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1. Serina  S, Radice  F, Maffioli  S, Donadio  S, Sosio  M,     ( 2004 )

Glycopeptide resistance determinants from the teicoplanin producer Actinoplanes teichomyceticus.

FEMS microbiology letters 240 (1)
PMID : 15500981  :   DOI  :   10.1016/j.femsle.2004.09.017    
Abstract >>
In enterococci and other pathogenic bacteria, high-level resistance to vancomycin and other glycopeptide antibiotics requires the action of the van genes, which direct the synthesis of peptidoglycan terminating in the depsipeptide D-alanyl-D-lactate, in place of the usual D-Ala-D-Ala. The Actinoplanes teichomyceticus tcp cluster, devoted to the biosynthesis of the glycopeptide antibiotic teicoplanin, contains van genes associated to a murF-like sequence (murF2). We show that A. teichomyceticus contains also a house-keeping murF1 gene, capable of complementing a temperature sensitive Escherichia coli murF mutant. MurF1, expressed in Streptomyces lividans, can catalyze the addition of either D-Ala-D-Ala or D-Ala-D-Lac to the UDP-N-acetyl-muramyl-L-Ala-D-Glu-d-Lys. However, similarly expressed MurF2 shows a small enzymatic activity only with D-Ala-D-lactate. Introduction of a single copy of the entire set of van genes confers resistance to teicoplanin-type glycopeptides to S. coelicolor.
KeywordMeSH Terms
2. Li  TL, Huang  F, Haydock  SF, Mironenko  T, Leadlay  PF, Spencer  JB,     ( 2004 )

Biosynthetic gene cluster of the glycopeptide antibiotic teicoplanin: characterization of two glycosyltransferases and the key acyltransferase.

Chemistry & biology 11 (1)
PMID : 15113000  :   DOI  :   10.1016/j.chembiol.2004.01.001    
Abstract >>
The gene cluster encoding biosynthesis of the clinically important glycopeptide antibiotic teicoplanin has been cloned from Actinoplanes teichomyceticus. Forty-nine putative open reading frames (ORFs) were identified within an 89 kbp genetic locus and assigned roles in teicoplanin biosynthesis, export, resistance, and regulation. Two ORFs, designated orfs 1 and 10*, showed significant homology to known glycosyltransferases. When heterologously expressed in Escherichia coli, these glycosyltransferases were shown to catalyze the transfer of UDP-(N-acetyl)-glucosamine onto, respectively, 3-chloro-beta-hydroxytyrosine-6 (3-Cl-6betaHty) and 4-hydroxyphenylglycine-4 (4Hpg) of the teicoplanin heptapeptide aglycone. The product of another ORF, orf11*, was demonstrated in vitro to transfer n-acetyl-, n-butyryl-, and n-octanoyl-groups from acyl-CoA donors either to a free UDP-aminosugar or to an aminosugar moiety in the teicoplanin pseudoaglycone, thus identifying Orf11* as the key acyltransferase in teicoplanin maturation. These findings should accelerate the combinatorial engineering of new and improved glycopeptide drugs.
KeywordMeSH Terms
Genes, Bacterial
3. Sosio  M, Kloosterman  H, Bianchi  A, de Vreugd  P, Dijkhuizen  L, Donadio  S,     ( 2004 )

Organization of the teicoplanin gene cluster in Actinoplanes teichomyceticus.

Microbiology (Reading, England) 150 (Pt 1)
PMID : 14702401  :   DOI  :   10.1099/mic.0.26507-0    
Abstract >>
The glycopeptide teicoplanin is used for the treatment of serious infections caused by Gram-positive pathogens. The tcp gene cluster, devoted to teicoplanin biosynthesis in the actinomycete Actinoplanes teichomyceticus, was isolated and characterized. From sequence analysis, the tcp cluster spans approximately 73 kb and includes 39 ORFs participating in teicoplanin biosynthesis, regulation, resistance and export. Of these, 34 ORFs find a match in at least one of the five glycopeptide gene clusters previously characterized. Putative roles could be assigned for most of the tcp genes. The two glycosyltransferases responsible for attaching amino sugars to amino acids 4 and 6 of the teicoplanin aglycon were overexpressed in Escherichia coli and characterized. They both recognize N-acetylglucosamine as the substrate. tGtfA can add a sugar residue in the presence or absence of N-acetylglucosamine at amino acid 4, while tGtfB can only glycosylate the teicoplanin aglycon.
KeywordMeSH Terms
Genes, Bacterial
Multigene Family
4. Li  Z, Rupasinghe  SG, Schuler  MA, Nair  SK,     ( 2011 )

Crystal structure of a phenol-coupling P450 monooxygenase involved in teicoplanin biosynthesis.

