1. |
Eustáquio AS,
Luft T,
Wang ZX,
Gust B,
Chater KF,
Li SM,
Heide L,
( 2003 ) Novobiocin biosynthesis: inactivation of the putative regulatory gene novE and heterologous expression of genes involved in aminocoumarin ring formation. PMID : 12736771 : DOI : 10.1007/s00203-003-0555-2 Abstract >>
The left ends of the biosynthetic gene clusters of novobiocin (nov), clorobiocin (clo) and coumermycin A(1) (cou) from Streptomyces spheroides (syn. S. caeruleus) NCIMB 11891, S. roseochromogenes var. oscitans DS 12.976 and S. rishiriensis DSM 40489 were cloned and sequenced. Sequence comparison suggested that novE, cloE and couE, respectively, represent the borders of these three clusters. Inactivation of novE proved that novE does not have an essential catalytic role in novobiocin biosynthesis, but is likely to have a regulatory function. The gene products of novF and cloF show sequence similarity to prephenate dehydrogenase and may produce 4-hydroxyphenylpyruvate (4HPP) as a precursor of the substituted benzoate moiety of novobiocin and clorobiocin. Coumermycin A(1) does not contain this benzoate moiety, and correspondingly the coumermycin cluster was found not to contain a functional novF homologue. The coumermycin biosynthetic gene cluster apparently evolved from an ancestral cluster similar to those of novobiocin and clorobiocin, and parts of the ancestral novF homologue have been deleted in this process. No homologue to novC was identified in the gene clusters of clorobiocin and coumermycin, questioning the postulated involvement of novC in aminocoumarin biosynthesis. Heterologous expression of novDEFGHIJK in Streptomyces lividans resulted in the formation of 2,4-dihydroxy-alpha-oxy-phenylacetic acid, suggesting that at least one of the proteins encoded by these genes may participate in a hydroxylation reaction.
|
2. |
Freel Meyers CL,
Oberthür M,
Anderson JW,
Kahne D,
Walsh CT,
( 2003 ) Initial characterization of novobiocic acid noviosyl transferase activity of NovM in biosynthesis of the antibiotic novobiocin. PMID : 12680772 : DOI : 10.1021/bi0340088 Abstract >>
The aminocoumarin class of antibiotics, exemplified by novobiocin, is composed of tripartite l-noviosylaminocoumarin prenylbenzoate natural products. The decorated noviosyl sugar component interacts with the target bacterial enzyme DNA gyrase. We have subcloned the putative 40 kDa l-noviosyl transferase from Streptomyces spheroides into Escherichia coli, expressed it in soluble form, and purified it to homogeneity as a C-terminal His(8) fusion protein. The aglycone novobiocic acid, obtained from selective degradation of novobiocin, and TDP-l-noviose, obtained by an 11-step chemical synthesis from l-rhamnose, were shown to be robust substrates for NovM to produce the desmethyldescarbamoyl novobiocin intermediate with a k(cat) of >300 min(-1). NovM displays activity with variant coumarin aglycones, suggesting it may be a promiscuous catalyst for noviosylation of a range of planar scaffolds. Conversely, NovM shows no activity with and is inhibited by TDP-l-rhamnose (K(i) = 83.5 +/- 5.5 microM), the sugar donor that most closely structurally resembles the natural substrate TDP-l-noviose. The NovM reaction products generated during the course of this work will serve as substrates for subsequent analysis of the NovP and NovN tailoring enzymes that impart the noviose decorations required for DNA gyrase binding and antibiotic activity.
|
3. |
Jakimowicz P,
Freel Meyers CL,
Walsh CT,
Buttner MJ,
Lawson DM,
( 2003 ) Crystallization and preliminary X-ray studies on the putative dTDP sugar epimerase NovW from the novobiocin biosynthetic cluster of Streptomyces spheroides. PMID : 12876368 : DOI : 10.1107/s0907444903012848 Abstract >>
Crystals of recombinant NovW (subunit MW = 22 289 Da), a putative dTDP sugar epimerase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallizes in space group P4(3)2(1)2, with unit-cell parameters a = b = 59.20, c = 109.23 A. Native data to a resolution of 2.0 A were collected from a single crystal at 100 K on a rotating-anode X-ray generator. Preliminary analysis of these data indicated that the asymmetric unit corresponded to a monomer, whilst dynamic light scattering (DLS) suggested that NovW was a dimer in solution. NovW is involved in the biosynthesis of the aminocoumarin antibiotic novobiocin, which targets the bacterial enzyme DNA gyrase, and represents the first enzyme to be crystallized from this biosynthetic pathway.
