immediately downstream of, and partially overlapped, jadL. It contained 786 nucleotides encoding an amino acid sequence like those of enzymes in the phosphopantetheinyl transferase family. The jadN ORF contained 1794 nucleotides and encoded an amino acid sequence resembling acyl-CoA decarboxylases, thus suggesting a role in polyketide condensation reactions. The jadX ORF was not identified, but the partial jadO showed marked similarities in its deduced amino acid sequence to NDP-hexose-2,3-dehydratases, indicating a role in forming the sugar component of jadomycin B. Expression of jadM in Escherichia coli and examination of the product by SDS-PAGE established that the ORF encoded a 29.1 kDa protein, corresponding in size to the 262 amino acid polypeptide deduced from the jadM sequence. Evidence from a Northern hybridization indicated that jadM expression is correlated with jadomycin B synthesis. Cultures of S. venezuelae ISP5230 disrupted in jadM produced only 2-5% of the wild-type titre of jadomycin B, but grew well and produced chloramphenicol normally. The authors conclude that jadM encodes a holo-ACP synthase needed primarily for jadomycin B biosynthesis.
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14. |
Wilson D,
Xue Y,
( 2000 ) Genetic architecture of the polyketide synthases for methymycin and pikromycin series macrolides. PMID : 10713461 : DOI : 10.1016/s0378-1119(00)00003-2 Abstract >>
The methymycin and pikromycin series of antibiotics are structurally related macrolides produced by several Streptomyces species, including Streptomyces venezuelae ATCC 15439, which produces both 12-membered ring macrolides methymycin, neomethymycin, and 14-membered ring macrolides pikromycin and narbomycin. Cloning and sequencing of the biosynthetic gene clusters for these macrolides from three selected Streptomyces strains revealed a common genetic architecture of their polyketide synthases (PKSs). Unlike PKS clusters of other 14-membered ring macrolides such as erythromycin and oleandomycin, each of the pikromycin series producers harbors a six module PKS cluster, in which modules 5 and 6 are encoded on two separate proteins instead of one bimodular protein, as well as a thioesterase II gene immediately downstream of the main PKS gene. The results shed new light on the evolution of modular PKSs and provide further evidence on the regulation of methymycin and pikromycin production in S. venezuelae ATCC 15439.
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15. |
Xue Y,
( 2000 ) Alternative modular polyketide synthase expression controls macrolactone structure. PMID : 10676969 : DOI : 10.1038/35000624 Abstract >>
Modular polyketide synthases are giant multifunctional enzymes that catalyse the condensation of small carboxylic acids such as acetate and propionate into structurally diverse polyketides that possess a spectrum of biological activities. In a modular polyketide synthase, an enzymatic domain catalyses a specific reaction, and three to six enzymatic domains involved in a condensation-processing cycle are organized into a module. A fundamental aspect of a modular polyketide synthase is that its module arrangement linearly specifies the structure of its polyketide product. Here we report a natural example in which alternative expression of the pikromycin polyketide synthase results in the generation of two macrolactone structures. Expression of the full-length modular polyketide synthase PikAIV in Streptomyces venezuelae generates the 14-membered ring macrolactone narbonolide, whereas expression of the amino-terminal truncated form of PikAIV leads to 'skipping' of the final condensation cycle in polyketide biosynthesis to generate the 12-membered ring macrolactone 10-deoxymethynolide. Our findings provide insight into the structure and function of modular polyketide synthases, as well as a new set of tools to generate structural diversity in polyketide natural products.
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16. |
Fu H,
Betlach MC,
McDaniel R,
( 1999 ) Elucidating the mechanism of chain termination switching in the picromycin/methymycin polyketide synthase. PMID : 10421766 : DOI : 10.1016/S1074-5521(99)80087-8 Abstract >>
A single modular polyketide synthase (PKS) gene cluster is responsible for production of both the 14-membered macrolide antibiotic picromycin and the 12-membered macrolide antibiotic methymycin in Streptomyces venezuelae. Building on the success of the heterologous expression system engineered using the erythromycin PKS, we have constructed an analogous system for the picromycin/methymycin PKS. Through heterologous expression and construction of a hybrid PKS, we have examined the contributions that the PKS, its internal thioesterase domain (pikTE) and the Pik TEII thioesterase domain make in termination and cyclization of the two polyketide intermediates. The picromycin/methymycin PKS genes were functionally expressed in the heterologous host Streptomyces lividans, resulting in production of both narbonolide and 10-deoxymethynolide (the precursors of picromycin and methymycin, respectively). Co-expression with the Pik TEII thioesterase led to increased production levels, but did not change the ratio of the two compounds produced, leaving the function of this protein largely unknown. Fusion of the PKS thioesterase domain (pikTE) to 6-deoxyerythronolide B synthase (DEBS) resulted in formation of only 14-membered macrolactones. These experiments demonstrate that the PKS alone is capable of catalyzing the synthesis of both 14- and 12-membered macrolactones and favor a model by which different macrolactone rings result from a combination of the arrangement between the module 5 and module 6 subunits in the picromycin PKS complex and the selectivity of the pikTE domain.
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17. |
Kittendorf JD,
Beck BJ,
Buchholz TJ,
Seufert W,
Sherman DH,
( 2007 ) Interrogating the molecular basis for multiple macrolactone ring formation by the pikromycin polyketide synthase. PMID : 17719493 : DOI : 10.1016/j.chembiol.2007.07.013 PMC : PMC2707933 Abstract >>
The pikromycin polyketide synthase (PKS) is unique in its ability to generate both 12 and 14 membered ring macrolactones. As such, dissection of the molecular basis for controlling metabolic diversity in this system remains an important objective for understanding modular PKS function and expanding chemical diversity. Here, we describe a series of experiments designed to probe the importance of the protein-protein interaction that occurs between the final two monomodules, PikAIII (module 5) and PikAIV (module 6), for the production of the 12 membered ring macrolactone 10-deoxymethynolide. The results obtained from these in vitro studies demonstrate that PikAIII and PikAIV generate the 12 membered ring macrocycle most efficiently when engaged in their native protein-protein interaction. Accordingly, the data are consistent with PikAIV adopting an alternative conformation that enables the terminal thioesterase domain to directly off-load the PikAIII-bound hexaketide intermediate for macrocyclization.
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18. |
Burgie ES,
Thoden JB,
Holden HM,
( 2007 ) Molecular architecture of DesV from Streptomyces venezuelae: a PLP-dependent transaminase involved in the biosynthesis of the unusual sugar desosamine. PMID : 17456741 : DOI : 10.1110/ps.062711007 PMC : PMC2206644 Abstract >>
Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found in certain macrolide antibiotics such as the commonly prescribed erythromycin. Six enzymes are required for its biosynthesis in Streptomyces venezuelae. The focus of this article is DesV, which catalyzes the PLP-dependent replacement of a 3-keto group with an amino functionality in the fifth step of the pathway. For this study the three-dimensional structures of both the internal aldimine and the ketimine intermediate with glutamate were determined to 2.05 A resolution. DesV is a homodimer with each subunit containing 12 alpha-helical regions and 12 beta-strands that together form three layers of sheet. The structure of the internal aldimine demonstrates that the PLP-cofactor is held in place by residues contributed from both subunits (Asp 164 and Gln 167 from Subunit I and Tyr 221 and Asn 235 from Subunit II). When the ketimine intermediate is present in the active site, the loop defined by Gln 225 to Ser 228 from Subunit II closes down upon the active site. The structure of DesV is similar to another sugar-modifying enzyme referred to as PseC. This enzyme is involved in the biosynthesis of pseudaminic acid, which is a sialic acid-like nonulosonate found in the flagellin of Helicobacter pylori. In the case of PseC, however, the amino group is transferred to the C-4 rather than the C-3 position. Details concerning the structural analysis of DesV and a comparison of its molecular architecture to that of PseC are presented.