Proteins 79 (6)
PMID : 21445994  :   DOI  :   10.1002/prot.22996     PMC  :   PMC3103887    
Abstract >>
The lipoglycopeptide antibiotic teicoplanin has proven efficacy against gram-positive pathogens. Teicoplanin is distinguished from the vancomycin-type glycopeptide antibiotics, by the presence of an additional cross-link between the aromatic amino acids 1 and 3 that is catalyzed by the cytochrome P450 monooxygenase Orf6* (CYP165D3). As a goal towards understanding the mechanism of this phenol-coupling reaction, we have characterized recombinant Orf6* and determined its crystal structure to 2.2-? resolution. Although the structure of Orf6* reveals the core fold common to other P450 monooxygenases, there are subtle differences in the disposition of secondary structure elements near the active site cavity necessary to accommodate its complex heptapeptide substrate. Specifically, the orientation of the F and G helices in Orf6* results in a more closed active site than found in the vancomycin oxidative enzymes OxyB and OxyC. In addition, Met226 in the I helix replaces the more typical Gly/Ala residue that is positioned above the heme porphyrin ring, where it forms a hydrogen bond with a heme iron-bound water molecule. Sequence comparisons with other phenol-coupling P450 monooxygenases suggest that Met226 plays a role in determining the substrate regiospecificity of Orf6*. These features provide further insights into the mechanism of the cross-linking mechanisms that occur during glycopeptide antibiotics biosynthesis.
KeywordMeSH Terms
5. Chan  HC, Huang  YT, Lyu  SY, Huang  CJ, Li  YS, Liu  YC, Chou  CC, Tsai  MD, Li  TL,     ( 2011 )

Regioselective deacetylation based on teicoplanin-complexed Orf2* crystal structures.

Molecular bioSystems 7 (4)
PMID : 21267472  :   DOI  :   10.1039/c0mb00320d    
Abstract >>
Lipoglycopeptide antibiotics are more effective than vancomycin against MRSA as they carry an extra aliphatic acyl side chain on glucosamine (Glm) at residue 4 (r4). The biosynthesis of the r4 N-acyl Glc moiety at teicoplanin (Tei) or A40926 has been elucidated, in which the primary amine nucleophile of Glm is freed from the r4 GlcNac pseudo-Tei precursor by Orf2* for the subsequent acylation reaction to occur. In this report, two Orf2* structures in complex with �]-D-octyl glucoside or Tei were solved. Of the complexed structures, the substrate binding site and a previously unknown hydrophobic cavity were revealed, wherein r4 GlcNac acts as the key signature for molecular recognition and the cavity allows substrates carrying longer acyl side chains in addition to the acetyl group. On the basis of the complexed structures, a triple-mutation mutant S98A/V121A/F193Y is able to regioselectively deacetylate r6 GlcNac pseudo-Tei instead of that at r4. Thereby, novel analogs can be made at the r6 sugar moiety.
KeywordMeSH Terms
Models, Molecular
Protein Engineering
Teicoplanin
6. Cryle  MJ, Staaden  J, Schlichting  I,     ( 2011 )

Structural characterization of CYP165D3, a cytochrome P450 involved in phenolic coupling in teicoplanin biosynthesis.

