Functional Characterization of a Dehydratase Domain from the Pikromycin Polyketide Synthase.

Metabolic engineering of polyketide synthase (PKS) pathways represents a promising approach to natural products discovery. The dehydratase (DH) domains of PKSs, which generate an α,β-unsaturated bond through a dehydration reaction, have been poorly studied compared with other domains, likely because of the simple nature of the chemical reaction they catalyze and the lack of a convenient assay to measure substrate turnover. Herein we report the first steady-state kinetic analysis of a PKS DH domain employing LC-MS/MS analysis for product quantitation. PikDH2 was selected as a model DH domain. Its substrate specificity and mechanism were interrogated with a systematic series of synthetic triketide substrates containing a nonhydrolyzable thioether linkage as well as by site-directed mutagenesis, evaluation of the pH dependence of the catalytic efficiency (V(max)/K(M)), and kinetic characterization of a mechanism-based inhibitor. These studies revealed that PikDH2 converts d-alcohol substrates to trans-olefin products. The reaction is reversible with equilibrium constants ranging from 1.2 to 2. Moreover, the enzyme activity is robust, and PikDH2 was used on a preparative scale for the chemoenzymatic synthesis of unsaturated triketide products. PikDH2 was shown to possess remarkably strict substrate specificity and is unable to turn over substrates that are epimeric at the β-, γ-, or δ-position. We also demonstrated that PikDH2 has a key ionizable group with a pK(a) of 7.0 and can be irreversibly inactivated through covalent modification by a mechanism-based inhibitor, which provides a foundation for future structural studies to elucidate substrate-protein interactions.

[1]  F. Hahn,et al.  A dehydratase domain in ambruticin biosynthesis displays additional activity as a pyran-forming cyclase. , 2014, Angewandte Chemie.

[2]  Janet L. Smith,et al.  Polyketide Intermediate Mimics as Probes for Revealing Cryptic Stereochemistry of Ketoreductase Domains , 2014, ACS chemical biology.

[3]  Steven J. Moss,et al.  Uncovering the origin of Z-configured double bonds in polyketides: intermediate E-double bond formation during borrelidin biosynthesis , 2014 .

[4]  Georgios Skiniotis,et al.  Structure of a modular polyketide synthase , 2014, Nature.

[5]  Adrian Keatinge-Clay,et al.  Structure and stereospecificity of the dehydratase domain from the terminal module of the rifamycin polyketide synthase. , 2013, Biochemistry.

[6]  M. Burkart,et al.  Sulfonyl 3-alkynyl pantetheinamides as mechanism-based cross-linkers of acyl carrier protein dehydratase. , 2013, Journal of the American Chemical Society.

[7]  A. Keatinge-Clay.  The structures of type I polyketide synthases. , 2012, Natural product reports.

[8]  M. Burkart,et al.  Dehydratase-specific probes for fatty acid and polyketide synthases. , 2012, Journal of the American Chemical Society.

[9]  P. Leadlay,et al.  Stereoselectivity of Isolated Dehydratase Domains of the Borrelidin Polyketide Synthase: Implications for cis Double Bond Formation , 2011, ChemBioChem.

[10]  Tiangang Liu,et al.  Mechanism and stereospecificity of a fully saturating polyketide synthase module: nanchangmycin synthase module 2 and its dehydratase domain. , 2010, Journal of the American Chemical Society.

[11]  Chaitan Khosla,et al.  Stereospecificity of the dehydratase domain of the erythromycin polyketide synthase. , 2010, Journal of the American Chemical Society.

[12]  Jing He,et al.  Exploiting enzymatic promiscuity to engineer a focused library of highly selective antifungal and antiproliferative aureothin analogues. , 2010, Journal of the American Chemical Society.

[13]  C. Hertweck,et al.  Functionally distinct modules operate two consecutive alpha,beta-->beta,gamma double-bond shifts in the rhizoxin polyketide assembly line. , 2010, Angewandte Chemie.

[14]  C. Hertweck,et al.  Functionally Distinct Modules Operate Two Consecutive α,β→β,γ Double‐Bond Shifts in the Rhizoxin Polyketide Assembly Line , 2010 .

[15]  David H Sherman,et al.  Crystal structures of dehydratase domains from the curacin polyketide biosynthetic pathway. , 2010, Structure.

[16]  D. Sherman,et al.  The methymycin/pikromycin pathway: a model for metabolic diversity in natural product biosynthesis. , 2009, Bioorganic & medicinal chemistry.

[17]  Adrian Keatinge-Clay,et al.  Crystal structure of the erythromycin polyketide synthase dehydratase. , 2008, Journal of molecular biology.

[18]  Janet L. Smith,et al.  An Enzyme Assembly Line , 2008, Science.

[19]  Shiou-Chuan Tsai,et al.  The type I fatty acid and polyketide synthases: a tale of two megasynthases. , 2007, Natural product reports.

[20]  D. Cane,et al.  Polyketide double bond biosynthesis. Mechanistic analysis of the dehydratase-containing module 2 of the picromycin/methymycin polyketide synthase. , 2005, Journal of the American Chemical Society.

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

[22]  K. Reynolds,et al.  Substrate recognition and channeling of monomodules from the pikromycin polyketide synthase. , 2003, Journal of the American Chemical Society.

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

[24]  D. Cane,et al.  Expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin/methymycin polyketide synthase. , 2003, Journal of the American Chemical Society.

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

[26]  D. Cane,et al.  Expression, site-directed mutagenesis, and steady state kinetic analysis of the terminal thioesterase domain of the methymycin/picromycin polyketide synthase. , 2002, Biochemistry.

[27]  Amos B. Smith,et al.  The relationship between Taxol and (+)-discodermolide: synthetic analogs and modeling studies. , 2001, Chemistry & biology.

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

[29]  C. Khosla,et al.  Cloning and heterologous expression of the epothilone gene cluster. , 2000, Science.

[30]  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.

[31]  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.

[32]  D. Sherman,et al.  A gene cluster for macrolide antibiotic biosynthesis in Streptomyces venezuelae: architecture of metabolic diversity. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[33]  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.

[34]  J. Richard,et al.  Generation and stability of a simple thiol ester enolate in aqueous solution , 1992 .

[35]  C. Townsend,et al.  Direct observation by carbon-13 NMR spectroscopy of the regioselectivity and stoichiometry of "suicide" enzyme inactivation , 1984 .

[36]  G. M. Helmkamp,et al.  Mode of inhibition of beta-hydroxydecanoyl thioester dehydrase by 3-decynoyl-N-acetylcysteamine. , 1970, The Journal of biological chemistry.

[37]  K. Bloch,et al.  Beta-hydroxydecanoyl thioester dehydrase. II. Mode of action. , 1967, The Journal of biological chemistry.

[38]  K. Bloch,et al.  On the enzymatic synthesis of unsaturated fatty acids in Escherichia coli. , 1967, Proceedings of the National Academy of Sciences of the United States of America.

[39]  R. Kitz,et al.  Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase. , 1962, The Journal of biological chemistry.

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

[41]  A. R. Fresht.  Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding , 1999 .

[42]  W. Gerwick,et al.  Structure-activity analysis of the interaction of curacin A, the potent colchicine site antimitotic agent, with tubulin and effects of analogs on the growth of MCF-7 breast cancer cells. , 1998, Molecular pharmacology.