title: Functional Characterization of a Dehydratase Domain from the Pikromycin Polyketide Synthase.
abstract: 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.
authors.affiliation: †Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455, United States.
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.affiliation: ‡Department of Biological Chemistry and Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, United States.
authors.forename: Greg J
authors.initials: GJ
authors.lastname: Dodge
authors.affiliation: †Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455, United States.
authors.forename: William D
authors.initials: WD
authors.lastname: Fiers
authors.affiliation: †Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455, United States.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Fecik
authors.affiliation: ‡Department of Biological Chemistry and Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, United States.
authors.forename: Janet L
authors.initials: JL
authors.lastname: Smith
authors.affiliation: †Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455, United States.
authors.forename: Courtney C
authors.initials: CC
authors.lastname: Aldrich
mesh_terms: D006836:Hydro-Lyases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D018942:Macrolides / Q000378:metabolism*; D048630:Polyketide Synthases / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D018942:Macrolides; D048630:Polyketide Synthases; D006836:Hydro-Lyases; C003145:picromycin