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19. |
Sherman DH,
Li S,
Yermalitskaya LV,
Kim Y,
Smith JA,
Waterman MR,
Podust LM,
( 2006 ) The structural basis for substrate anchoring, active site selectivity, and product formation by P450 PikC from Streptomyces venezuelae. PMID : 16825192 : DOI : 10.1074/jbc.M605478200 PMC : PMC2939096 Abstract >>
The pikromycin (Pik)/methymycin biosynthetic pathway of Streptomyces venezuelae represents a valuable system for dissecting the fundamental mechanisms of modular polyketide biosynthesis, aminodeoxysugar assembly, glycosyltransfer, and hydroxylation leading to the production of a series of macrolide antibiotics, including the natural ketolides narbomycin and pikromycin. In this study, we describe four x-ray crystal structures and allied functional studies for PikC, the remarkable P450 monooxygenase responsible for production of a number of related macrolide products from the Pik pathway. The results provide important new insights into the structural basis for the C10/C12 and C12/C14 hydroxylation patterns for the 12-(YC-17) and 14-membered ring (narbomycin) macrolides, respectively. This includes two different ligand-free structures in an asymmetric unit (resolution 2.1 A) and two co-crystal structures with bound endogenous substrates YC-17 (resolution 2.35 A)or narbomycin (resolution 1.7 A). A central feature of the enzyme-substrate interaction involves anchoring of the desosamine residue in two alternative binding pockets based on a series of distinct amino acid residues that form a salt bridge and a hydrogen-bonding network with the deoxysugar C3' dimethylamino group. Functional significance of the salt bridge was corroborated by site-directed mutagenesis that revealed a key role for Glu-94 in YC-17 binding and Glu-85 for narbomycin binding. Taken together, the x-ray structure analysis, site-directed mutagenesis, and corresponding product distribution studies reveal that PikC substrate tolerance and product diversity result from a combination of alternative anchoring modes rather than an induced fit mechanism.
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20. |
Hong JS,
Park SJ,
Parajuli N,
Park SR,
Koh HS,
Jung WS,
Choi CY,
Yoon YJ,
( 2007 ) Functional analysis of desVIII homologues involved in glycosylation of macrolide antibiotics by interspecies complementation. PMID : 17049185 : DOI : 10.1016/j.gene.2006.08.021 Abstract >>
The DesVIII is an auxiliary protein which enhances the transfer of TDP-d-desosamine catalyzed by DesVII glycosyltransferase in the biosynthesis of macrolide antibiotics, neomethymycin, methymycin and pikromycin, in Streptomyces venezuelae ATCC 15439. Homologues of the desVIII gene are present in a number of aminosugar-containing antibiotic biosynthetic gene clusters including eryCII from the erythromycin producer Saccharopolyspora erythraea, oleP1 from the oleandomycin producer Streptomyces antibioticus, dnrQ from the doxorubicin producer Streptomyces peucetius, and tylMIII from the tylosin producer Streptomyces fradiae. In order to gain further insight into the function of these DesVIII homologues, interspecies complementation experiments were carried out by expressing each gene in a desVIII deletion mutant strain of S. venezuelae. Complementation by expressing EryCII, OleP1, and DnrQ in this mutant strain restored the production of glycosylated macrolides to an approximate level of 66%, 26% and 26%, respectively, compared to self-complementation by DesVIII. However, expression of TylMIII did not restore the antibiotic production. These results suggest that the DesVIII homologues (except for TylMIII) can functionally replace the native DesVIII for glycosylation to proceed in vivo and their functions are similar in acting as glycosyltransferase auxiliary proteins. The requirement of glycosyltransferase auxiliary protein seems to be more widespread in polyketide biosynthetic pathways than previously known.
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21. |
Giraldes JW,
Akey DL,
Kittendorf JD,
Sherman DH,
Smith JL,
Fecik RA,
( 2006 ) Structural and mechanistic insights into polyketide macrolactonization from polyketide-based affinity labels. PMID : 16969373 : DOI : 10.1038/nchembio822 Abstract >>
Polyketides are a diverse class of natural products having important clinical properties, including antibiotic, immunosuppressive and anticancer activities. They are biosynthesized by polyketide synthases (PKSs), which are modular, multienzyme complexes that sequentially condense simple carboxylic acid derivatives. The final reaction in many PKSs involves thioesterase-catalyzed cyclization of linear chain elongation intermediates. As the substrate in PKSs is presented by a tethered acyl carrier protein, introduction of substrate by diffusion is problematic, and no substrate-bound type I PKS domain structure has been reported so far. We describe the chemical synthesis of polyketide-based affinity labels that covalently modify the active site serine of excised pikromycin thioesterase from Streptomyces venezuelae. Crystal structures reported here of the affinity label-pikromycin thioesterase adducts provide important mechanistic insights. These results suggest that affinity labels can be valuable tools for understanding the mechanisms of individual steps within multifunctional PKSs and for directing rational engineering of PKS domains for combinatorial biosynthesis.
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22. |
Akey DL,
Kittendorf JD,
Giraldes JW,
Fecik RA,
Sherman DH,
Smith JL,
( 2006 ) Structural basis for macrolactonization by the pikromycin thioesterase. PMID : 16969372 : DOI : 10.1038/nchembio824 Abstract >>
Polyketides are a class of biologically active microbial and plant-derived metabolites that possess a high degree of structural and functional diversity and include many human therapeutics, among them anti-infective and anti-cancer drugs, growth promoters and anti-parasitic agents. The macrolide antibiotics, characterized by a glycoside-linked macrolactone, constitute an important class of polyketides, including erythromycin and the natural ketolide anti-infective agent pikromycin. Here we describe new mechanistic details of macrolactone ring formation catalyzed by the pikromycin polyketide synthase thioesterase domain from Streptomyces venezuelae. A pentaketide phosphonate mimic of the final pikromycin linear chain-elongation intermediate was synthesized and shown to be an active site affinity label. The crystal structures of the affinity-labeled enzyme and of a 12-membered-ring macrolactone product complex suggest a mechanism for cyclization in which a hydrophilic barrier in the enzyme and structural restraints of the substrate induce a curled conformation to direct macrolactone ring formation.
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23. |
Borisova SA,
Zhang C,
Takahashi H,
Zhang H,
Wong AW,
Thorson JS,
Liu HW,
( 2006 ) Substrate specificity of the macrolide-glycosylating enzyme pair DesVII/DesVIII: opportunities, limitations, and mechanistic hypotheses. PMID : 16538696 : DOI : 10.1002/anie.200503195 Abstract >>
N/A
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24. |
Reuther J,
Wohlleben W,
Muth G,
( 2006 ) Modular architecture of the conjugative plasmid pSVH1 from Streptomyces venezuelae. PMID : 16439019 : DOI : 10.1016/j.plasmid.2005.11.007 Abstract >>
The conjugative rolling circle replication (RCR) type plasmid pSVH1 from the chloramphenicol producer Streptomyces venezuelae was characterized by DNA sequence analysis and insertion/deletion analysis. Nucleotide sequence of the 12,652 bp pSVH1 revealed 11 open reading frames with high coding probability for which putative functions could be assigned. Beside the replication initiator gene rep for RCR, pSVH1 contained only genes involved in conjugative transfer. The transfer gene traB encoding the septal DNA translocator TraB is regulated by the GntR-type transcriptional regulator TraR. Six spd genes involved in intra-mycelial plasmid spreading are organized in two operons, consisting of two and three translationally coupled genes. Subcloning experiments demonstrated that the transfer gene traB represents a kill function and localized the pSVH1 minimal replicon consisting of rep and the dso origin to a 2072-bp fragment. Plasmid pSVH1 showed a modular architecture. Its replication region resembled that of the Streptomyces natalensis plasmid pSNA1, while the transfer and spread regions involved in conjugative plasmid transfer were highly similar to the corresponding regions of the Streptomyces ghanaensis plasmid pSG5.
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25. |
Chen YH,
Wang CC,
Greenwell L,
Rix U,
Hoffmeister D,
Vining LC,
Rohr J,
Yang KQ,
( 2005 ) Functional analyses of oxygenases in jadomycin biosynthesis and identification of JadH as a bifunctional oxygenase/dehydrase. PMID : 15817470 : DOI : 10.1074/jbc.M414229200 PMC : PMC2883817 Abstract >>
A novel angucycline metabolite, 2,3-dehydro-UWM6, was identified in a jadH mutant of Streptomyces venezuelae ISP5230. Both UWM6 and 2,3-dehydro-UWM6 could be converted to jadomycin A or B by a ketosynthase alpha (jadA) mutant of S. venezuelae. These angucycline intermediates were also converted to jadomycin A by transformant of the heterologous host Streptomyces lividans expressing the jadFGH oxygenases in vivo and by its cell-free extracts in vitro; thus the three gene products JadFGH are implicated in catalysis of the post-polyketide synthase biosynthetic reactions converting UWM6 to jadomycin aglycone. Genetic and biochemical analyses indicate that JadH possesses dehydrase activity, not previously associated with polyketide-modifying oxygenase. Since the formation of aromatic polyketides often requires multiple dehydration steps, bifunctionality of oxygenases modifying aromatic polyketides may be a general phenomenon.