|
4. |
Albermann C,
Soriano A,
Jiang J,
Vollmer H,
Biggins JB,
Barton WA,
Lesniak J,
Nikolov DB,
Thorson JS,
( 2003 ) Substrate specificity of NovM: implications for novobiocin biosynthesis and glycorandomization. PMID : 12633109 : DOI : 10.1021/ol0341086 Abstract >>
[reaction: see text] In an effort to expand the scope of natural product in vitro glycorandomization (IVG), the substrate specificity of NovM was investigated. A test of four aglycon analogues and over 40 nucleotide sugars revealed NovM has a surprisingly stringent substrate specificity and provided only three new "unnatural" natural products. On the basis of the determined substrate specificity, an alternative to the sugar nucleotide biosynthetic dogma and a cautionary note for the general applicability of IVG are introduced.
|
5. |
Chen H,
Walsh CT,
( 2001 ) Coumarin formation in novobiocin biosynthesis: beta-hydroxylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450 NovI. PMID : 11325587 : Abstract >>
Coumarin group antibiotics, such as novobiocin, coumermycin A1 and clorobiocin, are potent inhibitors of DNA gyrase. These antibiotics have been isolated from various Streptomyces species and all possess a 3-amino-4-hydroxy-coumarin moiety as their structural core. Prior labeling experiments on novobiocin established that the coumarin moiety was derived from L-tyrosine, probably via a beta-hydroxy-tyrosine (beta-OH-Tyr) intermediate. Recently the novobiocin gene cluster from Streptomyces spheroides was cloned and sequenced and allows analysis of the biosynthesis of the coumarin at the biochemical level using overexpressed and purified proteins. Two open reading frames (ORFs), NovH and NovI, from the novobiocin producer S. spheroides have been overexpressed in Escherichia coli, purified and characterized for tyrosine activation and oxygenation which are the initial steps in coumarin formation. The 65 kDa NovH has two predicted domains, an adenylation (A) and a peptidyl carrier protein (PCP), reminiscent of non-ribosomal peptide synthetases. Purified NovH catalyzes L-tyrosyl-AMP formation by its A domain, can be posttranslationally phosphopantetheinylated on the PCP domain, and accumulates the covalent L-tyrosyl-S-enzyme intermediate on the holo PCP domain. The second enzyme in the pathway, NovI, is a 45 kDa heme protein that functions as a cytochrome P450-type monooxygenase with specificity for the tyrosyl-S-NovH acyl enzyme. The product beta-OH-tyrosyl-S-NovH was detected by alkaline release and high performance liquid chromatography analysis of radioactive [3H]beta-OH-Tyr and by mass spectrometry. Also detected was 4-OH-benzaldehyde, a retro aldol breakdown product of beta-OH-Tyr. The amino acid released was (3R,2S)-3-OH-Tyr by comparison with authentic standards. This work establishes that NovH and NovI are responsible for the formation of a beta-OH-Tyr intermediate that is covalently tethered to NovH in novobiocin biosynthesis. Comparable A-PCP/P450 pairs for amino acid beta-hydroxylation are found in various biosynthetic gene clusters, such as ORF19/ORF20 in the chloroeremomycin cluster for tyrosine, CumC/CumD in the coumermycin A1 cluster for tyrosine, and NikP1/NikQ in the nikkomycin cluster for histidine. This phenomenon of covalent docking of the amino acid in a kinetically stable thioester linkage prior to chemical modification by downstream tailoring enzymes, could represent a common strategy for controlling the partitioning of the amino acid for incorporation into secondary metabolites.