Archives of biochemistry and biophysics 507 (1)
PMID : 20974107  :   DOI  :   10.1016/j.abb.2010.10.017    
Abstract >>
Teicoplanin is a glycopeptide antibiotic with activity against Gram-positive bacteria and remains one of the last lines of clinical defense against certain bacterial infections. We have cloned, expressed, and purified the cytochrome P450 OxyE (CYP165D3) from the teicoplanin biosynthetic gene cluster of Actinoplanes teichomyceticus, which is responsible for the phenolic coupling of the aromatic side chains of the first and third peptide residues in the teicoplanin peptide. The crystal structure of OxyE has been determined to 2.5? resolution, revealing the probable binding surface for the carrier protein substrate and an extension of the active site into a pocket located above the �]-1 sheet. The binding of potential substrates to OxyE shows that peptidyl carrier protein-bound linear peptides bind to OxyE, albeit with low affinity in the absence of a phenolic cross-link that should normally be installed by another Oxy protein in the teicoplanin biosynthetic pathway. This result indicates that the carrier protein alone is not sufficient for tight substrate binding to OxyE and that the Oxy proteins sense the structure of the bound peptide in addition to the presence of the carrier protein, a feature distinct from other carrier protein/P450 systems.
KeywordMeSH Terms
7. Truman  AW, Dias  MV, Wu  S, Blundell  TL, Huang  F, Spencer  JB,     ( 2009 )

Chimeric glycosyltransferases for the generation of hybrid glycopeptides.

Chemistry & biology 16 (6)
PMID : 19549605  :   DOI  :   10.1016/j.chembiol.2009.04.013    
Abstract >>
Glycodiversification, an invaluable tool for generating biochemical diversity, can be catalyzed by glycosyltransferases, which attach activated sugar "donors" onto "acceptor" molecules. However, many glycosyltransferases can tolerate only minor modifications to their native substrates, thus making them unsuitable tools for current glycodiversification strategies. Here we report the production of functional chimeric glycosyltransferases by mixing and matching the N- and C-terminal domains of glycopeptide glycosyltransferases. Using this method we have generated hybrid glycopeptides and have demonstrated that domain swapping can result in a predictable switch of substrate specificity, illustrating that N- and C-terminal domains predominantly dictate acceptor and donor specificity, respectively. The determination of the structure of a chimera in complex with a sugar donor analog shows that almost all sugar-glycosyltransferase binding interactions occur in the C-terminal domain.
KeywordMeSH Terms
8. Zou  Y, Brunzelle  JS, Nair  SK,     ( 2008 )

Crystal structures of lipoglycopeptide antibiotic deacetylases: implications for the biosynthesis of A40926 and teicoplanin.

Chemistry & biology 15 (6)
PMID : 18559264  :   DOI  :   10.1016/j.chembiol.2008.05.009    
Abstract >>
The lipoglycopeptide antibiotics teicoplanin and A40926 have proven efficacy against Gram-positive pathogens. These drugs are distinguished from glycopeptide antibiotics by N-linked long chain acyl-D-glucosamine decorations that contribute to antibacterial efficacy. During the biosynthesis of lipoglycopeptides, tailoring glycosyltransferases attach an N-acetyl-D-glucosamine to the aglycone, and this N-acetyl-glucosaminyl pseudoaglycone is deacetylated prior to long chain hydrocarbon attachment. Here we present several high-resolution crystal structures of the pseudoaglycone deacetylases from the biosynthetic pathways of teicoplanin and A40926. The cocrystal structure of the teicoplanin pseudoaglycone deacetylase with a fatty acid product provides further insights into the roles of active-site residues, and suggests mechanistic similarities with structurally distinct zinc deacetylases, such as peptidoglycan deacetylase and LpxC. A unique, structurally mobile capping lid, located at the apex of these pseudoaglycone deacetylases, likely serves as a determinant of substrate specificity.
KeywordMeSH Terms
9. Ha  HS, Hwang  YI, Choi  SU,     ( 2008 )

Application of conjugation using phiC31 att/int system for Actinoplanes teichomyceticus, a producer of teicoplanin.

