Reversal of coenzyme specificity of 2,3-butanediol dehydrogenase from Saccharomyces cerevisae and in vivo functional analysis.
Saccharomyces cerevisiae NAD(H)-dependent 2,3-butanediol dehydrogenase (Bdh1), a medium chain dehydrogenase/reductase is the main enzyme catalyzing the reduction of acetoin to 2,3-butanediol. In this work we focused on altering the coenzyme specificity of Bdh1 from NAD(H) to NADP(H). Based on homology studies and the crystal structure of the NADP(H)-dependent yeast alcohol dehydrogenase Adh6, three adjacent residues (Glu(221), Ile(222), and Ala(223)) were predicted to be involved in the coenzyme specificity of Bdh1 and were altered by site-directed mutagenesis. Coenzyme reversal of Bdh1 was obtained with double Glu221Ser/Ile222Arg and triple Glu221Ser/Ile222Arg/Ala223Ser mutants. The performance of the triple mutant for NADPH was close to that of native Bdh1 for NADH. The three engineered mutants were able to restore the growth of a phosphoglucose isomerase deficient strain (pgi), which cannot grow on glucose unless an alternative NADPH oxidizing system is provided, thus demonstrating their in vivo functionality. These mutants are interesting tools to reduce the excess of acetoin produced by engineered brewing or wine yeasts overproducing glycerol. In addition, they represent promising tools for the manipulation of the NADP(H) metabolism and for the development of a powerful catalyst in biotransformations requiring NADPH regeneration.
Ehsani
Maryam
M
INRA, UMR 1083, 2 place Viala, F-34060 Montpellier, France.
Fernández
Maria R
MR
Biosca
Josep A
JA
Dequin
Sylvie
S
D000093:Acetoin / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002072:Butylene Glycols / Q000378:metabolism; D003067:Coenzymes / Q000494:pharmacology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
D002072:Butylene Glycols; D003067:Coenzymes; D050505:Mutant Proteins; D029701:Saccharomyces cerevisiae Proteins; C026978:2,3-butylene glycol; D000093:Acetoin; D000429:Alcohol Oxidoreductases; C019801:butanediol dehydrogenase