|
6. |
Li SM,
Steffensky M,
( 2000 ) Cloning, overexpression, and purification of novobiocic acid synthetase from Streptomyces spheroides NCIMB 11891. PMID : 10801869 : DOI : 10.1074/jbc.M003066200 Abstract >>
Novobiocic acid synthetase, a key enzyme in the biosynthesis of the antibiotic novobiocin, was cloned from the novobiocin producer Streptomyces spheroides NCIMB 11891. The enzyme is encoded by the gene novL, which codes for a protein of 527 amino acids with a calculated mass of 56,885 Da. The protein was overexpressed as a His(6) fusion protein in Escherichia coli and purified to apparent homogeneity by affinity chromatography and gel chromatography. The purified enzyme catalyzed the formation of an amide bond between 3-dimethylallyl-4-hydroxybenzoic acid (ring A of novobiocin) and 3-amino-4,7-dihydroxy-8-methyl coumarin (ring B of novobiocin) in an ATP-dependent reaction. NovL shows homology to the superfamily of adenylate-forming enzymes, and indeed the formation of an acyl adenylate from ring A and ATP was demonstrated by an ATP-PP(i) exchange assay. The purified enzyme exhibited both activation and transferase activity, i.e. it catalyzed both the activation of ring A as acyl adenylate and the subsequent transfer of the acyl group to the amino group of ring B. It is active as a monomer as determined by gel filtration chromatography. The reaction was specific for ATP as nucleotide triphosphate and dependent on the presence of Mg(2+) or Mn(2+). Apparent K(m) values for ring A and ring B were determined as 19 and 131 micrometer respectively. Of several analogues of ring A, only 3-geranyl-4-hydroxybenzoate and to a lesser extent 3-methyl-4-aminobenzoate were accepted as substrates.
|
7. |
Mühlenweg A,
Wang ZX,
Steffensky M,
( 2000 ) Identification of the novobiocin biosynthetic gene cluster of Streptomyces spheroides NCIB 11891. PMID : 10770754 : DOI : 10.1128/aac.44.5.1214-1222.2000 PMC : PMC89847 Abstract >>
The novobiocin biosynthetic gene cluster from Streptomyces spheroides NCIB 11891 was cloned by using homologous deoxynucleoside diphosphate (dNDP)-glucose 4,6-dehydratase gene fragments as probes. Double-stranded sequencing of 25.6 kb revealed the presence of 23 putative open reading frames (ORFs), including the gene for novobiocin resistance, gyrB(r), and at least 11 further ORFs to which a possible role in novobiocin biosynthesis could be assigned. An insertional inactivation experiment with a dNDP-glucose 4, 6-dehydratase fragment resulted in abolishment of novobiocin production, since biosynthesis of the deoxysugar moiety of novobiocin was blocked. Heterologous expression of a key enzyme of novobiocin biosynthesis, i.e., novobiocic acid synthetase, in Streptomyces lividans TK24 further confirmed the involvement of the analyzed genes in the biosynthesis of the antibiotic.
|
8. |
Keller S,
Pojer F,
Heide L,
Lawson DM,
( 2006 ) Molecular replacement in the 'twilight zone': structure determination of the non-haem iron oxygenase NovR from Streptomyces spheroides through repeated density modification of a poor molecular-replacement solution. PMID : 17139092 : DOI : 10.1107/S0907444906040169 Abstract >>
Crystals of recombinant NovR (subunit MW = 29 924 Da; 270 amino acids), a non-haem iron oxygenase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in space group C2, with unit-cell parameters a = 86.69, b = 139.38, c = 100.82 A, beta = 101.18 degrees . Native data were collected to a resolution of 2.1 A from a single crystal at a synchrotron and a molecular-replacement solution was obtained using the program AMoRe. The starting phase information was very poor and did not permit model building. Phases were subsequently improved using a combination of fourfold averaging and very gradual phase extension in the program DM to yield an interpretable map. NovR belongs to a novel class of non-haem iron oxygenases that share sequence similarity with class II aldolases. It is predicted to perform two consecutive oxidative decarboxylation steps in the biosynthesis of the prenylated hydroxybenzoic acid moiety of the aminocoumarin antibiotic novobiocin.