Biotechnology letters 30 (7)
PMID : 18317703  :   DOI  :   10.1007/s10529-008-9671-z    
Abstract >>
Actinoplanes teichomyceticus produces teicoplanin, which is a glycopeptide antibiotic for Gram-positive pathogenic bacteria and methicillin-resistant Staphylococcus aureus (MRSA). For a molecular genetic study of A. teichomyceticus, an effective transformation method using the conjugal transfer of DNA from E. coli to spores of A. teichomyceticus was established for the first time, based on the bacteriophage varphiC31 att/int system, in the genus of Actinoplanes. The high frequency of transconjugation was obtained on MS medium containing 40 mM MgCl(2), using 1.25 x 10(8) E. coli donor cells and 10(5) spores without a heat treatment. In addition, by cloning and sequencing the attB site A. teichomyceticus was shown to contain a single attB site within an ORF coding for a pirin homolog. Also, its attB site sequence showed high homology to that of Streptomyces lividans, unlike the case of Kitasatospora setae despite being a non-Streptomyces actinomycete, which seems to be closely related to the high transconjugation frequency of A. teichomyceticus.
KeywordMeSH Terms
Conjugation, Genetic
Teicoplanin
10. Sosio  M, Lorenzetti  R, Robbiati  F, Denaro  M,     ( 1991 )

Nucleotide sequence to a teicoplanin resistance gene from Actinoplanes teichomyceticus.

Biochimica et biophysica acta 1089 (3)
PMID : 1830492  :   DOI  :   10.1016/0167-4781(91)90185-o    
Abstract >>
A DNA fragment, isolated from A. teichomyceticus and able to confer teicoplanin resistance in a sensitive host, has been sequenced. It reveals the presence of two open reading frames (ORFs) positioned on opposite strands, named ORF1 and ORF2. ORF2 seems to be responsible for the acquisition of the resistance character.
KeywordMeSH Terms
Anti-Bacterial Agents
Genes, Bacterial
11. Horbal  L, Ostash  B, Luzhetskyy  A, Walker  S, Kalinowski  J, Fedorenko  V,     ( 2016 )

A gene cluster for the biosynthesis of moenomycin family antibiotics in the genome of teicoplanin producer Actinoplanes teichomyceticus.

Applied microbiology and biotechnology 100 (17)
PMID : 27344593  :   DOI  :   10.1007/s00253-016-7685-3    
Abstract >>
Moenomycins are phosphoglycolipid antibiotics notable for their extreme potency, unique mode of action, and proven record of use in animal nutrition without selection for resistant microflora. There is a keen interest in manipulation of structures of moenomycins in order to better understand their structure-activity relationships and to generate improved analogs. Only two almost identical moenomycin biosynthetic gene clusters are known, limiting our knowledge of the evolution of moenomycin pathways and our ability to genetically diversify them. Here, we report a novel gene cluster (tchm) that directs production of the phosphoglycolipid teichomycin in Actinoplanes teichomyceticus. Its overall genetic architecture is significantly different from that of the moenomycin biosynthesis (moe) gene clusters of Streptomyces ghanaensis and Streptomyces clavuligerus, featuring multiple gene rearrangements and two novel structural genes. Involvement of the tchm cluster in teichomycin biosynthesis was confirmed via heterologous co-expression of amidotransferase tchmH5 and moe genes. Our work sets the background for further engineering of moenomycins and for deeper inquiries into the evolution of this fascinating biosynthetic pathway.
KeywordMeSH Terms
Actinoplanes
Moenomycins
Nosokomycin
Teichomycin
12. Haslinger  K, Cryle  MJ,     ( 2016 )

Structure of OxyA tei: completing our picture of the glycopeptide antibiotic producing Cytochrome P450 cascade.

FEBS letters 590 (4)
PMID : 26820384  :   DOI  :   10.1002/1873-3468.12081    
Abstract >>
Cyclization of glycopeptide antibiotic precursors occurs in either three or four steps catalyzed by Cytochrome P450 enzymes. Three of these enzymes have been structurally characterized to date with the second enzyme along the pathway, OxyA, escaping structural analysis. We are now able to present the structure of OxyAtei involved in teicoplanin biosynthesis - the same enzyme recently shown to be the first active OxyA homolog. In spite of the hydrophobic character of the teicoplanin precursor, the polar active site of OxyAtei and its affinity for certain azole inhibitors hint at its preference for substrates with polar decorations.
KeywordMeSH Terms
Cytochrome P450
glycopeptide antibiotics
nonribosomal peptide synthesis
phenolic coupling
teicoplanin
13. Haslinger  K, Redfield  C, Cryle  MJ,     ( 2015 )

Structure of the terminal PCP domain of the non-ribosomal peptide synthetase in teicoplanin biosynthesis.

