title: Molecular determinants of the cofactor specificity of ribitol dehydrogenase, a short-chain dehydrogenase/reductase.
abstract: Ribitol dehydrogenase from Zymomonas mobilis (ZmRDH) catalyzes the conversion of ribitol to d-ribulose and concomitantly reduces NAD(P)(+) to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of ZmRDH. A homologous conserved amino acid, Ser156, in the substrate-binding pocket of the wild-type ZmRDH was identified as an important residue affecting the cofactor specificity of ZmRDH. Further insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD(+) (S156D, [k(cat)/K(m)(,NAD)]/[k(cat)/K(m)(,NADP)] = 10.9, where K(m)(,NAD) is the K(m) for NAD(+) and K(m)(,NADP) is the K(m) for NADP(+)). In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP(+) as the cofactor (S156H, [k(cat)/K(m)(,NAD)]/[k(cat)/K(m)(,NADP)] = 0.11). These data, in addition to those of molecular dynamics and isothermal titration calorimetry studies, suggest that the cofactor specificity of ZmRDH can be modulated by manipulating the amino acid residue at position 156.
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Gwangjin-Gu, Seoul, Republic of Korea. jkrhee@konkuk.ac.kr
authors.forename: Hee-Jung
authors.initials: HJ
authors.lastname: Moon
authors.forename: Manish Kumar
authors.initials: MK
authors.lastname: Tiwari
authors.forename: Ranjitha
authors.initials: R
authors.lastname: Singh
authors.forename: Yun Chan
authors.initials: YC
authors.lastname: Kang
authors.forename: Jung-Kul
authors.initials: JK
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003067:Coenzymes / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D010429:Pentoses / Q000378:metabolism; D011485:Protein Binding; D012255:Ribitol / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics* / Q000378:metabolism*; D017355:Zymomonas / Q000201:enzymology*
chemical_list: D003067:Coenzymes; D010429:Pentoses; D009243:NAD; D012255:Ribitol; D009249:NADP; C100182:ribulose; D013401:Sugar Alcohol Dehydrogenases; C022529:ribitol 2-dehydrogenase