Structure and mechanism of the 6-deoxyerythronolide B synthase.

6-Deoxyerythronolide B, the macrocyclic aglycone of the antibiotic erythromycin, is synthesized by a polyketide synthase (PKS) that has emerged as the prototypical modular megasynthase. A variety of molecular biological, protein chemical, and biosynthetic experiments over the past two decades have yielded insights into its mechanistic features. More recently, high-resolution structural images of portions of the 6-deoxyerythronolide B synthase have provided a platform for interpreting this wealth of biochemical data, while at the same time presenting a fundamentally new basis for the design of more detailed investigations into this remarkable enzyme. For example, the critical roles of domain-domain interactions and nonconserved linkers, as well as large interdomain movements in the structure and function of modular PKSs, have been highlighted. In turn, these insights point the way forward for more sophisticated and efficient biosynthetic engineering of complex polyketide natural products.

[1]  D. Cane,et al.  Precursor‐Directed Biosynthesis: Stereospecificity for Branched‐Chain Diketides of the β‐Ketoacyl‐ACP Synthase Domain 2 of 6‐Deoxyerythronolide B Synthase , 2003 .

[2]  D. Cane,et al.  Macrolactonization to 10-deoxymethynolide catalyzed by the recombinant thioesterase of the picromycin/methymycin polyketide synthase. , 2006, Bioorganic & medicinal chemistry letters.

[3]  D. Cane,et al.  Selective protein-protein interactions direct channeling of intermediates between polyketide synthase modules. , 2001, Biochemistry.

[4]  D. Cane,et al.  Intermodular communication in modular polyketide synthases: structural and mutational analysis of linker mediated protein-protein recognition. , 2003, Journal of the American Chemical Society.

[5]  P. Leadlay,et al.  Polyketide synthesis in vitro on a modular polyketide synthase. , 1995, Chemistry & biology.

[6]  K. Reynolds,et al.  Generation of multiple bioactive macrolides by hybrid modular polyketide synthases in Streptomyces venezuelae. , 2002, Chemistry & biology.

[7]  D. Cane,et al.  Functional orientation of the acyltransferase domain in a module of the erythromycin polyketide synthase. , 1998, Biochemistry.

[8]  P. Dorrestein,et al.  Characterization of a new tailoring domain in polyketide biogenesis: the amine transferase domain of MycA in the mycosubtilin gene cluster. , 2005, Journal of the American Chemical Society.

[9]  A. Witkowski,et al.  Mechanism of the β-Ketoacyl Synthase Reaction Catalyzed by the Animal Fatty Acid Synthase† , 2002 .

[10]  B A Pfeifer,et al.  Biosynthesis of Complex Polyketides in a Metabolically Engineered Strain of E. coli , 2001, Science.

[11]  D. Cane,et al.  Dissecting and exploiting intermodular communication in polyketide synthases. , 1999, Science.

[12]  C R Hutchinson,et al.  A multiplasmid approach to preparing large libraries of polyketides. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[13]  Mark Welch,et al.  Genetic approaches to polyketide antibiotics. 1. , 2005, Chemical reviews.

[14]  Kenji Watanabe,et al.  Understanding Substrate Specificity of Polyketide Synthase Modules by Generating Hybrid Multimodular Synthases* , 2003, Journal of Biological Chemistry.

[15]  D. Cane,et al.  Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase. , 1999, Chemistry & biology.

[16]  David E. Cane,et al.  Macrolide biosynthesis. 3. Stereochemistry of the chain-elongation steps of erythromycin biosynthesis , 1986 .

[17]  J. L. Smith,et al.  Structure of a dehydratase-isomerase from the bacterial pathway for biosynthesis of unsaturated fatty acids: two catalytic activities in one active site. , 1996, Structure.

[18]  P. Leadlay,et al.  Molecular basis of Celmer's rules: the role of two ketoreductase domains in the control of chirality by the erythromycin modular polyketide synthase. , 1999, Chemistry & biology.

[19]  Camilla M. Kao,et al.  GAIN OF FUNCTION MUTAGENESIS OF THE ERYTHROMYCIN POLYKETIDE SYNTHASE. 2. ENGINEERED BIOSYNTHESIS OF AN EIGHT-MEMBERED RING TETRAKETIDE LACTONE , 1997 .

[20]  Chaitan Khosla,et al.  Biochemical analysis of the substrate specificity of the beta-ketoacyl-acyl carrier protein synthase domain of module 2 of the erythromycin polyketide synthase. , 2004, Biochemistry.

[21]  Robert M Stroud,et al.  The structure of a ketoreductase determines the organization of the beta-carbon processing enzymes of modular polyketide synthases. , 2006, Structure.

