title: Alteration of coenzyme specificity of malate dehydrogenase from Streptomyces coelicolor A3(2) by site-directed mutagenesis.
abstract: We describe here for the first time the alteration of coenzyme specificity of malate dehydrogenase (MDH) from Streptomyces coelicolor A3(2) (ScMDH). In the present study, we replaced four amino acid residues in the Rossmann fold (βB-αC) region of NADH-dependent ScMDH by site-directed mutagenesis with those of NADPH-dependent MDH (Glu42Gly, Ile43Ser, Pro45Arg, and Ala46Ser). The coenzyme specificity of the mutant enzyme (ScMDH-T4) was examined. Coenzyme specificity of ScMDH-T4 was shifted 2231.3-fold toward NADPH using kcat/Km(coenzyme) as the measurement of coenzyme specificity. Accordingly, the effect of the replacements on coenzyme specificity is discussed. Our work provides further insight into the coenzyme specificity of ScMDH.
authors.affiliation: Institute of Molecular Biology and Biotechnology, Key Laboratory of Molecular Evolution and Biodiversity, Key Laboratory of the Biotic Environment and Ecological Safety in Anhui Province, Anhui Normal University, Wuhu, Anhui, China.
authors.forename: Y D
authors.initials: YD
authors.lastname: Ge
authors.affiliation: Institute of Molecular Biology and Biotechnology, Key Laboratory of Molecular Evolution and Biodiversity, Key Laboratory of the Biotic Environment and Ecological Safety in Anhui Province, Anhui Normal University, Wuhu, Anhui, China.
authors.forename: P
authors.initials: P
authors.lastname: Song
authors.affiliation: Institute of Molecular Biology and Biotechnology, Key Laboratory of Molecular Evolution and Biodiversity, Key Laboratory of the Biotic Environment and Ecological Safety in Anhui Province, Anhui Normal University, Wuhu, Anhui, China.
authors.forename: Z Y
authors.initials: ZY
authors.lastname: Cao
authors.affiliation: Institute of Molecular Biology and Biotechnology, Key Laboratory of Molecular Evolution and Biodiversity, Key Laboratory of the Biotic Environment and Ecological Safety in Anhui Province, Anhui Normal University, Wuhu, Anhui, China.
authors.forename: P
authors.initials: P
authors.lastname: Wang
authors.affiliation: Institute of Molecular Biology and Biotechnology, Key Laboratory of Molecular Evolution and Biodiversity, Key Laboratory of the Biotic Environment and Ecological Safety in Anhui Province, Anhui Normal University, Wuhu, Anhui, China gpz1996@yahoo.com.
authors.forename: G P
authors.initials: GP
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D003067:Coenzymes / Q000378:metabolism*; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D003067:Coenzymes; D008291:Malate Dehydrogenase