Proteins 83 (4)
PMID : 25586301  :   DOI  :   10.1002/prot.24758    
Abstract >>
The biosynthesis of the glycopeptide antibiotics, of which teicoplanin and vancomycin are representative members, relies on the combination of non-ribosomal peptide synthesis and modification of the peptide by cytochrome P450 (Oxy) enzymes while the peptide remains bound to the peptide synthesis machinery. We have structurally characterized the final peptidyl carrier protein domain of the teicoplanin non-ribosomal peptide synthetase machinery: this domain is believed to mediate the interactions with tailoring Oxy enzymes in addition to its function as a shuttle for intermediates between multiple non-ribosomal peptide synthetase domains. Using solution state NMR, we have determined structures of this PCP domain in two states, the apo and the post-translationally modified holo state, both of which conform to a four-helix bundle assembly. The structures exhibit the same general fold as the majority of known carrier protein structures, in spite of the complex biosynthetic role that PCP domains from the final non-ribosomal peptide synthetase module must play in glycopeptide antibiotic biosynthesis. These structures thus support the hypothesis that it is subtle rearrangements, rather than dramatic conformational changes, which govern carrier protein interactions and selectivity during non-ribosomal peptide synthesis.
KeywordMeSH Terms
4-helix bundle
carrier protein
glycopeptide antibiotic biosynthesis
peptide biosynthesis
posttranslational modification
secondary metabolism
14. Haslinger  K, Peschke  M, Brieke  C, Maximowitsch  E, Cryle  MJ,     ( 2015 )

X-domain of peptide synthetases recruits oxygenases crucial for glycopeptide biosynthesis.

Nature 521 (7550)
PMID : 25686610  :   DOI  :   10.1038/nature14141    
Abstract >>
Non-ribosomal peptide synthetase (NRPS) mega-enzyme complexes are modular assembly lines that are involved in the biosynthesis of numerous peptide metabolites independently of the ribosome. The multiple interactions between catalytic domains within the NRPS machinery are further complemented by additional interactions with external enzymes, particularly focused on the final peptide maturation process. An important class of NRPS metabolites that require extensive external modification of the NRPS-bound peptide are the glycopeptide antibiotics (GPAs), which include vancomycin and teicoplanin. These clinically relevant peptide antibiotics undergo cytochrome P450-catalysed oxidative crosslinking of aromatic side chains to achieve their final, active conformation. However, the mechanism underlying the recruitment of the cytochrome P450 oxygenases to the NRPS-bound peptide was previously unknown. Here we show, through in vitro studies, that the X-domain, a conserved domain of unknown function present in the final module of all GPA NRPS machineries, is responsible for the recruitment of oxygenases to the NRPS-bound peptide to perform the essential side-chain crosslinking. X-ray crystallography shows that the X-domain is structurally related to condensation domains, but that its amino acid substitutions render it catalytically inactive. We found that the X-domain recruits cytochrome P450 oxygenases to the NRPS and determined the interface by solving the structure of a P450-X-domain complex. Additionally, we demonstrated that the modification of peptide precursors by oxygenases in vitro--in particular the installation of the second crosslink in GPA biosynthesis--occurs only in the presence of the X-domain. Our results indicate that the presentation of peptidyl carrier protein (PCP)-bound substrates for oxidation in GPA biosynthesis requires the presence of the NRPS X-domain to ensure conversion of the precursor peptide into a mature aglycone, and that the carrier protein domain alone is not always sufficient to generate a competent substrate for external cytochrome P450 oxygenases.
KeywordMeSH Terms
15. Ostash  B, Yushchuk  O, Tistechok  S, Mutenko  H, Horbal  L, Muryn  A, Dacyuk  Y, Kalinowski  J, Luzhetskyy  A, Fedorenko  V,     ( 2015 )

The adpA-like regulatory gene from Actinoplanes teichomyceticus: in silico analysis and heterologous expression.