[22]  Chu-Young Kim,et al.  Extender unit and acyl carrier protein specificity of ketosynthase domains of the 6-deoxyerythronolide B synthase. , 2006, Journal of the American Chemical Society.

[23]  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.

[24]  C. Kao,et al.  Evidence for two catalytically independent clusters of active sites in a functional modular polyketide synthase. , 1996, Biochemistry.

[25]  D. Cane,et al.  Erythromycin biosynthesis. The 4-pro-S hydride of NADPH is utilized for ketoreduction by both module 5 and module 6 of the 6-deoxyerythronolide B synthase. , 2001, Bioorganic & medicinal chemistry letters.

[26]  David H Sherman,et al.  An unexpected interaction between the modular polyketide synthases, erythromycin DEBS1 and pikromycin PikAIV, leads to efficient triketide lactone synthesis. , 2002, Biochemistry.

[27]  L. Miercke,et al.  Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase: Versatility from a unique substrate channel , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Ziwei Gu,et al.  Human fatty acid synthase: Role of interdomain in the formation of catalytically active synthase dimer , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[29]  P. Vagelos,et al.  Characterization of a malonyl-enzyme intermediate and identification of the malonyl binding site in malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli. , 1973, The Journal of biological chemistry.

[30]  B Wilkinson,et al.  Engineering broader specificity into an antibiotic-producing polyketide synthase. , 1998, Science.

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

[32]  J B McAlpine,et al.  Modular organization of genes required for complex polyketide biosynthesis. , 1991, Science.

[33]  C. Kao,et al.  Gain-of-Function Mutagenesis of a Modular Polyketide Synthase , 1997 .

[34]  D. Cane,et al.  Quantitative analysis of loading and extender acyltransferases of modular polyketide synthases. , 2003, Biochemistry.

[35]  Camilla M. Kao,et al.  Manipulation of macrolide ring size by directed mutagenesis of a modular polyketide synthase , 1995 .

[36]  D. Cane,et al.  Analysis of the Molecular Recognition Features of Individual Modules Derived from the Erythromycin Polyketide Synthase , 2000 .

[37]  C. Walsh.  Enabling the chemistry of life , 2001, Nature.

[38]  C. Khosla,et al.  Metabolic engineering of a methylmalonyl-CoA mutase-epimerase pathway for complex polyketide biosynthesis in Escherichia coli. , 2002, Biochemistry.

[39]  B. Sedgwick,et al.  Stereochemical course of dehydration catalysed by the yeast fatty acid synthetase , 1978 .

[40]  A. Henriksen,et al.  Fatty acid synthesis , 2006, The FEBS journal.

[41]  D. Cane,et al.  Macrolide biosynthesis. 4. Intact incorporation of a chain-elongation intermediate into erythromycin. , 1987 .

[42]  D. Cane,et al.  Assessing the balance between protein-protein interactions and enzyme-substrate interactions in the channeling of intermediates between polyketide synthase modules. , 2001, Journal of the American Chemical Society.

[43]  D. Cane,et al.  Analysis of covalently bound polyketide intermediates on 6-deoxyerythronolide B synthase by tandem proteolysis-mass spectrometry. , 2005, Biochemistry.

[44]  S. Donadio,et al.  Organization of the enzymatic domains in the multifunctional polyketide synthase involved in erythromycin formation in Saccharopolyspora erythraea. , 1992, Gene.

[45]  Shiou-Chuan Tsai,et al.  Structural Analysis of Actinorhodin Polyketide Ketoreductase: Cofactor Binding and Substrate Specificity , 2004, Biochemistry.

[46]  J R Jacobsen,et al.  Precursor-directed biosynthesis of erythromycin analogs by an engineered polyketide synthase. , 1997, Science.

[47]  P. Leadlay,et al.  Stereospecific acyl transfers on the erythromycin-producing polyketide synthase. , 1994, Science.

[48]  K. Fiebig,et al.  The Structure of (3R)-Hydroxyacyl-Acyl Carrier Protein Dehydratase (FabZ) from Pseudomonas aeruginosa* , 2004, Journal of Biological Chemistry.

[49]  P. Vagelos,et al.  The isolation and general properties of Escherichia coli malonyl coenzyme A-acyl carrier protein transacylase. , 1973, The Journal of biological chemistry.

[50]  Robert Finking,et al.  Biosynthesis of nonribosomal peptides , 2003 .

[51]  P. Leadlay,et al.  A hybrid modular polyketide synthase obtained by domain swapping. , 1996, Chemistry & biology.

[52]  Chu-Young Kim,et al.  The 2.7-Å crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B synthase , 2006 .

