title: A new NAD(H)-dependent meso-2,3-butanediol dehydrogenase from an industrially potential strain Serratia marcescens H30.
abstract: The budC gene coding for a new meso-2,3-butanediol dehydrogenase (BDH) from Serratia marcescens H30 was cloned and expressed in Escherichia coli BL21(DE3), purified, and characterized for its properties. The recombinant BDH with a molecular weight of 27.4 kDa exhibited a reversible transformation between acetoin and 2,3-butanediol. In the presence of NADH, BDH could catalyze the reduction of diacetyl and (3R)-acetoin to (3S)-acetoin and meso-2,3-butanediol, respectively, while (3S)-acetoin as a substrate could be further transformed into (2S, 3S)-2,3-butanediol at pH 9.0. For diol oxidation reactions, (3R)-acetoin and (3S)-acetoin were obtained when meso-2,3-butanediol and (2S,3S)-2,3-butanediol were used as the substrates with BDH and NAD(+). (2R,3R)-2,3-butanediol was not a substrate for the BDH at all. The low K m value (4.1 mM) in meso-2,3-butanediol oxidation reaction and no activity for diacetyl, acetoin, and 2,3-butanediol as the substrates with NADP(+)/NADPH suggested that the budC gene product belongs to a NAD(H)-dependent meso-2,3-BDH. Maximum activities for diacetyl and (3S/3R)-acetoin reduction were observed at pH 8.0 and pH 5.0 while for meso-2,3-butanediol oxidation it was pH 8.0. However, the optimum temperature for oxidation and reduction reactions was about 40 °C. In addition, the BDH activity for meso-2,3-butanediol oxidation was enhanced in the presence of Fe(2+) and for diacetyl and (3S/3R)-acetoin reduction in the presence of Mg(2+) and Mn(2+), while several metal ions inhibited its activity, particularly Fe(3+) for reduction of diacetyl and acetoin. Sequence analysis showed that the BDH from S. marcescens H30 possessed two conserved sequences including the coenzyme binding motif (GxxxGxG) and the active-site motif (YxxxK), which are present in the short-chain dehydrogenase/reductase superfamily.
authors.affiliation: Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, College of Life Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian Province, 350002, People's Republic of China.
authors.forename: Liaoyuan
authors.initials: L
authors.lastname: Zhang
authors.forename: Quanming
authors.initials: Q
authors.lastname: Xu
authors.forename: Senran
authors.initials: S
authors.lastname: Zhan
authors.forename: Yongyu
authors.initials: Y
authors.lastname: Li
authors.forename: Hui
authors.initials: H
authors.lastname: Lin
authors.forename: Shujing
authors.initials: S
authors.lastname: Sun
authors.forename: Li
authors.initials: L
authors.lastname: Sha
authors.forename: Kaihui
authors.initials: K
authors.lastname: Hu
authors.forename: Xiong
authors.initials: X
authors.lastname: Guan
authors.forename: Yaling
authors.initials: Y
authors.lastname: Shen
mesh_terms: D000093:Acetoin / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002072:Butylene Glycols / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism*; D020536:Enzyme Activators / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002072:Butylene Glycols; D002412:Cations; D003067:Coenzymes; D020536:Enzyme Activators; D008670:Metals; D011994:Recombinant Proteins; D009243:NAD; C026978:2,3-butylene glycol; D000093:Acetoin; D000429:Alcohol Oxidoreductases; C019801:butanediol dehydrogenase