World journal of microbiology & biotechnology 31 (8)
PMID : 26041369  :   DOI  :   10.1007/s11274-015-1882-6    
Abstract >>
Analysis of the draft sequence of the genome of teicoplanin producer Actinoplanes teichomyceticus (NRRL-B16726) led to identification of several genes encoding AraC-family regulators that resemble AdpA, master regulator of transcription in Streptomyces. We elucidated possible regulatory functions of one of the identified genes, adpA19(at), most similar to archetypal adpA from model Streptomyces species, in a series of expression experiments. Introduction of adpA19 at under control of its own promoter on moderate copy number vector pKC1139 into NRRL-B16726 had no influence on antibiotic production and sporulation. Introduction of adpA19 at into Streptomyces coelicolor M145 and several S. ghanaensis strains had major influence on antibiotic production by these bacteria. Finally, adpA19 at expression in a set of soil actinomycete isolates led to induction of synthesis of antibiotic compounds. Our data point to pleiotropic regulatory role of adpA19(at), warranting its use as a tool to manipulate secondary metabolome of actinomycetes.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Genes, Regulator
16. Haslinger  K, Maximowitsch  E, Brieke  C, Koch  A, Cryle  MJ,     ( 2014 )

Cytochrome P450 OxyBtei catalyzes the first phenolic coupling step in teicoplanin biosynthesis.

Chembiochem : a European journal of chemical biology 15 (18)
PMID : 25358800  :   DOI  :   10.1002/cbic.201402441    
Abstract >>
Bacterial cytochrome P450s form a remarkable clade of the P450 superfamily of oxidative hemoproteins, and are often involved in the biosynthesis of complex natural products. Those in a subgroup known as "Oxy enzymes" play a crucial role in the biosynthesis of glycopeptide antibiotics, including vancomycin and teicoplanin. The Oxy enzymes catalyze crosslinking of aromatic residues in the non-ribosomal antibiotic precursor peptide while it remains bound to the non-ribosomal peptide synthetase (NRPS); this crosslinking secures the three-dimensional structure of the glycopeptide, crucial for antibiotic activity. We have characterized OxyBtei , the first of the Oxy enzymes in teicoplanin biosynthesis. Our results reveal that OxyBtei possesses a structure similar to those of other Oxy proteins and is active in crosslinking NRPS-bound peptide substrates. However, OxyBtei displays a significantly altered activity spectrum against peptide substrates compared to its well-studied vancomycin homologue.
KeywordMeSH Terms
biocatalysis
cytochromes
peptide biosynthesis
secondary metabolism
teicoplanin
17. Lyu  SY, Liu  YC, Chang  CY, Huang  CJ, Chiu  YH, Huang  CM, Hsu  NS, Lin  KH, Wu  CJ, Tsai  MD, Li  TL,     ( 2014 )

Multiple complexes of long aliphatic N-acyltransferases lead to synthesis of 2,6-diacylated/2-acyl-substituted glycopeptide antibiotics, effectively killing vancomycin-resistant enterococcus.

Journal of the American Chemical Society 136 (31)
PMID : 25095906  :   DOI  :   10.1021/ja504125v    
Abstract >>
Teicoplanin A2-2 (Tei)/A40926 is the last-line antibiotic to treat multidrug-resistant Gram-positive bacterial infections, e.g., methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE). This class of antibiotics is powered by the N-acyltransferase (NAT) Orf11*/Dbv8 through N-acylation on glucosamine at the central residue of Tei/A40926 pseudoaglycone. The NAT enzyme possesses enormous value in untapped applications; its advanced development is hampered largely due to a lack of structural information. In this report, we present eight high-resolution X-ray crystallographic unary, binary, and ternary complexes in order to decipher the molecular basis for NAT's functionality. The enzyme undergoes a multistage conformational change upon binding of acyl-CoA, thus allowing the uploading of Tei pseudoaglycone to enable the acyl-transfer reaction to take place in the occlusion between the N- and C-halves of the protein. The acyl moiety of acyl-CoA can be bulky or lengthy, allowing a large extent of diversity in new derivatives that can be formed upon its transfer. Vancomycin/synthetic acyl-N-acetyl cysteamine was not expected to be able to serve as a surrogate for an acyl acceptor/donor, respectively. Most strikingly, NAT can catalyze formation of 2-N,6-O-diacylated or C6��C2 acyl-substituted Tei analogues through an unusual 1,4-migration mechanism under stoichiometric/solvational reaction control, wherein selected representatives showed excellent biological activities, effectively counteracting major types (VanABC) of VRE.
KeywordMeSH Terms
18. Kim  DY, Hwang  YI, Choi  SU,     ( 2011 )

Cloning of metK from Actinoplanes teichomyceticus ATCC31121 and effect of its high expression on antibiotic production.

