Reprogramming a module of the 6-deoxyerythronolide B synthase for iterative chain elongation

Multimodular polyketide synthases (PKSs) have an assembly line architecture in which a set of protein domains, known as a module, participates in one round of polyketide chain elongation and associated chemical modifications, after which the growing chain is translocated to the next PKS module. The ability to rationally reprogram these assembly lines to enable efficient synthesis of new polyketide antibiotics has been a long-standing goal in natural products biosynthesis. We have identified a ratchet mechanism that can explain the observed unidirectional translocation of the growing polyketide chain along the 6-deoxyerythronolide B synthase. As a test of this model, module 3 of the 6-deoxyerythronolide B synthase has been reengineered to catalyze two successive rounds of chain elongation. Our results suggest that high selectivity has been evolutionarily programmed at three types of protein–protein interfaces that are present repetitively along naturally occurring PKS assembly lines.

[1]  Chaitan Khosla,et al.  Revisiting the modularity of modular polyketide synthases. , 2009, Current opinion in chemical biology.

[2]  Chu-Young Kim,et al.  Structural and mechanistic analysis of protein interactions in module 3 of the 6-deoxyerythronolide B synthase. , 2007, Chemistry & biology.

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

[4]  C. Hertweck,et al.  Functional Analysis of the Aureothin Iterative Type I Polyketide Synthase , 2005, Chembiochem : a European journal of chemical biology.

[5]  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.

[6]  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.

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

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

[9]  L. Katz,et al.  Biosynthesis of the anti‐parasitic agent megalomicin: transformation of erythromycin to megalomicin in Saccharopolyspora erythraea , 2000, Molecular microbiology.

[10]  Ruth Nussinov,et al.  FireDock: Fast interaction refinement in molecular docking , 2007, Proteins.

[11]  Pieter C Dorrestein,et al.  Facile detection of acyl and peptidyl intermediates on thiotemplate carrier domains via phosphopantetheinyl elimination reactions during tandem mass spectrometry. , 2006, Biochemistry.

[12]  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.

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

[14]  J. Turner,et al.  Production of a novel polyketide through the construction of a hybrid polyketide synthase. , 1996, Gene.

[15]  Ruth Nussinov,et al.  PatchDock and SymmDock: servers for rigid and symmetric docking , 2005, Nucleic Acids Res..

[16]  Chaitan Khosla,et al.  Molecular recognition between ketosynthase and acyl carrier protein domains of the 6-deoxyerythronolide B synthase , 2010, Proceedings of the National Academy of Sciences.

[17]  Chaitan Khosla,et al.  Solution structure and proposed domain–domain recognition interface of an acyl carrier protein domain from a modular polyketide synthase , 2007, Protein science : a publication of the Protein Society.

[18]  P. Leadlay,et al.  Evidence from engineered gene fusions for the repeated use of a module in a modular polyketide synthase. , 2003, Chemical communications.

[19]  Chaitan Khosla,et al.  Structure and mechanism of the 6-deoxyerythronolide B synthase. , 2007, Annual review of biochemistry.

[20]  Camilla M. Kao,et al.  Engineered Biosynthesis of Structurally Diverse Tetraketides by a Trimodular Polyketide Synthase , 1996 .

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

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

[23]  Katalin F Medzihradszky,et al.  An antibiotic factory caught in action , 2004, Nature Structural &Molecular Biology.

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

[25]  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.

[26]  Ruth Nussinov,et al.  FireDock: a web server for fast interaction refinement in molecular docking† , 2008, Nucleic Acids Res..

[27]  Chu-Young Kim,et al.  The 2.7-Angstrom crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B synthase. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

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

[29]  Chaitan Khosla,et al.  The biochemical basis for stereochemical control in polyketide biosynthesis. , 2009, Journal of the American Chemical Society.

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

[31]  Chaitan Khosla,et al.  Stereospecificity of ketoreductase domains of the 6-deoxyerythronolide B synthase. , 2007, Journal of the American Chemical Society.