title: Epimerase and Reductase Activities of Polyketide Synthase Ketoreductase Domains Utilize the Same Conserved Tyrosine and Serine Residues.
abstract: The role of the conserved active site tyrosine and serine residues in epimerization catalyzed by polyketide synthase ketoreductase (PKS KR) domains has been investigated. Both mutant and wild-type forms of epimerase-active KR domains, including the intrinsically redox-inactive EryKR3° and PicKR3° as well as redox-inactive mutants of EryKR1, were incubated with [2-(2)H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-(2)H]-2) and 0.05 equiv of NADP(+) in the presence of the redox-active, epimerase-inactive EryKR6 domain. The residual epimerase activity of each mutant was determined by tandem equilibrium isotope exchange, in which the first-order, time-dependent washout of isotope from 2 was monitored by liquid chromatography-tandem mass spectrometry with quantitation of the deuterium content of the diagnostic pantetheinate ejection fragment (4). Replacement of the active site Tyr or Ser residues, alone or together, significantly reduced the observed epimerase activity of each KR domain with minimal effect on substrate binding. Our results demonstrate that the epimerase and reductase activities of PKS KR domains share a common active site, with both reactions utilizing the same pair of Tyr and Ser residues.
authors: Department of Chemistry, Box H, Brown University , Providence, Rhode Island 02912-9108, United States.
authors: Xinqiang
authors: X
authors: Xie
authors: Department of Chemistry, Box H, Brown University , Providence, Rhode Island 02912-9108, United States.
authors: Ashish
authors: A
authors: Garg
authors: Departments of Molecular Biosciences and Chemistry, The University of Texas at Austin , 1 University Station A5300, Austin, Texas 78712-0165, United States.
authors: Adrian T
authors: AT
authors: Keatinge-Clay
authors: Departments of Chemical Engineering, Chemistry, and Biochemistry, Stanford University , Stanford, California 94305, United States.
authors: Chaitan
authors: C
authors: Khosla
authors: Department of Chemistry, Box H, Brown University , Providence, Rhode Island 02912-9108, United States.
authors: David E
authors: DE
authors: Cane
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001419:Bacteria / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D017124:Conserved Sequence; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D019998:Racemases and Epimerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D000429:Alcohol Oxidoreductases; C087192:polyketide synthase ketoreductase; D019998:Racemases and Epimerases