Myxovirescin A Biosynthesis is Directed by Hybrid Polyketide Synthases/Nonribosomal Peptide Synthetase, 3‐Hydroxy‐3‐Methylglutaryl–CoA Synthases, and trans‐Acting Acyltransferases

Myxococcus xanthus DK1622 is shown to be a producer of myxovirescin (antibiotic TA) antibiotics. The myxovirescin biosynthetic gene cluster spans at least 21 open reading frames (ORFs) and covers a chromosomal region of approximately 83 kb. In silico analysis of myxovirescin ORFs in conjunction with genetic studies suggests the involvement of four type I polyketide synthases (PKSs; TaI, TaL, TaO, and TaP), one major hybrid PKS/NRPS (Ta‐1), and a number of monofunctional enzymes similar to the ones involved in type II fatty‐acid biosyntesis (FAB). Whereas deletion of either taI or taL causes a dramatic drop in myxovirescin production, deletion of both genes (ΔtaIL) leads to the complete loss of myxovirescin production. These results suggest that both TaI and TaL PKSs might act in conjunction with a methyltransferase, reductases, and a monooxygenase to produce the 2‐hydroxyvaleryl–S–ACP starter that is proposed to act as the biosynthetic primer in the initial condensation reaction with glycine. Polymerization of the remaining 11 acetates required for lactone formation is directed by 12 modules of Ta‐1, TaO, and TaP megasynthetases. All modules, except for the first module of TaL, lack cognate acyltransferase (AT) domains. Furthermore, deletion of a discrete tandem AT—encoded by taV—blocks myxovirescin production; this suggests an “in trans” mode of action. To embellish the macrocycle with methyl and ethyl moieties, assembly of the myxovirescin scaffold is proposed to switch twice from PKS to 3‐hydroxy‐3‐methylglutaryl–CoA (HMG–CoA)‐like biochemistry during biosynthesis. Disruption of the S‐adenosylmethionine (SAM)‐dependent methyltransferase, TaQ, shifts production toward two novel myxovirescin analogues, designated myxovirescin Qa and myxovirescin Qc. NMR analysis of purified myxovirescin Qa revealed the loss of the methoxy carbon atom. This novel analogue lacks bioactivity against E. coli.

[1]  Rolf Müller,et al.  Nichtribosomale Peptidbiosynthese: Punktmutationen und Überspringen eines Moduls führen zu chemischer Diversität , 2006 .

[2]  R. Müller,et al.  Identification and analysis of the chivosazol biosynthetic gene cluster from the myxobacterial model strain Sorangium cellulosum So ce56. , 2006, Journal of biotechnology.

[3]  Rolf Müller,et al.  Der Einfluss bakterieller Genomik auf die Naturstoff-Forschung , 2005 .

[4]  R. Müller,et al.  Production of the Tubulin Destabilizer Disorazol in Sorangium cellulosum: Biosynthetic Machinery and Regulatory Genes , 2005, Chembiochem : a European journal of chemical biology.

[5]  Jie J. Zheng,et al.  The structural biology of type II fatty acid biosynthesis. , 2005, Annual review of biochemistry.

[6]  M. Marahiel,et al.  Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics. , 2005, Chemical reviews.

[7]  Matthias Platzer,et al.  Antitumor polyketide biosynthesis by an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Christine J. Martin,et al.  Loss of co-linearity by modular polyketide synthases: a mechanism for the evolution of chemical diversity. , 2004, Natural product reports.

[9]  Mehul M. Patel,et al.  Staphylococcus aureus 3-Hydroxy-3-methylglutaryl-CoA Synthase , 2004, Journal of Biological Chemistry.

[10]  David H Sherman,et al.  Biosynthetic pathway and gene cluster analysis of curacin A, an antitubulin natural product from the tropical marine cyanobacterium Lyngbya majuscula. , 2004, Journal of natural products.

[11]  William H Gerwick,et al.  Structure and biosynthesis of the jamaicamides, new mixed polyketide-peptide neurotoxins from the marine cyanobacterium Lyngbya majuscula. , 2004, Chemistry & biology.

[12]  Christopher T Walsh,et al.  Polyketide and Nonribosomal Peptide Antibiotics: Modularity and Versatility , 2004, Science.

[13]  M. Platzer,et al.  Unprecedented Diversity of Catalytic Domains in the First Four Modules of the Putative Pederin Polyketide Synthase , 2004, Chembiochem : a European journal of chemical biology.

[14]  B. Shen,et al.  Leinamycin biosynthesis revealing unprecedented architectural complexity for a hybrid polyketide synthase and nonribosomal peptide synthetase. , 2004, Chemistry & biology.

[15]  L. Shimkets,et al.  Membrane Localization of Motility, Signaling, and Polyketide Synthetase Proteins in Myxococcus xanthus , 2003, Journal of bacteriology.

[16]  Andrzej Witkowski,et al.  Structural and functional organization of the animal fatty acid synthase. , 2003, Progress in lipid research.

[17]  Christopher M Thomas,et al.  Characterization of the mupirocin biosynthesis gene cluster from Pseudomonas fluorescens NCIMB 10586. , 2003, Chemistry & biology.

[18]  B. Shen,et al.  Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Zihe Rao,et al.  Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for beta-lactam acetylation. , 2003, Journal of molecular biology.

[20]  C Richard Hutchinson,et al.  A model of structure and catalysis for ketoreductase domains in modular polyketide synthases. , 2003, Biochemistry.

[21]  Jörn Piel,et al.  A polyketide synthase-peptide synthetase gene cluster from an uncultured bacterial symbiont of Paederus beetles , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[22]  K. Reynolds,et al.  The hidden steps of domain skipping: macrolactone ring size determination in the pikromycin modular polyketide synthase. , 2002, Chemistry & biology.