|
9. |
Li SM,
Heide L,
( 2006 ) The biosynthetic gene clusters of aminocoumarin antibiotics. PMID : 16868863 : DOI : 10.1055/s-2006-946699 Abstract >>
Plants and microorganisms are the most important sources of secondary metabolites in nature. For research in the functional genomics of secondary metabolism, and for the biotechnological application of such research by genetic engineering and combinatorial biosynthesis, most microorganisms offer a unique advantage to the researcher: the biosynthetic genes for a specific secondary metabolite are not scattered over the genome, but rather are clustered in a well-defined, contiguous region - the biosynthetic gene cluster of that metabolite. This is exemplified in this review for the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A (1), which are potent inhibitors of DNA gyrase. Cloning, sequencing and analysis of the biosynthetic gene clusters of these three antibiotics revealed that the structural differences and similarities of the compounds are perfectly reflected by the genetic organisation of the biosynthetic gene clusters. The function of most biosynthetic genes could be identified by gene inactivation experiments as well as by heterologous expression and biochemical investigation. The prenylated benzoic acid moiety of novobiocin and clorobiocin, involved in the interaction with gyrase, is structurally similar to metabolites found in plants. However, detailed investigations of the biosynthesis revealed that the biosynthetic pathway and the enzymes involved are totally different from those identified in plants.
|
10. |
Tello M,
Jakimowicz P,
Errey JC,
Freel Meyers CL,
Walsh CT,
Buttner MJ,
Lawson DM,
Field RA,
( 2006 ) Characterisation of Streptomyces spheroides NovW and revision of its functional assignment to a dTDP-6-deoxy-D-xylo-4-hexulose 3-epimerase. PMID : 16514445 : DOI : 10.1039/b515763c Abstract >>
Characterisation of recombinant Streptomyces spheroides NovW in vitro suggests that it is not a kinetically competent dual action dTDP-6-deoxy-d-xylo-4-hexulose 3,5-epimerase, but possesses only significant 3-epimerase activity.
|
11. |
Eustáquio AS,
Li SM,
Heide L,
( 2005 ) NovG, a DNA-binding protein acting as a positive regulator of novobiocin biosynthesis. PMID : 15942002 : DOI : 10.1099/mic.0.27669-0 Abstract >>
The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin contains two putative regulatory genes, i.e. novE and novG. The predicted gene product of novG shows a putative helix-turn-helix DNA-binding motif and shares sequence similarity with StrR, a well-studied pathway-specific transcriptional activator of streptomycin biosynthesis. Here functional proof is provided, by genetic and biochemical approaches, for the role of NovG as a positive regulator of novobiocin biosynthesis. The entire novobiocin cluster of the producer organism Streptomyces spheroides was expressed in the heterologous host Streptomyces coelicolor M512, and additional strains were produced which lacked the novG gene within the heterologously expressed cluster. These Delta novG strains produced only 2% of the novobiocin formed by the S. coelicolor M512 strains carrying the intact novobiocin cluster. The production could be restored by introducing an intact copy of novG into the mutant. The presence of novG on a multicopy plasmid in the strain containing the intact cluster led to almost threefold overproduction of the antibiotic, suggesting that novobiocin biosynthesis is limited by the availability of NovG protein. Furthermore, purified N-terminal His(6)-tagged NovG showed specific DNA-binding activity for the novG-novH and the cloG-cloY intergenic regions of the novobiocin and clorobiocin biosynthetic gene clusters, respectively. By comparing the DNA sequences of the fragments binding NovG, conserved inverted repeats were identified in both fragments, similar to those identified as the binding sites for StrR. The consensus sequence for the StrR and the putative NovG binding sites was GTTCRACTG(N)(11)CRGTYGAAC. Therefore, NovG and StrR apparently belong to the same family of DNA-binding regulatory proteins.