[53]  P. Leadlay,et al.  The molecular basis of Celmer's rules: the stereochemistry of the condensation step in chain extension on the erythromycin polyketide synthase. , 1997, Biochemistry.

[54]  P. Leadlay,et al.  Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl‐CoA:acyl carrier protein transacylase domains in modular polyketide synthases , 1995, FEBS letters.

[55]  A. Hill.  The biosynthesis, molecular genetics and enzymology of the polyketide-derived metabolites. , 2006, Natural product reports.

[56]  P. Leadlay,et al.  Directed mutagenesis alters the stereochemistry of catalysis by isolated ketoreductase domains from the erythromycin polyketide synthase. , 2006, Chemistry & biology.

[57]  C. Gustafsson,et al.  Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel "unnatural" natural products. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[58]  D. Cane,et al.  Substrate specificity of the loading didomain of the erythromycin polyketide synthase. , 2000, Biochemistry.

[59]  Kira J. Weissman,et al.  Combinatorial biosynthesis of reduced polyketides , 2005, Nature Reviews Microbiology.

[60]  Rembert Pieper,et al.  Cell-free synthesis of polyketides by recombinant erythromycin polyketide synthases , 1995, Nature.

[61]  G. Challis,et al.  Mechanisms for incorporation of glycerol-derived precursors into polyketide metabolites , 2006, Journal of Industrial Microbiology and Biotechnology.

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

[63]  J R Jacobsen,et al.  Tolerance and specificity of polyketide synthases. , 1999, Annual review of biochemistry.

[64]  P. Leadlay,et al.  High-throughput mutagenesis to evaluate models of stereochemical control in ketoreductase domains from the erythromycin polyketide synthase. , 2006, Chemistry & biology.

[65]  C. Khosla,et al.  Mechanistic analysis of a type II polyketide synthase. Role of conserved residues in the beta-ketoacyl synthase-chain length factor heterodimer. , 2000, Biochemistry.

[66]  John Crosby,et al.  The crystal structure of the actIII actinorhodin polyketide reductase: proposed mechanism for ACP and polyketide binding. , 2004, Structure.

[67]  Kira J Weissman,et al.  The structure of docking domains in modular polyketide synthases. , 2003, Chemistry & biology.

[68]  Chaitan Khosla,et al.  Mechanistic analysis of acyl transferase domain exchange in polyketide synthase modules. , 2003, Journal of the American Chemical Society.

[69]  P. Leadlay,et al.  Molecular basis of Celmer's rules: role of the ketosynthase domain in epimerisation and demonstration that ketoreductase domains can have altered product specificity with unnatural substrates. , 2001, Chemistry & biology.

[70]  P. Leadlay,et al.  An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea , 1990, Nature.

[71]  J B McAlpine,et al.  An erythromycin analog produced by reprogramming of polyketide synthesis. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[72]  P. Leadlay,et al.  Stereochemistry of catalysis by the ketoreductase activity in the first extension module of the erythromycin polyketide synthase. , 2002, Biochemistry.

[73]  James Staunton,et al.  Engineering specificity of starter unit selection by the erythromycin‐producing polyketide synthase , 2002, Molecular microbiology.

[74]  Camilla M. Kao,et al.  ALCOHOL STEREOCHEMISTRY IN POLYKETIDE BACKBONES IS CONTROLLED BY THE BETA -KETOREDUCTASE DOMAINS OF MODULAR POLYKETIDE SYNTHASES , 1998 .

[75]  Christine J. Martin,et al.  Skipping in a hybrid polyketide synthase. Evidence for ACP-to-ACP chain transfer. , 2002, Chemistry & biology.

[76]  P. Leadlay,et al.  Engineering of a minimal modular polyketide synthase, and targeted alteration of the stereospecificity of polyketide chain extension. , 1998, Chemistry & biology.

[77]  D. Cane,et al.  Precursor-directed biosynthesis: biochemical basis of the remarkable selectivity of the erythromycin polyketide synthase toward unsaturated triketides. , 2002, Chemistry & biology.

[78]  R. T. Gray,et al.  The biosynthesis of long-chain fatty acids. Incorporation of radioactivity from stereospecifically tritiated malonyl thiol esters, and the stereochemistry of the acetyl-CoA carboxylase reaction. , 1977, European journal of biochemistry.

[79]  Patrick Caffrey,et al.  Conserved Amino Acid Residues Correlating With Ketoreductase Stereospecificity in Modular Polyketide Synthases , 2003, Chembiochem : a European journal of chemical biology.

[80]  Kira J Weissman,et al.  The Structural Basis for Docking in Modular Polyketide Biosynthesis , 2006, Chembiochem : a European journal of chemical biology.