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26. |
Szu PH,
He X,
Zhao L,
Liu HW,
( 2005 ) Biosynthesis of TDP-D-desosamine: identification of a strategy for C4 deoxygenation. PMID : 16187386 : DOI : 10.1002/anie.200501998 Abstract >>
N/A
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27. |
Borisova SA,
Zhao L,
Melançon III CE,
Kao CL,
Liu HW,
( 2004 ) Characterization of the glycosyltransferase activity of desVII: analysis of and implications for the biosynthesis of macrolide antibiotics. PMID : 15161264 : DOI : 10.1021/ja049967j Abstract >>
In vitro catalytic activity of DesVII, the glycosyltransferase involved in the biosynthesis of methymycin, neomethymycin, narbomycin, and pikromycin in Streptomyces venezuelae, is described. This is the first report of demonstrated in vitro activity of a glycosyltransferase involved in the biosynthesis of macrolide antibiotics. DesVII is unique among glycosyltransferases in that it requires an additional protein component, DesVIII, as well as basic pH for its full activity.
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28. |
Yanai K,
Sumida N,
Okakura K,
Moriya T,
Watanabe M,
Murakami T,
( 2004 ) Para-position derivatives of fungal anthelmintic cyclodepsipeptides engineered with Streptomyces venezuelae antibiotic biosynthetic genes. PMID : 15184904 : DOI : 10.1038/nbt978 Abstract >>
PF1022A, a cyclooctadepsipeptide possessing strong anthelmintic properties and produced by the filamentous fungus Rosellinia sp. PF1022, consists of four alternating residues of N-methyl-L-leucine and four residues of D-lactate or D-phenyllactate. PF1022A derivatives obtained through modification of their benzene ring at the para-position with nitro or amino groups act as valuable starting materials for the synthesis of compounds with improved anthelmintic activities. Here we describe the production of such derivatives by fermentation through metabolic engineering of the PF1022A biosynthetic pathway in Rosellinia sp. PF1022. Three genes cloned from Streptomyces venezuelae, and required for the biosynthesis of p-aminophenylpyruvate from chorismate in the chloramphenicol biosynthetic pathway, were expressed in a chorismate mutase-deficient strain derived from Rosellinia sp. PF1022. Liquid chromatography-mass spectrometry and NMR analyses confirmed that this approach facilitated the production of PF1022A derivatives specifically modified at the para-position. This fermentation method is environmentally safe and can be used for the industrial scale production of PF1022A derivatives.
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29. |
Zheng J,
Keatinge-Clay AT,
( 2011 ) Structural and functional analysis of C2-type ketoreductases from modular polyketide synthases. PMID : 21570406 : DOI : 10.1016/j.jmb.2011.04.065 Abstract >>
The process by which �\-stereocenters of polyketide intermediates are set by modular polyketide synthases (PKSs) when condensation is not immediately followed by reduction is mysterious. However, the reductase-incompetent ketoreductase (KR) from the third module of 6-deoxyerythronolide B synthase has been proposed to operate as a racemase, aiding in the epimerization process that reverses the orientation of the �\-methyl group of the polyketide intermediate generated by the ketosynthase to the configuration observed in the 6-deoxyerythronolide B final product. To learn more about the epimerization process, the structure of the C2-type KR from the third module of the pikromycin synthase, analogous to the KR from the third module of 6-deoxyerythronolide B synthase, was determined to 1.88 ? resolution. This first structural analysis of this KR-type reveals differences from reductase-competent KRs such as that the site NADPH binds to reductase-competent KRs is occluded by side chains and the putative catalytic tyrosine possesses more degrees of freedom. The active-site geometry may enable C2-type KRs to align the thioester and �]-keto groups of a polyketide intermediate to reduce the pK(a) of the �\-proton and accelerate its abstraction. Results from in vivo assays of engineered PKSs support that C2-type KRs cooperate with epimer-specific ketosynthases to set the configurations of substituent-bearing �\-carbons.
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30. |
Ruszczycky MW,
Choi SH,
Mansoorabadi SO,
Liu HW,
( 2011 ) Mechanistic studies of the radical S-adenosyl-L-methionine enzyme DesII: EPR characterization of a radical intermediate generated during its catalyzed dehydrogenation of TDP-D-quinovose. PMID : 21513273 : DOI : 10.1021/ja201212f PMC : PMC3099232 Abstract >>
DesII, a radical S-adenosyl-l-methionine (SAM) enzyme from Streptomyces venezuelae, catalyzes the deamination of TDP-4-amino-4,6-dideoxy-D-glucose to TDP-3-keto-4,6-dideoxy-D-glucose in the desosamine biosynthetic pathway. DesII can also catalyze the dehydrogenation of TDP-D-quinovose to the corresponding 3-keto sugar. Similar to other radical SAM enzymes, DesII catalysis has been proposed to proceed via a radical mechanism. This hypothesis is now confirmed by EPR spectroscopy with the detection of a TDP-D-quinovose radical intermediate having a g-value of 2.0025 with hyperfine coupling to two spin 1/2 nuclei, each with a splitting constant of 33.6 G. A significant decrease in the EPR line width is observed when the radical is generated in reactions conducted in D(2)O versus H(2)O. These results are consistent with a C3 �\-hydroxyalkyl radical in which the p-orbital harboring the unpaired electron spin at C3 is periplanar with the C-H bonds at both C2 and C4.
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31. |
Borisova SA,
Liu HW,
( 2010 ) Characterization of glycosyltransferase DesVII and its auxiliary partner protein DesVIII in the methymycin/picromycin biosynthetic pathway. PMID : 20695498 : DOI : 10.1021/bi1007657 PMC : PMC2939310 Abstract >>
The in vitro characterization of the catalytic activity of DesVII, the glycosyltransferase involved in the biosynthesis of the macrolide antibiotics methymycin, neomethymycin, narbomycin, and pikromycin in Streptomyces venezuelae, is described. DesVII is unique among glycosyltransferases in that it requires an additional protein component, DesVIII, for activity. Characterization of the metabolites produced by a S. venezuelae mutant lacking the desVIII gene confirmed that desVIII is important for the biosynthesis of glycosylated macrolides but can be replaced by at least one of the homologous genes from other pathways. The addition of recombinant DesVIII protein significantly improves the glycosylation efficiency of DesVII in the in vitro assay. When affinity-tagged DesVII and DesVIII proteins were coproduced in Escherichia coli, they formed a tight (�\�])(3) complex that is at least 10(3)-fold more active than DesVII alone. The formation of the DesVII/DesVIII complex requires coexpression of both genes in vivo and cannot be fully achieved by mixing the individual protein components in vitro. The ability of the DesVII/DesVIII system to catalyze the reverse reaction with the formation of TDP-desosamine was also demonstrated in a transglycosylation experiment. Taken together, our data suggest that DesVIII assists the folding of DesVII during protein production and remains tightly bound during catalysis. This requirement must be taken into consideration in the design of combinatorial biosynthetic experiments with new glycosylated macrolides.
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32. |
Ruszczycky MW,
Choi SH,
Liu HW,
( 2010 ) Stoichiometry of the redox neutral deamination and oxidative dehydrogenation reactions catalyzed by the radical SAM enzyme DesII. PMID : 20121093 : DOI : 10.1021/ja909451a PMC : PMC2833275 Abstract >>
DesII from Streptomyces venezuelae is a radical SAM (S-adenosyl-l-methionine) enzyme that catalyzes the deamination of TDP-4-amino-4,6-dideoxy-d-glucose to form TDP-3-keto-4,6-dideoxy-d-glucose in the biosynthesis of TDP-d-desosamine. DesII also catalyzes the dehydrogenation of the nonphysiological substrate TDP-D-quinovose to TDP-3-keto-6-deoxy-d-glucose. These properties prompted an investigation of how DesII handles SAM in the redox neutral deamination versus the oxidative dehydrogenation reactions. This work was facilitated by the development of an enzymatic synthesis of TDP-4-amino-4,6-dideoxy-d-glucose that couples a transamination equilibrium to the thermodynamically favorable oxidation of formate. In this study, DesII is found to consume SAM versus TDP-sugar with stoichiometries of 0.96 +/- 0.05 and 1.01 +/- 0.05 in the deamination and dehydrogenation reactions, respectively, using Na(2)S(2)O(4) as the reductant. Importantly, no significant change in stoichiometry is observed when the flavodoxin/flavodoxin NADP(+) oxidoreductase/NADPH reducing system is used in place of Na(2)S(2)O(4). Moreover, there is no evidence of an uncoupled or abortive process in the deamination reaction, as indicated by the observation that dehydrogenation can take place in the absence of an external source of reductant whereas deamination cannot. Mechanistic and biochemical implications of these results are discussed.