|
12. |
Jakimowicz P,
Tello M,
Meyers CL,
Walsh CT,
Buttner MJ,
Field RA,
Lawson DM,
( 2006 ) The 1.6-A resolution crystal structure of NovW: a 4-keto-6-deoxy sugar epimerase from the novobiocin biosynthetic gene cluster of Streptomyces spheroides. PMID : 16411240 : DOI : 10.1002/prot.20818 Abstract >>
N/A
|
13. |
Eustáquio AS,
Gust B,
Galm U,
Li SM,
Chater KF,
Heide L,
( 2005 ) Heterologous expression of novobiocin and clorobiocin biosynthetic gene clusters. PMID : 15870333 : DOI : 10.1128/AEM.71.5.2452-2459.2005 PMC : PMC1087579 Abstract >>
A method was developed for the heterologous expression of biosynthetic gene clusters in different Streptomyces strains and for the modification of these clusters by single or multiple gene replacements or gene deletions with unprecedented speed and versatility. Lambda-Red-mediated homologous recombination was used for genetic modification of the gene clusters, and the attachment site and integrase of phage phiC31 were employed for the integration of these clusters into the heterologous hosts. This method was used to express the gene clusters of the aminocoumarin antibiotics novobiocin and clorobiocin in the well-studied strains Streptomyces coelicolor and Streptomyces lividans, which, in contrast to the natural producers, can be easily genetically manipulated. S. coelicolor M512 derivatives produced the respective antibiotic in yields comparable to those of natural producer strains, whereas S. lividans TK24 derivatives were at least five times less productive. This method could also be used to carry out functional investigations. Shortening of the cosmids' inserts showed which genes are essential for antibiotic production.
|
14. |
Pacholec M,
Tao J,
Walsh CT,
( 2005 ) CouO and NovO: C-methyltransferases for tailoring the aminocoumarin scaffold in coumermycin and novobiocin antibiotic biosynthesis. PMID : 16274243 : DOI : 10.1021/bi051599o Abstract >>
During the biosynthesis of the streptomycete aminocoumarin antibiotics novobiocin and the dimeric coumermycin A(1), the bicyclic coumarin scaffold is C-methylated adjacent to the phenolic oxygen. The SAM-dependent C-methyltransferases NovO and CouO have been heterologously expressed and purified from Escherichia coli and shown to act after the aminocoumarin ring has been constructed by prior action of Nov/CouHIJK. Neither C-methyltransferase works on the tyrosyl-derived S-pantetheinyl intermediates tethered to NovH or on the subsequently released free aminocoumarin. NovL ligates the aminocoumarin to prenylhydroxybenzoate to yield novobiocic acid, which is the substrate for NovO before it is O-glycosylated by NovM. In coumermycin assembly, the corresponding ligase CouL makes the bis-amide by tandem ligation of two aminocoumarins to a dicarboxypyrrole. CouO works on both the mono- and bis-amides for mono- and di-C-methylation adjacent to the phenolic hydroxyl before it is glycosylated by CouM. Thus, the specific timing of C-methylation in the aminocoumarin antibiotic pathways is established.
|
15. |
Thuy TT,
Lee HC,
Kim CG,
Heide L,
Sohng JK,
( 2005 ) Functional characterizations of novWUS involved in novobiocin biosynthesis from Streptomyces spheroides. PMID : 15752721 : DOI : 10.1016/j.abb.2005.01.012 Abstract >>
NovW, novU, and novS gene products represent dTDP-4-keto-6-deoxy-D-glucose 3,5 epimarase, C-methyltransferase and dTDP-glucose-4-ketoreductase involved in noviose biosynthetic pathway, respectively. We have expressed three genes to elucidate the functions of NovW, NovU, and NovS in Escherichia coli. NovW and NovU catalyze the formation of dTDP-4-keto-6-deoxy-5-C-methyl-L-lyxo-hexose from dTDP-4-keto-6-deoxy-D-glucose. NovS reduces the product formed from the reaction of NovW with dTDP-4-keto-6-deoxy-D-glucose in the presence of NADH to result in dTDP-l-rhamnose. Furthermore, a pathway for the biosynthesis of noviose is proposed.