[81]  Hui Hong,et al.  Identification of a Phosphopantetheinyl Transferase for Erythromycin Biosynthesis in Saccharopolyspora erythraea , 2004, Chembiochem : a European journal of chemical biology.

[82]  A. D. Buss,et al.  Novel octaketide macrolides related to 6-deoxyerythronolide B provide evidence for iterative operation of the erythromycin polyketide synthase. , 2000, Chemistry & biology.

[83]  Chu-Young Kim,et al.  Reconstituting modular activity from separated domains of 6-deoxyerythronolide B synthase. , 2004, Biochemistry.

[84]  P. Leadlay,et al.  Limited proteolysis and active-site studies of the first multienzyme component of the erythromycin-producing polyketide synthase. , 1994, Journal of Biological Chemistry.

[85]  D. Cane,et al.  Erythromycin Biosynthesis: The β-Ketoreductase Domains Catalyze the Stereospecific Transfer of the 4-pro-S Hydride of NADPH , 1998 .

[86]  D. Cane,et al.  Dissecting the role of acyltransferase domains of modular polyketide synthases in the choice and stereochemical fate of extender units. , 1999, Biochemistry.

[87]  P. Leadlay,et al.  Molecular basis of Celmer's rules: stereochemistry of catalysis by isolated ketoreductase domains from modular polyketide synthases. , 2005, Chemistry & biology.

[88]  Linquan Bai,et al.  Functional expression of genes involved in the biosynthesis of the novel polyketide chain extension unit, methoxymalonyl-acyl carrier protein, and engineered biosynthesis of 2-desmethyl-2-methoxy-6-deoxyerythronolide B. , 2002, Journal of the American Chemical Society.

[89]  John R Carney,et al.  Precursor‐Directed Biosynthesis of Novel Triketide Lactones , 2008, Biotechnology progress.

[90]  Hui Hong,et al.  Chain initiation on type I modular polyketide synthases revealed by limited proteolysis and ion‐trap mass spectrometry , 2005, The FEBS journal.

[91]  C M Kao,et al.  Engineered biosynthesis of a complete macrolactone in a heterologous host. , 1994, Science.

[92]  D. Cane,et al.  Remarkably broad substrate tolerance of malonyl-CoA synthetase, an enzyme capable of intracellular synthesis of polyketide precursors. , 2001, Journal of the American Chemical Society.

[93]  K A Reynolds,et al.  Ethyl-substituted erythromycin derivatives produced by directed metabolic engineering. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[94]  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.

[95]  D. Cane,et al.  Chain elongation, macrolactonization, and hydrolysis of natural and reduced hexaketide substrates by the picromycin/methymycin polyketide synthase. , 2005, Angewandte Chemie.

[96]  John R Carney,et al.  Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes , 2005, Nature Biotechnology.

[97]  Chaitan Khosla,et al.  Quantitative analysis of the relative contributions of donor acyl carrier proteins, acceptor ketosynthases, and linker regions to intermodular transfer of intermediates in hybrid polyketide synthases. , 2002, Biochemistry.

[98]  C. Walsh,et al.  Harnessing the biosynthetic code: combinations, permutations, and mutations. , 1998, Science.

[99]  R. Stroud,et al.  Insights into channel architecture and substrate specificity from crystal structures of two macrocycle-forming thioesterases of modular polyketide synthases. , 2002, Biochemistry.

[100]  J. Staunton,et al.  Active-site residue, domain and module swaps in modular polyketide synthases , 2003, Journal of Industrial Microbiology and Biotechnology.

[101]  Arinthip Thamchaipenet,et al.  Biosynthesis of 2-Nor-6-deoxyerythronolide B by Rationally Designed Domain Substitution , 1997 .

[102]  Chaitan Khosla,et al.  Catalysis, specificity, and ACP docking site of Streptomyces coelicolor malonyl-CoA:ACP transacylase. , 2003, Structure.

[103]  A. D. Buss,et al.  Engineering a polyketide with a longer chain by insertion of an extra module into the erythromycin-producing polyketide synthase. , 2001, Chemistry & biology.

[104]  B. Sedgwick,et al.  The biosynthesis of long-chain fatty acids. Stereochemical differentiation in the enzymic incorporation of chiral acetates. , 1977, European journal of biochemistry.

[105]  Timm Maier,et al.  Architecture of Mammalian Fatty Acid Synthase at 4.5 Å Resolution , 2006, Science.

[106]  Bradley S Moore,et al.  Biosynthesis and attachment of novel bacterial polyketide synthase starter units. , 2002, Natural product reports.

[107]  H. O’Hare,et al.  Broad Substrate Specificity of Ketoreductases Derived from Modular Polyketide Synthases , 2006, Chembiochem : a European journal of chemical biology.