title: Coupled methyl group epimerization and reduction by polyketide synthase ketoreductase domains. Ketoreductase-catalyzed equilibrium isotope exchange.
abstract: Incubation of [2-(2)H]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SACP ([2-(2)H]-1a) with the epimerizing ketoreductase domain EryKR1 in the presence of a catalytic amount NADP(+) (0.05 equiv) resulted in time- and cofactor-dependent washout of deuterium from 1a, as a result of equilibrium isotope exchange of transiently generated [2-(2)H]-2-methyl-3-ketopentanoyl-ACP. Incubations of [2-(2)H]-(2S,3S)-2-methyl-3-hydroxy-pentanoyl-SACP with RifKR7 and with NysKR1 also resulted in time-dependent loss of deuterium. By contrast, incubations of [2-(2)H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP and [2-(2)H]-(2R,3R)-2-methyl-3-hydroxypentanoyl-SACP with the non-epimerizing ketoreductase domains EryKR6 and TylKR1, respectively, did not result in any significant washout of deuterium. The isotope exchange assay directly establishes that specific polyketide synthase ketoreductase domains also have an intrinsic epimerase activity, thus enabling mechanistic analysis of a key determinant of polyketide stereocomplexity.
authors.affiliation: Department of Chemistry, Brown University , Providence, Rhode Island 02912-9108, United States.
authors.forename: Ashish
authors.initials: A
authors.lastname: Garg
authors.forename: Chaitan
authors.initials: C
authors.lastname: Khosla
authors.forename: David E
authors.initials: DE
authors.lastname: Cane
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D003903:Deuterium / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D010084:Oxidation-Reduction; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D003903:Deuterium; D000429:Alcohol Oxidoreductases; C087192:polyketide synthase ketoreductase