|
16. |
Pacholec M,
Hillson NJ,
Walsh CT,
( 2005 ) NovJ/NovK catalyze benzylic oxidation of a beta-hydroxyl tyrosyl-S-pantetheinyl enzyme during aminocoumarin ring formation in novobiocin biosynthesis. PMID : 16171397 : DOI : 10.1021/bi051297m Abstract >>
The bicyclic coumarin ring in the aminocoumarin natural product antibiotics that target bacterial DNA gyrase is assembled from tyrosine by nonribosomal peptide synthetase logic. Tyrosine has previously been shown to be activated and installed as a phosphopantetheinyl thioester on the thiolation domain of NovH and then hydroxylated on the benzylic carbon by the heme protein NovI, generating beta-OH-Tyr-S-NovH. This aminoacyl-S-protein is the substrate for the next two orfs, Streptomyces sphaeroides NovJ and NovK, that have now been expressed in and purified from Escherichia coli as a J2K2 heterotetramer. NovJ/NovK use NADP as an electron acceptor to oxidize the beta-OH of the tyrosyl moiety to yield the tethered beta-ketotyrosyl-S-NovH. The enol tautomer is the form that predominates in the subsequently cyclized aminocoumarin scaffold. The labile beta-ketotyrosyl thioester moiety was identified by hydrolytic release from NovH, analysis by mass spectroscopy, and comparison with a synthetic sample. We also have identified a residue in NovJ that when mutated results in a 50-fold reduction in catalytic activity.
|
17. |
Pi N,
Meyers CL,
Pacholec M,
Walsh CT,
Leary JA,
( 2004 ) Mass spectrometric characterization of a three-enzyme tandem reaction for assembly and modification of the novobiocin skeleton. PMID : 15218104 : DOI : 10.1073/pnas.0403526101 PMC : PMC454160 Abstract >>
The tripartite scaffold of the natural product antibiotic novobiocin is assembled by the tandem action of novobiocin ligase (NovL) and novobiocic acid noviosyl transferase (NovM). The noviosyl ring of the tripartite scaffold is further decorated by a methyltransferase (NovP) and a carbamoyltransferase (NovN), resulting in the formation of novobiocin. To facilitate kinetic evaluation of alternate substrate usage by NovL and NovM toward the creation of variant antibiotic scaffolds, an electrospray ionization/MS assay for obtaining kinetic measurements is presented for NovL and NovM separately, in each case with natural substrate and the 3-methyl-4-hydroxybenzoic acid analog. Additionally, assays of tandem two-enzyme (NovL/NovM) and three-enzyme (NovL/NovM/NovP) incubations were developed. The development of these assays allows for the direct detection of each intermediate followed by its utilization as substrate for the next enzyme, as well as the subsequent formation of final product as a function of time. This MS tandem assay is useful for optimization of conditions for chemoenzymatic generation of novobiocin and is also suitable for evaluation of competitive usage of variant substrate analogs by multiple enzymes. The studies presented here serve as a platform for the subsequent expansion of the repertoire of coumarin-based antibiotics.