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33. |
Podzelinska K,
Latimer R,
Bhattacharya A,
Vining LC,
Zechel DL,
Jia Z,
( 2010 ) Chloramphenicol biosynthesis: the structure of CmlS, a flavin-dependent halogenase showing a covalent flavin-aspartate bond. PMID : 20080101 : DOI : 10.1016/j.jmb.2010.01.020 Abstract >>
Chloramphenicol is a halogenated natural product bearing an unusual dichloroacetyl moiety that is critical for its antibiotic activity. The operon for chloramphenicol biosynthesis in Streptomyces venezuelae encodes the chloramphenicol halogenase CmlS, which belongs to the large and diverse family of flavin-dependent halogenases (FDH's). CmlS was previously shown to be essential for the formation of the dichloroacetyl group. Here we report the X-ray crystal structure of CmlS determined at 2.2 A resolution, revealing a flavin monooxygenase domain shared by all FDHs, but also a unique 'winged-helix' C-terminal domain that creates a T-shaped tunnel leading to the halogenation active site. Intriguingly, the C-terminal tail of this domain blocks access to the halogenation active site, suggesting a structurally dynamic role during catalysis. The halogenation active site is notably nonpolar and shares nearly identical residues with Chondromyces crocatus tyrosyl halogenase (CndH), including the conserved Lys (K71) that forms the reactive chloramine intermediate. The exception is Y350, which could be used to stabilize enolate formation during substrate halogenation. The strictly conserved residue E44, located near the isoalloxazine ring of the bound flavin adenine dinucleotide (FAD) cofactor, is optimally positioned to function as a remote general acid, through a water-mediated proton relay, which could accelerate the reaction of the chloramine intermediate during substrate halogenation, or the oxidation of chloride by the FAD(C4alpha)-OOH intermediate. Strikingly, the 8alpha carbon of the FAD cofactor is observed to be covalently attached to D277 of CmlS, a residue that is highly conserved in the FDH family. In addition to representing a new type of flavin modification, this has intriguing implications for the mechanism of FDHs. Based on the crystal structure and in analogy to known halogenases, we propose a reaction mechanism for CmlS.
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34. |
Szu PH,
Ruszczycky MW,
Choi SH,
Yan F,
Liu HW,
( 2009 ) Characterization and mechanistic studies of DesII: a radical S-adenosyl-L-methionine enzyme involved in the biosynthesis of TDP-D-desosamine. PMID : 19746907 : DOI : 10.1021/ja903354k PMC : PMC2780582 Abstract >>
D-desosamine (1) is a 3-(N,N-dimethylamino)-3,4,6-trideoxyhexose found in a number of macrolide antibiotics including methymycin (2), neomethymycin (3), pikromycin (4), and narbomycin (5) produced by Streptomyces venezuelae . It plays an essential role in conferring biological activities to its parent aglycones. Previous genetic and biochemical studies of the biosynthesis of desosamine in S. venezuelae showed that the conversion of TDP-4-amino-4,6-dideoxy-D-glucose (8) to TDP-3-keto-4,6-dideoxy-D-glucose (9) is catalyzed by DesII, which is a member of the radical S-adenosyl-L-methionine (SAM) enzyme superfamily. Here, we report the purification and reconstitution of His(6)-tagged DesII, characterization of its [4Fe-4S] cluster using UV-vis and EPR spectroscopies, and the capability of flavodoxin, flavodoxin reductase, and NADPH to reduce the [4Fe-4S](2+) cluster. Also included are a steady-state kinetic analysis of DesII-catalyzed reaction and an investigation of the substrate flexibility of DesII. Studies of deuterium incorporation into SAM using TDP-[3-(2)H]-4-amino-4,6-dideoxy-D-glucose as the substrate provides strong evidence for direct hydrogen atom transfer to a 5'-deoxyadenosyl radical in the catalytic cycle. The fact that hydrogen atom abstraction occurs at C-3 also sheds light on the mechanism of this intriguing deamination reaction.
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35. |
Li S,
Chaulagain MR,
Knauff AR,
Podust LM,
Montgomery J,
Sherman DH,
( 2009 ) Selective oxidation of carbolide C-H bonds by an engineered macrolide P450 mono-oxygenase. PMID : 19833867 : DOI : 10.1073/pnas.0907203106 PMC : PMC2774028 Abstract >>
Regio- and stereoselective oxidation of an unactivated C-H bond remains a central challenge in organic chemistry. Considerable effort has been devoted to identifying transition metal complexes, biological catalysts, or simplified mimics, but limited success has been achieved. Cytochrome P450 mono-oxygenases are involved in diverse types of regio- and stereoselective oxidations, and represent a promising biocatalyst to address this challenge. The application of this class of enzymes is particularly significant if their substrate spectra can be broadened, selectivity controlled, and reactions catalyzed in the absence of expensive heterologous redox partners. In this study, we engineered a macrolide biosynthetic P450 mono-oxygenase PikC (PikC(D50N)-RhFRED) with remarkable substrate flexibility, significantly increased activity compared to wild-type enzyme, and self-sufficiency. By harnessing its unique desosamine-anchoring functionality via a heretofore under-explored "substrate engineering" strategy, we demonstrated the ability of PikC to hydroxylate a series of carbocyclic rings linked to the desosamine glycoside via an acetal linkage (referred to as "carbolides") in a regioselective manner. Complementary analysis of a number of high-resolution enzyme-substrate cocrystal structures provided significant insights into the function of the aminosugar-derived anchoring group for control of reaction site selectivity. Moreover, unexpected biological activity of a select number of these carbolide systems revealed their potential as a previously unrecorded class of antibiotics.
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36. |
Yan J,
Gupta S,
Sherman DH,
Reynolds KA,
( 2009 ) Functional dissection of a multimodular polypeptide of the pikromycin polyketide synthase into monomodules by using a matched pair of heterologous docking domains. PMID : 19437523 : DOI : 10.1002/cbic.200900098 PMC : PMC4652847 Abstract >>
The pikromyin polyketide synthase (PKS) in Streptomyces venezulae is comprised of a loading module and six extension modules, which generate the corresponding 14-membered macrolactone product. PikAI is a multimodular component of this PKS and houses both the loading domain and the first two extension modules, joined by short intraprotein linkers. We have shown that PikAI can be separated into two proteins at either of these linkers, only when matched pairs of docking domains (DDs) from a heterologous modular phoslactomycin PKS are used in place of the intraprotein linker. In both cases the yields of pikromycin produced by the S. venezuelae mutant were 50% of that of a S. venezuelae strain expressing the native trimodular PikAI. This observation provides the first demonstration that such separations do not dramatically impact the efficiency of the entire in vivo biosynthetic process. Expression of module 2 as a monomodular protein fused to a heterologous N-terminal docking domain was also observed to give almost a tenfold improvement in the in vivo generation of pikromycin from a synthetic diketide intermediate. These results demonstrate the utility of DDs to manipulate biosynthetic processes catalyzed by modular PKSs and the quest to generate novel polyketide products.
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37. |
Wang L,
Tian X,
Wang J,
Yang H,
Fan K,
Xu G,
Yang K,
Tan H,
( 2009 ) Autoregulation of antibiotic biosynthesis by binding of the end product to an atypical response regulator. PMID : 19423672 : DOI : 10.1073/pnas.0900592106 PMC : PMC2688989 Abstract >>
In bacteria, many "atypical" response regulators (ARRs) lack the conserved residues important for phosphorylation by which typical response regulators switch their output response, suggesting the existence of alternative regulatory mechanisms. However, such mechanisms have not been established. JadR1, an OmpR-type ARR of Streptomyces venezuelae, appears to activate the transcription of jadomycin B (JdB) biosynthetic genes while repressing its own gene. JadR1 activities were inhibited in cells induced to produce JdB, which was found to bind directly to the N-terminal receiver domain of JadR1, causing JadR1 to dissociate from target promoters. The activity of a NarL-type ARR, RedZ, that regulates production of another antibiotic was likewise modulated by the end product (undecylprodigisines), implying that end-product-mediated control of antibiotic pathway-specific ARRs may be widespread. These results could prove relevant to knowledge-based improvements in yield of commercially important antibiotics.
