title: Effects of the cofactor binding sites on the activities of secondary alcohol dehydrogenase (SADH).
abstract: SADHs from Thermoanaerobacter ethanolicus are enzymes that, together with various cofactors, catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. To explore how cofactors bind to SADH, TeSADH was cloned in this study, and Ser(199) and Arg(200) were replaced by Tyr and Asp, respectively. Both sites were expected to be inside or adjacent to the cofactor-binding domain according to computational a prediction. Analysis of TeSADH activities revealed that the enzymatic efficiency (kcat/Km) of the S199Y mutant was noticeably enhanced using by NADH, NADPH as cofactors, and similar with that of wild-type using by NADP(+), NAD(+). Conversely, the activity of the R200D mutant significantly decreased with all cofactors. Furthermore, in yeast, the S199Y mutant substantially elevated the ethanol concentration compared with the wild type. Molecular dynamics simulation results indicated the H-bonding network between TeSADH and the cofactors was stronger for the S199Y mutant and the binding energy was simultaneously increased. Moreover, the fluorescence results indicated the S199Y mutant exhibited an increased preference for NAD(P)H, binding with NAD(P)H more compactly compared with wild type.
authors.affiliation: Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Membrane Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Xiangjun
authors.initials: X
authors.lastname: Chen
authors.affiliation: Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Han
authors.affiliation: Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China; Biogas Institute of Ministry of Agriculture, Chengdu 610041, China.
authors.forename: Sichun
authors.initials: S
authors.lastname: Ma
authors.affiliation: Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China.
authors.forename: Jianmei
authors.initials: J
authors.lastname: Wang
authors.affiliation: Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China.
authors.forename: Xufeng
authors.initials: X
authors.lastname: Li
authors.affiliation: Biogas Institute of Ministry of Agriculture, Chengdu 610041, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China. Electronic address: liuzhibin@scu.edu.cn.
authors.forename: Zhibin
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, China. Electronic address: yangyi528@scu.edu.cn.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry*; D000438:Alcohols / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D003067:Coenzymes / Q000737:chemistry*; D007700:Kinetics; D009249:NADP / Q000737:chemistry; D013237:Stereoisomerism; D013379:Substrate Specificity; D046968:Thermoanaerobacter / Q000201:enzymology*
chemical_list: D000438:Alcohols; D003067:Coenzymes; D009249:NADP; D000429:Alcohol Oxidoreductases; C025192:isopropanol dehydrogenase (NADP)
keywords: Bioethanol; Cofactors; TeSADH