|
18. |
Freel Meyers CL,
Oberthür M,
Xu H,
Heide L,
Kahne D,
Walsh CT,
( 2004 ) Characterization of NovP and NovN: completion of novobiocin biosynthesis by sequential tailoring of the noviosyl ring. PMID : 14694473 : DOI : 10.1002/anie.200352626 Abstract >>
N/A
|
19. |
Ozaki T,
Mishima S,
Nishiyama M,
Kuzuyama T,
( 2009 ) NovQ is a prenyltransferase capable of catalyzing the addition of a dimethylallyl group to both phenylpropanoids and flavonoids. PMID : 19557032 : DOI : 10.1038/ja.2009.48 Abstract >>
NovQ is a member of a recently identified CloQ/NphB class of prenyltransferases. Although NphB has been well characterized as a prenyltransferase with flexibility against aromatic substrates, few studies have been carried out on characterization of NovQ. Hence, in this study, we investigate the kinetics, substrate specificity and regiospecificity of NovQ. The corresponding novQ gene was cloned from Streptomyces niveus, which produces an aminocoumarin antibiotic, novobiocin. Recombinant NovQ was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme was a soluble monomeric 40-kDa protein that catalyzed the transfer of a dimethylallyl group to 4-hydroxyphenylpyruvate (4-HPP) independently of divalent cations to yield 3-dimethylallyl-4-HPP, an intermediate of novobiocin. Steady-state kinetic constants for NovQ with the two substrates, 4-HPP and dimethylallyl diphosphate, were also calculated. In addition to the prenylation of 4-HPP, NovQ catalyzed carbon-carbon-based and carbon-oxygen-based prenylations of a diverse collection of phenylpropanoids, flavonoids and dihydroxynaphthalenes. Despite its catalytic promiscuity, the NovQ-catalyzed prenylation occurred in a regiospecific manner. NovQ is the first reported prenyltransferase capable of catalyzing the transfer of a dimethylallyl group to both phenylpropanoids, such as p-coumaric acid and caffeic acid, and the B-ring of flavonoids. This study shows that NovQ can serve as a useful biocatalyst for the synthesis of prenylated phenylpropanoids and prenylated flavonoids.
|
20. |
Gómez García I,
Stevenson CE,
Usón I,
Freel Meyers CL,
Walsh CT,
Lawson DM,
( 2010 ) The crystal structure of the novobiocin biosynthetic enzyme NovP: the first representative structure for the TylF O-methyltransferase superfamily. PMID : 19857499 : DOI : 10.1016/j.jmb.2009.10.045 PMC : PMC2813333 Abstract >>
NovP is an S-adenosyl-l-methionine-dependent O-methyltransferase that catalyzes the penultimate step in the biosynthesis of the aminocoumarin antibiotic novobiocin. Specifically, it methylates at 4-OH of the noviose moiety, and the resultant methoxy group is important for the potency of the mature antibiotic: previous crystallographic studies have shown that this group interacts directly with the target enzyme DNA gyrase, which is a validated drug target. We have determined the high-resolution crystal structure of NovP from Streptomyces spheroides as a binary complex with its desmethylated cosubstrate S-adenosyl-l-homocysteine. The structure displays a typical class I methyltransferase fold, in addition to motifs that are consistent with a divalent-metal-dependent mechanism. This is the first representative structure of a methyltransferase from the TylF superfamily, which includes a number of enzymes implicated in the biosynthesis of antibiotics and other therapeutics. The NovP structure reveals a number of distinctive structural features that, based on sequence conservation, are likely to be characteristic of the superfamily. These include a helical 'lid' region that gates access to the cosubstrate binding pocket and an active center that contains a 3-Asp putative metal binding site. A further conserved Asp likely acts as the general base that initiates the reaction by deprotonating the 4-OH group of the noviose unit. Using in silico docking, we have generated models of the enzyme-substrate complex that are consistent with the proposed mechanism. Furthermore, these models suggest that NovP is unlikely to tolerate significant modifications at the noviose moiety, but could show increasing substrate promiscuity as a function of the distance of the modification from the methylation site. These observations could inform future attempts to utilize NovP for methylating a range of glycosylated compounds.