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38. |
Maharjan S,
Oh TJ,
Lee HC,
Sohng JK,
( 2009 ) Identification and functional characterization of an afsR homolog regulatory gene from Streptomyces venezuelae ATCC 15439. PMID : 19307759 : Abstract >>
Sequencing analysis of a 5-kb DNA fragment from Streptomyces venezuelae ATCC 15439 revealed the presence of one 3.1-kb open reading frame (ORF), designated afsRsv. The deduced product of afsR-sv (1,056 aa) was found to have high homology with the global regulatory protein AfsR. Homology-based analysis showed that afsR-sv represents a transcriptional activator belonging to the Streptomyces antibiotic regulatory protein (SARP) family that includes an Nterminal SARP domain containing a bacterial transcriptional activation domain (BTAD), an NB-ARC domain, and a Cterminal tetratricopeptide repeat domain. Gene expression analysis by reverse transcriptase PCR (RT-PCR) demonstrated the activation of transcription of genes belonging to pikromycin production, when afsR-sv was overexpressed in S. venezuelae. Heterologous expression of the afsR-sv in different Streptomyces strains resulted in increased production of the respective antibiotics, suggesting that afsR-sv is a positive regulator of antibiotics biosynthesis.
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39. |
Kittendorf JD,
Sherman DH,
( 2009 ) The methymycin/pikromycin pathway: a model for metabolic diversity in natural product biosynthesis. PMID : 19027305 : DOI : 10.1016/j.bmc.2008.10.082 PMC : PMC2843759 Abstract >>
The methymycin/pikromycin (Pik) macrolide pathway represents a robust metabolic system for analysis of modular polyketide biosynthesis. The enzymes that comprise this biosynthetic pathway display unprecedented substrate flexibility, combining to produce six structurally diverse macrolide antibiotics in Streptomyces venezuelae. Thus, it is appealing to consider that the pikromycin biosynthetic enzymes could be leveraged for high-throughput production of novel macrolide antibiotics. Accordingly, efforts over the past decade have focused on the detailed investigation of the six-module polyketide synthase, desosamine sugar assembly and glycosyl transfer, and the cytochrome P450 monooxygenase that is responsible for hydroxylation. This review summarizes the advances in understanding of pikromycin biosynthesis that have been gained during the course of these investigations.
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40. |
Li S,
Ouellet H,
Sherman DH,
Podust LM,
( 2009 ) Analysis of transient and catalytic desosamine-binding pockets in cytochrome P-450 PikC from Streptomyces venezuelae. PMID : 19124459 : DOI : 10.1074/jbc.M807592200 PMC : PMC2931524 Abstract >>
The cytochrome P-450 PikC from Streptomyces venezuelae exhibits significant substrate tolerance and performs multiple hydroxylation reactions on structurally variant macrolides bearing the deoxyamino sugar desosamine. In previously determined co-crystal structures (Sherman, D. H., Li, S., Yermalitskaya, L. V., Kim, Y., Smith, J. A., Waterman, M. R., and Podust, L. M. (2006) J. Biol. Chem. 281, 26289-26297), the desosamine moiety of the native substrates YC-17 and narbomycin is bound in two distinct buried and surface-exposed binding pockets, mediated by specific interactions between the protonated dimethylamino group and the acidic amino acid residues Asp(50), Glu(85), and Glu(94). Although the Glu(85) and Glu(94) negative charges are essential for maximal catalytic activity of native enzyme, elimination of the surface-exposed negative charge at Asp(50) results in significantly enhanced catalytic activity. Nevertheless, the D50N substitution could not rescue catalytic activity of PikC(E94Q) based on lack of activity in the corresponding double mutant PikC(D50N/E94Q). To address the specific role for each desosamine-binding pocket, we analyzed the x-ray structures of the PikC(D50N) mutant co-crystallized with narbomycin (1.85A resolution) and YC-17 (3.2A resolution). In PikC(D50N), the desosamine moiety of both YC-17 and narbomycin was bound in a catalytically productive "buried site." This finding suggested a two-step substrate binding mechanism, whereby desosamine is recognized in the two subsites to allow the macrolide substrate to sequentially progress toward a catalytically favorable orientation. Collectively, the binding, mutagenesis, kinetic, and x-ray structural data suggest that enhancement of the catalytic activity of PikC(D50N) is due to the facilitated relocation of substrate to the buried site, which has higher binding affinity, as opposed to dissociation in solution from the transient "surface-exposed site."
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41. |
Buchholz TJ,
Geders TW,
Bartley FE,
Reynolds KA,
Smith JL,
Sherman DH,
( 2009 ) Structural basis for binding specificity between subclasses of modular polyketide synthase docking domains. PMID : 19146481 : DOI : 10.1021/cb8002607 PMC : PMC2853223 Abstract >>
Bacterial type I polyketide synthases (PKSs) assemble structurally diverse natural products of significant clinical value from simple metabolic building blocks. The synthesis of these compounds occurs in a processive fashion along a large multiprotein complex. Transfer of the acyl intermediate across interpolypeptide junctions is mediated, at least in large part, by N- and C-terminal docking domains. We report here a comprehensive analysis of the binding affinity and selectivity for the complete set of discrete docking domain pairs in the pikromycin and erythromycin PKS systems. Despite disconnection from their parent module, each cognate pair of docking domains retained exquisite binding selectivity. Further insights were obtained by X-ray crystallographic analysis of the PikAIII/PikAIV docking domain interface. This new information revealed a series of key interacting residues that enabled development of a structural model for the recently proposed H2-T2 class of polypeptides involved in PKS intermodular molecular recognition.
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42. |
Borisova SA,
Kim HJ,
Pu X,
Liu HW,
( 2008 ) Glycosylation of acyclic and cyclic aglycone substrates by macrolide glycosyltransferase DesVII/DesVIII: analysis and implications. PMID : 18548476 : DOI : 10.1002/cbic.200800155 PMC : PMC4400176 Abstract >>
N/A
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43. |
Gupta S,
Lakshmanan V,
Kim BS,
Fecik R,
Reynolds KA,
( 2008 ) Generation of novel pikromycin antibiotic products through mutasynthesis. PMID : 18512859 : DOI : 10.1002/cbic.200700635 PMC : PMC2614871 Abstract >>
The pikromycin polyketide synthase (PKS) of S. venezuelae, which consists of one loading module and six extension modules, is responsible for the formation of the hexaketide narbonolide, a key intermediate in the biosynthesis of the antibiotic pikromycin. S. venezuelae strains in which PikAI, which houses the loading domain and first two modules of the PKS, is either absent or catalytically inactive, produce no pikromycin product. When these strains are grown in the presence of a synthetically prepared triketide product, activated as the N-acetylcysteamine thioester, pikromycin yields are restored to as much as 11 % of that seen in the wild-type strain. Feeding analogues of the triketide intermediate provides pikromycin analogues bearing different alkyl substituents at C13 and C14. One of these analogues, Delta(15,16)-dehydropikromycin, exhibits improved antimicrobial activity relative to pikromycin.
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44. |
Burgie ES,
Holden HM,
( 2008 ) Three-dimensional structure of DesVI from Streptomyces venezuelae: a sugar N,N-dimethyltransferase required for dTDP-desosamine biosynthesis. PMID : 18327916 : DOI : 10.1021/bi800063j Abstract >>
D-Desosamine, or 3-(dimethylamino)-3,4,6-trideoxyglucose, is an unusual sugar found on the macrolide antibiotic erythromycin, and it has been shown to play a critical role in the biological activity of the drug. Desosamine is added to the parent aglycone via the action of a glycosyltransferase that utilizes dTDP-desosamine as its substrate. Six enzymes are required for the biosynthesis of dTDP-desosamine in Streptomyces venezuelae, with the last step catalyzed by DesVI, an N, N-dimethyltransferase. Here we describe the X-ray crystal structure determined to 2.0 A resolution of DesVI complexed with S-adenosylmethionine (SAM) and the substrate analogue UDP-benzene. Each subunit of the DesVI dimer contains a seven-stranded mixed beta-sheet flanked on either side by alpha-helices. In addition to this major tertiary structural element, there is a four-stranded antiparallel beta-sheet that provides the platform necessary for subunit-subunit assembly. On the basis of the UDP-benzene binding mode, the DesVI substrate, dTDP-3-(methylamino)-3,4,6-trideoxyglucose, has been modeled into the active site. This model places the C-6' methyl group of the sugar into a hydrophobic patch that is well-conserved among putative nucleotide-linked sugar dimethyltransferases. It is formed by Trp 140, Met 178, and Ile 200. The sugar C-2' hydroxyl sits near Tyr 14, and its C-3' amino group is properly positioned for direct in-line attack of the cofactor's reactive methyl group. While methyltransferases that catalyze single alkylations at carbons, oxygens, sulfurs, and nitrogens have been well characterized, little is known regarding enzymes capable of N,N-dimethylation reactions. As such, the ternary structure of DesVI reported here serves as a structural paradigm for a new family of dimethyltransferases that function on nucleotide-linked sugars.