Journal of microbiology and biotechnology 21 (12)
PMID : 22210616  :  
Abstract >>
A metK gene encoding S-adenosyl-L-methionine synthetase was cloned from the non-Streptomyces actinomycetes, Actinoplanes teichomyceticus ATCC31121. In order to evaluate the effect of the metK expression on antibiotic production in actinomycetes, an expression vector harboring the metK gene was constructed and introduced into Streptomyces lividans TK24 and A. teichomyceticus, and the antibiotic production of the exconjugants was assessed. As a result, it was determined that the expression of metK induced 17-fold and 2.2-fold increases in actinorhodin production from S. lividans TK24 and teicoplanin production from A. teichomyceticus, respectively, compared with the control strains.
KeywordMeSH Terms
Cloning, Molecular
19. Castelli  P, Donadio  S, Marinelli  F, Borghi  A, Sosio  M,     ( 1995 )

Complementation of a Streptomyces lividans Leu- mutant by the Actinoplanes teichomyceticus leuC gene.

Gene 158 (1)
PMID : 7789819  :   DOI  :   10.1016/0378-1119(95)00142-s    
Abstract >>
A leucine auxotroph of Streptomyces lividans (Sl), designated PC196, was unable to convert alpha-isopropylmalate into the beta-isomer. A DNA fragment from Actinoplanes teichomyceticus (At) cloned into the Streptomyces vector pIJ702 complemented PC196. Sequence analysis of the 3.0-kb insert revealed one complete ORF with high similarity to other leuC genes encoding the large subunit of isopropylmalate isomerase (IPMI), and the 5' end of a second ORF corresponding to leuD, which encodes the smaller subunit of IPMI. Further subcloning established that Sl strain PC196 is defective in the large subunit of IPMI.
KeywordMeSH Terms
Genes, Bacterial
Genetic Complementation Test
Hydro-Lyases
Mutation
20. Brieke  C, Tarnawski  M, Greule  A, Cryle  MJ,     ( 2018 )

Investigating Cytochrome P450 specificity during glycopeptide antibiotic biosynthesis through a homologue hybridization approach.

Journal of inorganic biochemistry 185 (N/A)
PMID : 29751197  :   DOI  :   10.1016/j.jinorgbio.2018.05.001    
Abstract >>
Cytochrome P450 enzymes perform an impressive range of oxidation reactions against diverse substrate scaffolds whilst generally maintaining a conserved tertiary structure and active site chemistry. Within secondary metabolism, P450 enzymes play widespread and important roles in performing crucial modifications of precursor molecules, with one example of the importance of such reactions being found in the biosynthesis of the glycopeptide antibiotics (GPAs). In GPA biosynthesis P450s, known as Oxy enzymes, are key players in the cyclization of the linear GPA peptide precursor, which is a process that is both essential for their antibiotic activity and is the source of the synthetic challenge of these important antibiotics. In this work, we developed chimeric P450 enzymes from GPA biosynthesis based on two homologues from different GPA biosynthesis pathways - vancomycin and teicoplanin - as an approach to explore the divergent catalytic behavior of the two parental homologues. We could generate, crystalize and explore the activity of new hybrid P450 enzymes from GPA biosynthesis and show that the unusual in vitro behavior of the vancomycin OxyB homologue does not stem from the major regions of the P450 active site, and that additional regions in and around the P450 active site must contribute to the unusual properties of this P450 enzyme. Our results further show that it is possible to successfully transplant entire regions of secondary structure between such P450s and retain P450 expression and activity, which opens the door to use such targeted approaches to generate and explore novel biosynthetic P450 enzymes.
KeywordMeSH Terms

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