|
21. |
Dangel V,
Härle J,
Goerke C,
Wolz C,
Gust B,
Pernodet JL,
Heide L,
( 2009 ) Transcriptional regulation of the novobiocin biosynthetic gene cluster. PMID : 19762445 : DOI : 10.1099/mic.0.032649-0 Abstract >>
The aminocoumarin antibiotic novobiocin is a gyrase inhibitor formed by a Streptomyces strain. The biosynthetic gene cluster of novobiocin spans 23.4 kb and contains 20 coding sequences, among them the two regulatory genes novE and novG. We investigated the location of transcriptional promoters within this cluster by insertion of transcriptional terminator cassettes and RT-PCR analysis of the resulting mutants. The cluster was found to contain eight DNA regions with promoter activity. The regulatory protein NovG binds to a previously identified binding site within the promoter region located upstream of novH, but apparently not to any of the other seven promoters. Quantitative real-time PCR was used to compare the number of transcripts in a strain carrying an intact novobiocin cluster with strains carrying mutated clusters. Both in-frame deletion of the regulatory gene novG and insertion of a terminator cassette into the biosynthetic gene novH led to a strong reduction of the number of transcripts of the genes located between novH and novW. This suggested that these 16 biosynthetic genes form a single operon. Three internal promoters are located within this operon but appear to be of minor importance, if any, under our experimental conditions. Transcription of novG was found to depend on the presence of NovE, suggesting that the two regulatory genes, novE and novG, act in a cascade-like mechanism. The resistance gene gyrB(R), encoding an aminocoumarin-resistant gyrase B subunit, may initially be co-transcribed with the genes from novH to novW. However, when the gyrase inhibitor novobiocin accumulates in the cultures, gyrB(R) is transcribed from its own promoter. Previous work has suggested that this promoter is controlled by the superhelical density of chromosomal DNA.
|
22. |
Dangel V,
Eustáquio AS,
Gust B,
Heide L,
( 2008 ) novE and novG act as positive regulators of novobiocin biosynthesis. PMID : 18568336 : DOI : 10.1007/s00203-008-0396-0 Abstract >>
The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin contains two putative regulatory genes, i.e., novE and novG. Functional proof for the role of NovG as a positive regulator of novobiocin biosynthesis had been provided previously, and we now investigated the role of novE. Heterologous expression experiments with the novobiocin biosynthetic gene cluster showed that the entire putative promoter region of novE is required to achieve optimal novobiocin production. Overexpression of novE, using a replicative vector, resulted in an increase of novobiocin formation. In contrast, inactivation of novE by in frame deletion resulted in a strong reduction of novobiocin biosynthesis. Novobiocin production could be restored by an intact copy of novE, but also by the regulatory gene novG. These observations suggest that novE is a positive regulator of novobiocin biosynthesis. NovE was expressed in E. coli and purified. However, in contrast to parallel experiments with NovG, no DNA-binding properties could be shown for NovE. RT-PCR experiments showed that expression of novG was detectable in the absence of NovE, and also that expression of novE occurred in absence of NovG.
|
23. |
Gómez García I,
Freel Meyers CL,
Walsh CT,
Lawson DM,
( 2008 ) Crystallization and preliminary X-ray analysis of the O-carbamoyltransferase NovN from the novobiocin-biosynthetic cluster of Streptomyces spheroides. PMID : 18997325 : DOI : 10.1107/S1744309108030145 PMC : PMC2581686 Abstract >>
Crystals of recombinant NovN, an O-carbamoyltransferase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in two different crystal forms. Crystal form I belonged to space group C2 and native data were collected to 2.9 A resolution in-house. Crystal form II had I-centred orthorhombic symmetry and native data were recorded to a resolution of 2.3 A at a synchrotron. NovN catalyses the final step in the biosynthesis of the aminocoumarin antibiotic novobiocin that targets the essential bacterial enzyme DNA gyrase.
|
24. |
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.
|
25. |
( 1993 ) Expression and analysis of two gyrB genes from the novobiocin producer, Streptomyces sphaeroides. PMID : 8392138 : DOI : 10.1111/j.1365-2958.1993.tb01593.x Abstract >>
A novobiocin producer, Streptomyces sphaeroides, contains two genes, designated gyrBS and gyrBR, that encode novobiocin-sensitive and -resistant DNA gyrase B proteins, respectively. The cloning of gyrBR was reported earlier; here, we describe the cloning of gyrBS. Both genes have been sequenced (the deduced products of gyrBS and gyrBR have M(r) values of 87.6K and 86.5K, respectively) and their transcripts have been mapped. Downstream of gyrBS, and co-transcribed with it, is the sole gyrA gene (encoding DNA gyrase A protein). By constructing hybrid gyrB genes, using fragments of gyrBS and gyrBR, a specific portion of the N-terminal domain of the gyrase B protein (corresponding to amino acid residues 134-256 of Escherichia coli gyrase B) has been implicated in the binding of novobiocin.
|