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45. |
Guo Y,
Zheng W,
Rong X,
Huang Y,
( 2008 ) A multilocus phylogeny of the Streptomyces griseus 16S rRNA gene clade: use of multilocus sequence analysis for streptomycete systematics. PMID : 18175701 : DOI : 10.1099/ijs.0.65224-0 Abstract >>
Streptomycetes are a complex group of actinomycetes that produce diverse bioactive metabolites of commercial significance. Systematics can provide a useful framework for identifying species that may produce novel metabolites. However, previously proposed approaches to the systematics of Streptomyces have suffered from either poor interlaboratory comparability or insufficient resolution. In particular, the Streptomyces griseus 16S rRNA gene clade is the most challenging and least defined group within the genus Streptomyces in terms of phylogeny. Here we report the results of a multilocus sequence analysis scheme developed to address the phylogeny of this clade. Sequence fragments of six housekeeping genes, atpD, gyrB, recA, rpoB, trpB and 16S rRNA, were obtained for 53 reference strains that represent 45 valid species and subspecies. Analysis of each individual locus confirmed the suitability of loci and the congruence of single-gene trees for concatenation. Concatenated trees of three, four, five and all six genes were constructed, and the stability of the topology and discriminatory power of each tree were analysed. It can be concluded from the results that phylogenetic analysis based on multilocus sequences is more accurate and robust for species delineation within Streptomyces. A multilocus phylogeny of six genes proved to be optimal for elucidating the interspecies relationships within the S. griseus 16S rRNA gene clade. Our multilocus sequence analysis scheme provides a valuable tool that can be applied to other Streptomyces clades for refining the systematic framework of this genus.
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46. |
Burgie ES,
Holden HM,
( 2007 ) Molecular architecture of DesI: a key enzyme in the biosynthesis of desosamine. PMID : 17630700 : DOI : 10.1021/bi700751d PMC : PMC2528198 Abstract >>
Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found, for example, in such macrolide antibiotics as erthyromycin, azithromycin, and clarithromycin. The efficacies of these macrolide antibiotics are markedly reduced in the absence of desosamine. In the bacterium Streptomyces venezuelae, six enzymes are required for the production of dTDP-desosamine. The focus of this X-ray crystallographic analysis is the third enzyme in the pathway, a PLP-dependent aminotransferase referred to as DesI. The structure of DesI was solved in complex with its product, dTDP-4-amino-4,6-dideoxyglucose, to a nominal resolution of 2.1 A. Each subunit of the dimeric enzyme contains 12 alpha-helices and 14 beta-strands. Three cis-peptides are observed in each subunit, Phe 330, Pro 332, and Pro 339. The two active sites of the enzyme are located in clefts at the subunit/subunit interface. Electron density corresponding to the bound product clearly demonstrates a covalent bond between the amino group of the product and C-4' of the PLP cofactor. Interestingly, there are no hydrogen-bonding interactions between the protein and the dideoxyglucosyl group of the product (within 3.2 A). The only other sugar-modifying aminotransferase whose structure is known in the presence of product is PseC from Helicobacter pylori. This enzyme, as opposed to DesI, catalyzes amino transfer to the axial position of the sugar. A superposition of the two active sites for these proteins reveals that the major differences in ligand binding occur in the orientations of the deoxyglucosyl and phosphoryl groups. Indeed, the nearly 180 degrees difference in hexose orientation explains the equatorial versus axial amino transfer exhibited by DesI and PseC, respectively.
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47. |
Negretti S,
Narayan AR,
Chiou KC,
Kells PM,
Stachowski JL,
Hansen DA,
Podust LM,
Montgomery J,
Sherman DH,
( 2014 ) Directing group-controlled regioselectivity in an enzymatic C-H bond oxygenation. PMID : 24627965 : DOI : 10.1021/ja5016052 PMC : PMC4012894 Abstract >>
Highly regioselective remote hydroxylation of a natural product scaffold is demonstrated by exploiting the anchoring mechanism of the biosynthetic P450 monooxygenase PikCD50N-RhFRED. Previous studies have revealed structural and biochemical evidence for the role of a salt bridge between the desosamine N,N-dimethylamino functionality of the natural substrate YC-17 and carboxylate residues within the active site of the enzyme, and selectivity in subsequent C-H bond functionalization. In the present study, a substrate-engineering approach was conducted that involves replacing desosamine with varied synthetic N,N-dimethylamino anchoring groups. We then determined their ability to mediate enzymatic total turnover numbers approaching or exceeding that of the natural sugar, while enabling ready introduction and removal of these amino anchoring groups from the substrate. The data establish that the size, stereochemistry, and rigidity of the anchoring group influence the regioselectivity of enzymatic hydroxylation. The natural anchoring group desosamine affords a 1:1 mixture of regioisomers, while synthetic anchors shift YC-17 analogue C-10/C-12 hydroxylation from 20:1 to 1:4. The work demonstrates the utility of substrate engineering as an orthogonal approach to protein engineering for modulation of regioselective C-H functionalization in biocatalysis.
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48. |
Jung WS,
Kim E,
Yoo YJ,
Ban YH,
Kim EJ,
Yoon YJ,
( 2014 ) Characterization and engineering of the ethylmalonyl-CoA pathway towards the improved heterologous production of polyketides in Streptomyces venezuelae. PMID : 24413979 : DOI : 10.1007/s00253-013-5503-8 Abstract >>
Streptomyces venezuelae has an inherent advantage as a heterologous host for polyketide production due to its fast rate of growth that cannot be endowed easily through metabolic engineering. However, the utility of S. venezuelae as a host has been limited thus far due to its inadequate intracellular reserves of the (2S)-ethylmalonyl-CoA building block needed to support the biosynthesis of polyketides preventing the efficient production of the desired metabolite, such as tylactone. Here, via precursor supply engineering, we demonstrated that S. venezuelae can be developed into a more efficient general heterologous host for the quick production of polyketides. We first identified and functionally characterized the ethylmalonyl-CoA pathway which plays a major role in supplying the (2S)-ethylmalonyl-CoA extender unit in S. venezuelae. Next, S. venezuelae was successfully engineered to increase the intracellular ethylmalonyl-CoA concentration by the deletion of the meaA gene encoding coenzyme B??-dependent ethylmalonyl-CoA mutase in combination with ethylmalonate supplementation and was engineered to upregulate the expression of the heterologous tylosin PKS by overexpression of the pathway specific regulatory gene pikD. Thus, a dramatic increase (?10-fold) in tylactone production was achieved. In addition, the detailed insights into the role of the ethylmalonyl-CoA pathway, which is present in most streptomycetes, provides a general strategy to increase the ethylmalonyl-CoA supply for polyketide biosynthesis in the most prolific family of polyketide-producing bacteria.
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49. |
Whicher JR,
Dutta S,
Hansen DA,
Hale WA,
Chemler JA,
Dosey AM,
Narayan AR,
Håkansson K,
Sherman DH,
Smith JL,
Skiniotis G,
( 2014 ) Structural rearrangements of a polyketide synthase module during its catalytic cycle. PMID : 24965656 : DOI : 10.1038/nature13409 PMC : PMC4074775 Abstract >>
The polyketide synthase (PKS) mega-enzyme assembly line uses a modular architecture to synthesize diverse and bioactive natural products that often constitute the core structures or complete chemical entities for many clinically approved therapeutic agents. The architecture of a full-length PKS module from the pikromycin pathway of Streptomyces venezuelae creates a reaction chamber for the intramodule acyl carrier protein (ACP) domain that carries building blocks and intermediates between acyltransferase, ketosynthase and ketoreductase active sites (see accompanying paper). Here we determine electron cryo-microscopy structures of a full-length pikromycin PKS module in three key biochemical states of its catalytic cycle. Each biochemical state was confirmed by bottom-up liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry. The ACP domain is differentially and precisely positioned after polyketide chain substrate loading on the active site of the ketosynthase, after extension to the �]-keto intermediate, and after �]-hydroxy product generation. The structures reveal the ACP dynamics for sequential interactions with catalytic domains within the reaction chamber, and for transferring the elongated and processed polyketide substrate to the next module in the PKS pathway. During the enzymatic cycle the ketoreductase domain undergoes dramatic conformational rearrangements that enable optimal positioning for reductive processing of the ACP-bound polyketide chain elongation intermediate. These findings have crucial implications for the design of functional PKS modules, and for the engineering of pathways to generate pharmacologically relevant molecules.