[23]  C R Hutchinson,et al.  Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations. , 2001, Biochemistry.

[24]  E. Ron,et al.  An unusual beta-ketoacyl:acyl carrier protein synthase and acyltransferase motifs in TaK, a putative protein required for biosynthesis of the antibiotic TA in Myxococcus xanthus. , 2001, FEMS microbiology letters.

[25]  Satoshi Omura,et al.  Organization of biosynthetic gene cluster for avermectin in Streptomyces avermitilis: analysis of enzymatic domains in four polyketide synthases , 2001, Journal of Industrial Microbiology and Biotechnology.

[26]  J. Olsen,et al.  beta-Ketoacyl-[acyl carrier protein] synthase I of Escherichia coli: aspects of the condensation mechanism revealed by analyses of mutations in the active site pocket. , 2001, Biochemistry.

[27]  Jorge F. Reyes-Spindola,et al.  Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods. , 2001, Nucleic acids research.

[28]  J. Staunton,et al.  Polyketide biosynthesis: a millennium review. , 2001, Natural product reports.

[29]  H. Blöcker,et al.  Novel features in a combined polyketide synthase/non-ribosomal peptide synthetase: the myxalamid biosynthetic gene cluster of the myxobacterium Stigmatella aurantiaca Sga15. , 2001, Chemistry & biology.

[30]  R. Heath,et al.  The 1.8 A crystal structure and active-site architecture of beta-ketoacyl-acyl carrier protein synthase III (FabH) from escherichia coli. , 2000, Structure.

[31]  J. Rafferty,et al.  Crystallization of the NADP-dependent beta-keto acyl-carrier protein reductase from Brassica napus. , 2000, Acta crystallographica. Section D, Biological crystallography.

[32]  H. Blöcker,et al.  New Lessons for Combinatorial Biosynthesis from Myxobacteria , 1999, The Journal of Biological Chemistry.

[33]  P. Leadlay,et al.  A chain initiation factor common to both modular and aromatic polyketide synthases , 1999, Nature.

[34]  E. Ron,et al.  A Nonessential Signal Peptidase II (Lsp) of Myxococcus xanthus Might Be Involved in Biosynthesis of the Polyketide Antibiotic TA , 1999, Journal of bacteriology.

[35]  Y. Lindqvist,et al.  Conversion of a beta-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine. , 1999, Biochemistry.

[36]  T. Stachelhaus,et al.  The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. , 1999, Chemistry & biology.

[37]  E. Ron,et al.  Cloning and characterization of a Myxococcus xanthus cytochrome P-450 hydroxylase required for biosynthesis of the polyketide antibiotic TA. , 1999, Gene.

[38]  E. Ron,et al.  The first gene in the biosynthesis of the polyketide antibiotic TA of Myxococcus xanthus codes for a unique PKS module coupled to a peptide synthetase. , 1999, Journal of molecular biology.

[39]  E. Ron,et al.  A NusG-like transcription anti-terminator is involved in the biosynthesis of the polyketide antibiotic TA of Myxococcus xanthus. , 1999, FEMS microbiology letters.

[40]  T. Nakatsu,et al.  Crystal structures of two tropinone reductases: different reaction stereospecificities in the same protein fold. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[41]  A. Goffeau,et al.  The complete genome sequence of the Gram-positive bacterium Bacillus subtilis , 1997, Nature.

[42]  K. Kashefi,et al.  Genetic suppression and phenotypic masking of a Myxococcus xanthus frzF‐ defect , 1995, Molecular microbiology.

[43]  R. Kagan,et al.  Widespread occurrence of three sequence motifs in diverse S-adenosylmethionine-dependent methyltransferases suggests a common structure for these enzymes. , 1994, Archives of biochemistry and biophysics.

[44]  C. Rock,et al.  Regulation of fatty acid biosynthesis in Escherichia coli. , 1993, Microbiological reviews.

[45]  E. Rosenberg,et al.  Effect of adhesive antibiotic TA on plaque and gingivitis in man. , 1989, Journal of clinical periodontology.

[46]  H. Reichenbach,et al.  Antibiotika aus Gleitenden Bakterien, XLI. Zur Konstitution der Myxovirescine B ‐ T und Biogenese des Myxovirescins A , 1989 .

[47]  O. Harumi,et al.  Effects of the temperature range and the lack of β-ketoacyl acyl-carrier protein synthase II on fatty acid synthesis in Escherichia coli K12 after shifts in temperature , 1986 .

[48]  E. Rosenberg,et al.  Adsorption of Antibiotic TA to Dental Hard Tissues , 1985, Journal of dental research.

[49]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[50]  V. Wray,et al.  On the biosynthesis of the antibiotic myxovirescin A1 by Myxococcus virescens , 1983 .

[51]  H. Reichenbach,et al.  The myxovirescins, a family of antibiotics from Myxococcus virescens (Myxobacterales). , 1982, The Journal of antibiotics.

[52]  S. Carmeli,et al.  Chemical properties of Myxococcus xanthus antibiotic TA. , 1982, The Journal of antibiotics.

[53]  V. Wray,et al.  Structure of myxovirescin A, a new macrocylic antibiotic from gliding bacteria , 1982 .

[54]  J. Cronan,et al.  Structural, enzymatic, and genetic studies of beta-ketoacyl-acyl carrier protein synthases I and II of Escherichia coli. , 1980, The Journal of biological chemistry.

[55]  J Davies,et al.  Bacterial resistance to aminoglycoside antibiotics. , 1997, The Journal of infectious diseases.