Characterization of a (2R,3R)-2,3-butanediol dehydrogenase as the Saccharomyces cerevisiae YAL060W gene product -: Disruption and induction of the gene

被引:111
作者
González, E
Fernández, MR
Larroy, C
Solà, L
Pericàs, MA
Parés, X [1 ]
Biosca, JA
机构
[1] Univ Autonoma Barcelona, Fac Sci, Dept Biochem & Mol Biol, E-08193 Bellaterra, Barcelona, Spain
[2] Univ Barcelona, Dept Quim Organ, Unitat Recerca Sintesi Asimetr, E-08028 Barcelona, Spain
关键词
D O I
10.1074/jbc.M003035200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The completion of the Saccharomyces cerevisiae genome project-in 1996 showed that almost 60% of the potential open reading frames of the genome had no experimentally determined function. Using a conserved sequence motif present in the zinc-containing medium-chain alcohol dehydrogenases, we found several potential alcohol dehydrogenase genes with no defined function. One of these, YAL060W, was overexpressed using a multicopy inducible vector, and its protein product was purified to homogeneity. The enzyme was found to be a homodimer that, in the presence of NAD(+), but not of NADP, could catalyze the stereospecific oxidation of (2R,3R)-2,3-butanediol (K(m) = 14 mM, k(cat) = 78,000 min(-1)) and meso-butanediol (K(m) = 65 mM, k(cat) = 46,000 min(-1)) to (3R)-acetoin and (SS)-acetoin, respectively. It was unable, however, to further oxidize these acetoins to diacetyl, In the presence of NADH, it could catalyze the stereospecific reduction of racemic acetoin ((3R/3S)acetoin K(m) = 4.5 mM, k(cat) = 98,000 min(-1)) to (2R,3R)-2,3-butanediol and meso-butanediol, respectively. The substrate stereospecificity was determined by analysis of products by gas-liquid chromatography, The YALOGOW gene product can therefore be classified as an NAD-dependent (2R,3R)-2,3-butanediol dehydrogenase (BDH). S. cerevisiae could grow on 2,3-butanediol as the sole carbon and energy source. hTnder these conditions, a 3.5-fold: increase in (2R,3R)-2,3-butanediol dehydrogenase activity was observed in the total cell extracts. The isoelectric focusing pattern of the induced enzyme coincided with that of the pure BDH (pI 6.9). The disruption of the YALOGOW gene was not lethal for the yeast under laboratory conditions. The disrupted strain could also grow on 2,3-butanediol, although attaining a lesser cell density than the wild-type strain. Taking into consideration the substrate specificity of the YALOGOW gene product, we propose the name of BDH for this gene. The corresponding enzyme is the first eukaryotic (2R,3R)2,3-butanediol dehydrogenase characterized of the medium-chain dehydrogenase/reductase family.
引用
收藏
页码:35876 / 35885
页数:10
相关论文
共 60 条
[1]  
ARONSON BD, 1989, J BIOL CHEM, V264, P5226
[2]   SYNTHESIS OF DROSOPHILA-MELANOGASTER ALCOHOL-DEHYDROGENASE IN YEAST [J].
ATRIAN, S ;
GONZALEZDUARTE, R ;
FOTHERGILLGILMORE, LA .
GENE, 1990, 93 (02) :205-212
[3]  
Ausubel FM., 2006, ENZYMATIC MANIPULATI
[4]  
BENNETZEN JL, 1982, J BIOL CHEM, V257, P3018
[5]   THE YDP PLASMIDS - A UNIFORM SET OF VECTORS BEARING VERSATILE GENE DISRUPTION CASSETTES FOR SACCHAROMYCES-CEREVISIAE [J].
BERBEN, G ;
DUMONT, J ;
GILLIQUET, V ;
BOLLE, PA ;
HILGER, F .
YEAST, 1991, 7 (05) :475-477
[6]   CHROMOSOMAL INTEGRATION AND EXPRESSION OF 2 BACTERIAL ALPHA-ACETOLACTATE DECARBOXYLASE GENES IN BREWERS-YEAST [J].
BLOMQVIST, K ;
SUIHKO, ML ;
KNOWLES, J ;
PENTTILA, M .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1991, 57 (10) :2796-2803
[7]   CHARACTERIZATION OF THE GENES OF THE 2,3-BUTANEDIOL OPERONS FROM KLEBSIELLA-TERRIGENA AND ENTEROBACTER-AEROGENES [J].
BLOMQVIST, K ;
NIKKOLA, M ;
LEHTOVAARA, P ;
SUIHKO, ML ;
AIRAKSINEN, U ;
STRABY, KB ;
KNOWLES, JKC ;
PENTTILA, ME .
JOURNAL OF BACTERIOLOGY, 1993, 175 (05) :1392-1404
[8]   Genetics - Yeast as a model organism [J].
Botstein, D ;
Chervitz, SA ;
Cherry, JM .
SCIENCE, 1997, 277 (5330) :1259-1260
[9]  
CHAOCHEN G, 1984, BIOCHEMISTRY-US, V23, P3576
[10]   GENETICS OF ALCOHOL-DEHYDROGENASE IN SACCHAROMYCES-CEREVISIAE .2 . 2 LOCI CONTROLLING SYNTHESIS OF GLUCOSE-REPRESSIBLE ADH-II [J].
CIRIACY, M .
MOLECULAR & GENERAL GENETICS, 1975, 138 (02) :157-164