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50. |
Santos M,
Prieto MA,
Gangoiti J,
Llama MJ,
( 2012 ) Characterization of a novel subgroup of extracellular medium-chain-length polyhydroxyalkanoate depolymerases from actinobacteria. PMID : 22865072 : DOI : 10.1128/AEM.01707-12 PMC : PMC3457088 Abstract >>
Nineteen medium-chain-length (mcl) poly(3-hydroxyalkanoate) (PHA)-degrading microorganisms were isolated from natural sources. From them, seven Gram-positive and three Gram-negative bacteria were identified. The ability of these microorganisms to hydrolyze other biodegradable plastics, such as short-chain-length (scl) PHA, poly(�`-caprolactone) (PCL), poly(ethylene succinate) (PES), and poly(l-lactide) (PLA), has been studied. On the basis of the great ability to degrade different polyesters, Streptomyces roseolus SL3 was selected, and its extracellular depolymerase was biochemically characterized. The enzyme consisted of one polypeptide chain of 28 kDa with a pI value of 5.2. Its maximum activity was observed at pH 9.5 with chromogenic substrates. The purified enzyme hydrolyzed mcl PHA and PCL but not scl PHA, PES, and PLA. Moreover, the mcl PHA depolymerase can hydrolyze various substrates for esterases, such as tributyrin and p-nitrophenyl (pNP)-alkanoates, with its maximum activity being measured with pNP-octanoate. Interestingly, when poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate [11%]) was used as the substrate, the main hydrolysis product was the monomer (R)-3-hydroxyoctanoate. In addition, the genes of several Actinobacteria strains, including S. roseolus SL3, were identified on the basis of the peptide de novo sequencing of the Streptomyces venezuelae SO1 mcl PHA depolymerase by tandem mass spectrometry. These enzymes did not show significant similarity to mcl PHA depolymerases characterized previously. Our results suggest that these distinct enzymes might represent a new subgroup of mcl PHA depolymerases.
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51. |
( 1997 ) Acyltransferase domain substitutions in erythromycin polyketide synthase yield novel erythromycin derivatives. PMID : 9335291 : DOI : 10.1128/jb.179.20.6416-6425.1997 PMC : PMC179558 Abstract >>
The methylmalonyl coenzyme A (methylmalonyl-CoA)-specific acyltransferase (AT) domains of modules 1 and 2 of the 6-deoxyerythronolide B synthase (DEBS1) of Saccharopolyspora erythraea ER720 were replaced with three heterologous AT domains that are believed, based on sequence comparisons, to be specific for malonyl-CoA. The three substituted AT domains were "Hyg" AT2 from module 2 of a type I polyketide synthase (PKS)-like gene cluster isolated from the rapamycin producer Streptomyces hygroscopicus ATCC 29253, "Ven" AT isolated from a PKS-like gene cluster of the pikromycin producer Streptomyces venezuelae ATCC 15439, and RAPS AT14 from module 14 of the rapamycin PKS gene cluster of S. hygroscopicus ATCC 29253. These changes led to the production of novel erythromycin derivatives by the engineered strains of S. erythraea ER720. Specifically, 12-desmethyl-12-deoxyerythromycin A, which lacks the methyl group at C-12 of the macrolactone ring, was produced by the strains in which the resident AT1 domain was replaced, and 10-desmethylerythromycin A and 10-desmethyl-12-deoxyerythromycin A, both of which lack the methyl group at C-10 of the macrolactone ring, were produced by the recombinant strains in which the resident AT2 domain was replaced. All of the novel erythromycin derivatives exhibited antibiotic activity against Staphylococcus aureus. The production of the erythromycin derivatives through AT replacements confirms the computer predicted substrate specificities of "Hyg" AT2 and "Ven" AT and the substrate specificity of RAPS AT14 deduced from the structure of rapamycin. Moreover, these experiments demonstrate that at least some AT domains of the complete 6-deoxyerythronolide B synthase of S. erythraea can be replaced by functionally related domains from different organisms to make novel, bioactive compounds.
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52. |
( 1996 ) A role for pabAB, a p-aminobenzoate synthase gene of Streptomyces venezuelae ISP5230, in chloramphenicol biosynthesis. PMID : 8704974 : DOI : 10.1099/13500872-142-6-1345 Abstract >>
Mutagenesis of Streptomyces venezuelae ISP5230 and selection for P-aminobenzoic acid-dependent growth in the presence of sulfanilamide yielded pab mutants (VS519 and VS620) that continued to produce chloramphenicol (Cm), although with increased medium dependence. Transforming the mutants with pDQ102 or pDQ103, which carried a pab-complementing fragment from S. venezuelae ISP5230 in alternative orientations, restored uniformly high Cm production in VS620, but did not alter the medium dependence of Cm production in VS519. The cloned S. venezuelae DNA fragment was subcloned and trimmed to the minimum size conferring pab complementation. The resulting 2.8 kb BamHl-Sacl fragment was sequenced. Codon preference analysis showed one complete ORF encoding a polypeptide of 670 amino acids. Comparison of the deduced amino acid sequence with database proteins indicated that the N- and C-terminal regions resembled PabA and PabB, respectively, of numerous bacteria. The gene product showed overall sequence similarity to the product of a fused pabAB gene associated with secondary metabolism in Streptomyces griseus. Insertion of an apramycin resistance gene into pabAB cloned in a segregationally unstable vector and replacement of the S. venezuelae chromosomal pabAB with the disrupted copy lowered sulfanilamide resistance from 25 to 5 micrograms mL-1 and blocked Cm production.
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53. |
( 1996 ) Accumulation of the angucycline antibiotic rabelomycin after disruption of an oxygenase gene in the jadomycin B biosynthetic gene cluster of Streptomyces venezuelae. PMID : 8581159 : DOI : 10.1099/13500872-142-1-123 Abstract >>
DNA from a region downstream of and overlapping the polyketide synthase (PKS) gene cluster for jadomycin B biosynthesis in Streptomyces venezuelae was cloned and sequenced. Analysis of the nucleotide sequence located one complete ORF (ORF6), an incomplete one representing the 3' region of ORF4 in the PKS cluster, and a second incomplete one (ORF7). The deduced amino acid sequences for ORFs 6 and 7 resemble those of oxygenases. Since a plausible biosynthetic pathway for jadomycin B includes an angular polyketide intermediate that undergoes oxidative ring fission before condensation with an amino acid, we subcloned one of the presumptive oxygenase genes (ORF6) in a segregationally unstable shuttle vector (pHJL400) and disrupted it by inserting the gene for apramycin resistance. Transformation of S. venezuelae with the disruption vector and selection for apramycin resistance gave mutants blocked in jadomycin biosynthesis. Southern hybridization confirmed that gene replacement had occurred. Cultures of the mutants accumulated a metabolite identified by comparison with an authentic sample as rabelomycin, a non-nitrogenous polyketide-derived antibiotic originally isolated from Streptomyces olivaceus.
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54. |
Yang K,
Han L,
Vining LC,
( 1995 ) Regulation of jadomycin B production in Streptomyces venezuelae ISP5230: involvement of a repressor gene, jadR2. PMID : 7592375 : DOI : 10.1128/jb.177.21.6111-6117.1995 PMC : PMC177450 Abstract >>
The nucleotide sequence of a region upstream of the type II polyketide synthase genes in the cluster for biosynthesis of the polyketide antibiotic jadomycin B in Streptomyces venezuelae contained an open reading frame encoding a sequence of 196 amino acids that resembeled sequences deduced for a group of repressor proteins. The strongest similarity was to EnvR of Escherichia coli, but the sequence also resembled MtrR, AcrR, TetC, and TcmR, all of which are involved in regulating resistance to antibiotics or toxic hydrophobic substances in the environment. Disruption of the nucleotide sequence of this putative S. venezuelae repressor gene (jadR2), by insertion of an apramycin resistance gene at an internal MluI site, and replacement of the chromosomal gene generated mutants that produced jadomycin B without the stress treatments (exposure to heat shock or to toxic concentrations of ethanol) required for jadomycin B production by the wild type. When cultures of the disruption mutants were ethanol stressed, they overproduced the antibiotic. From these results it was concluded that expression of the jadomycin B biosynthesis genes are negatively regulated by jadR2.
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55. |
( 2013 ) The structure of DesR from Streptomyces venezuelae, a �]-glucosidase involved in macrolide activation. PMID : 23225731 : DOI : 10.1002/pro.2204 PMC : PMC3719083 Abstract >>
Antibiotics have, indeed, altered the course of human history as is evidenced by the increase in human life expectancy since the 1940s. Many of these natural compounds are produced by bacteria that, by necessity, must have efficient self-resistance mechanisms. The methymycin/pikromycin producing species Streptomyces venezuelae, for example, utilizes �]-glucosylation of its macrolide products to neutralize their effects within the confines of the cell. Once released into the environment, these compounds are activated by the removal of the glucose moiety. In S. venezuelae, the enzyme responsible for removal of the sugar from the parent compound is encoded by the desR gene and referred to as DesR. It is a secreted enzyme containing 828 amino acid residues, and it is known to be a retaining glycosidase. Here, we describe the structure of the DesR/D-glucose complex determined to 1.4-? resolution. The overall architecture of the enzyme can be envisioned in terms of three regions: a catalytic core and two auxiliary domains. The catalytic core harbors the binding platform for the glucose ligand. The first auxiliary domain adopts a "PA14 fold," whereas the second auxiliary domain contains an immunoglobulin-like fold. Asp 273 and Glu 578 are in the proper orientation to function as the catalytic base and proton donor, respectively, required for catalysis. The overall fold of the core region places DesR into the GH3 glycoside hydrolase family of enzymes. Comparison of the DesR structure with the �]-glucosidase from Kluyveromyces marxianus shows that their PA14 domains assume remarkably different orientations.
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56. |
( 1998 ) Regulation of an anthranilate synthase gene in Streptomyces venezuelae by a trp attenuator. PMID : 9695930 : DOI : 10.1099/00221287-144-7-1971 Abstract >>
The nucleotide sequence of a 2-4 kb BamHI-SalI fragment of Streptomyces venezuelae ISP5230 DNA that complements trpE and trpG mutations in Escherichia coli contains two ORFs. The larger of these (ORF2) encodes a 624 amino acid sequence similar to the overall sequence of the two subunits of anthranilate synthase. The two-thirds nearest the amino terminus resembles the aminase subunit; the remaining one-third resembles the glutamine amidotransferase subunit. Upstream of ORF2 is a small ORF encoding 18 amino acids that include three adjacent Trp residues; in addition the ORF contains inverted repeats with sequence and positional similarity to the products of attenuator (trpL) regions that regulate tryptophan biosynthesis in other bacteria. In cultures of a trpC mutant of S. venezuelae, increasing the concentration of exogenous tryptophan decreased the formation of anthranilate synthase; similar evidence of endproduct repression was obtained in a trpCER mutant of E. coli transformed with a vector containing the cloned DNA fragment from S. venezuelae. The anthranilate synthase activity in S. venezuelae cell extracts was inhibited by tryptophan, although only at high concentrations of the amino acid. A two-base deletion introduced into the cloned S. venezuelae DNA fragment prevented complementation of a trpE mutation in E. coli. However, S. venezuelae transformants in which the two-base deletion had been introduced by replacement of homologous chromosomal DNA did not exhibit a Trp- phenotype. The result implies that S. venezuelae has one or more additional genes for anthranilate synthase. In alignments with anthranilate synthase genes from other organisms, ORF2 from S. venezuelae most closely resembled genes for phenazine biosynthesis in Pseudomonas. The results bear on the function of the gene in S. venezuelae.
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57. |
( 1998 ) A gene cluster for macrolide antibiotic biosynthesis in Streptomyces venezuelae: architecture of metabolic diversity. PMID : 9770448 : DOI : 10.1073/pnas.95.21.12111 PMC : PMC22793 Abstract >>
In a survey of microbial systems capable of generating unusual metabolite structural variability, Streptomyces venezuelae ATCC 15439 is notable in its ability to produce two distinct groups of macrolide antibiotics. Methymycin and neomethymycin are derived from the 12-membered ring macrolactone 10-deoxymethynolide, whereas narbomycin and pikromycin are derived from the 14-membered ring macrolactone, narbonolide. This report describes the cloning and characterization of the biosynthetic gene cluster for these antibiotics. Central to the cluster is a polyketide synthase locus (pikA) that encodes a six-module system comprised of four multifunctional proteins, in addition to a type II thioesterase (TEII). Immediately downstream is a set of genes for desosamine biosynthesis (des) and macrolide ring hydroxylation. The study suggests that Pik TEII plays a role in forming a metabolic branch through which polyketides of different chain length are generated, and the glycosyl transferase (encoded by desVII) has the ability to catalyze glycosylation of both the 12- and 14-membered ring macrolactones. Moreover, the pikC-encoded P450 hydroxylase provides yet another layer of structural variability by introducing regiochemical diversity into the macrolide ring systems. The data support the notion that the architecture of the pik gene cluster as well as the unusual substrate specificity of particular enzymes contributes to its ability to generate four macrolide antibiotics.
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58. |
( 1998 ) Characterization of the macrolide P-450 hydroxylase from Streptomyces venezuelae which converts narbomycin to picromycin. PMID : 9778370 : DOI : 10.1021/bi981699c Abstract >>
The post-polyketide synthase (PKS) biosynthetic tailoring of macrolide antibiotics usually involves one or more oxidation reactions catalyzed by cytochrome P450 monooxygenases. As the specificities of members from this class of enzymes vary significantly among PKS gene clusters, the identification and study of new macrolide P450s are important to the growing field of combinatorial biosynthesis. We have isolated the cytochrome P450 gene picK from Streptomyces venezuelae which is responsible for the C-12 hydroxylation of narbomycin to picromycin. The gene was located by searching regions proximal to modular PKS genes with a probe for macrolide P450 monooxygenases. The overproduction of PicK with a C-terminal six-His affinity tag (PicK/6-His) in Escherichia coli aided the purification of the enzyme for kinetic analysis. PicK/6-His was shown to catalyze the in vitro C-12 hydroxylation of narbomycin with a kcat of 1.4 s-1, which is similar to the value reported for the related C-12 hydroxylation of erythromycin D by the EryK hydroxylase. The unique specificity of this enzyme should be useful for the modification of novel macrolide substrates similar to narbomycin, in particular, ketolides, a promising class of semisynthetic macrolides with activity against erythromycin-resistant pathogens.
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59. |
( 1998 ) Hydroxylation of macrolactones YC-17 and narbomycin is mediated by the pikC-encoded cytochrome P450 in Streptomyces venezuelae. PMID : 9831532 : Abstract >>
. Streptomyces venezuelae produces two groups of antibiotics that include the 12-membered ring macrolides methymycin and neomethymycin, and the 14-membered ring macrolide pikromycin. Methymycin and pikromycin are derived from the corresponding precursors, YC-17 and narbomycin, respectively, by hydroxylation of the tertiary carbon position (C-10 in YC-17 or C-12 in narbomycin) on the macrolactone ring. In contrast, neomethymycin is derived from YC-17 by hydroxylation of the secondary carbon (C-12) of the propionyl starter unit sidechain. . Using a genetic and biochemical approach we have characterized a single P450 hydroxylase (PikC) in the methymycin/pikromycin biosynthetic gene cluster (pik) from S. venezuelae. Inactivation of pikC abolished production of all hydroxylated macrolides, with corresponding accumulation of YC-17 and narbomycin in the culture medium. The enzyme was produced efficiently and purified as a His-tagged protein from recombinant Escherichia coli cells. Purified PikC effectively converts YC-17 into methymycin and neomethymycin and narbomycin into pikromycin in vitro. . These results demonstrate that PikC is responsible for the conversion of YC-17 to methymycin and neomethymycin, and narbomycin to pikromycin in S. venezuelae. This substrate flexibility is unique and represents the first example of a P450 hydroxylase that can accept 12- and 14-membered ring macrolides as substrates, as well as functionalize at two positions on the macrolactone system. The broad substrate specificity of PikC provides a potentially valuable entry into the construction of novel macrolide- and ketolide-based antibiotics.
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