Molecular characterization of an NADPH-dependent acetoin reductase/2,3-butanediol dehydrogenase from Clostridium beijerinckii NCIMB 8052.
Acetoin reductase is an important enzyme for the fermentative production of 2,3-butanediol, a chemical compound with a very broad industrial use. Here, we report on the discovery and characterization of an acetoin reductase from Clostridium beijerinckii NCIMB 8052. An in silico screen of the C. beijerinckii genome revealed eight potential acetoin reductases. One of them (CBEI_1464) showed substantial acetoin reductase activity after expression in Escherichia coli. The purified enzyme (C. beijerinckii acetoin reductase [Cb-ACR]) was found to exist predominantly as a homodimer. In addition to acetoin (or 2,3-butanediol), other secondary alcohols and corresponding ketones were converted as well, provided that another electronegative group was attached to the adjacent C-3 carbon. Optimal activity was at pH 6.5 (reduction) and 9.5 (oxidation) and around 68°C. Cb-ACR accepts both NADH and NADPH as electron donors; however, unlike closely related enzymes, NADPH is preferred (Km, 32 μM). Cb-ACR was compared to characterized close homologs, all belonging to the "threonine dehydrogenase and related Zn-dependent dehydrogenases" (COG1063). Metal analysis confirmed the presence of 2 Zn(2+) atoms. To gain insight into the substrate and cofactor specificity, a structural model was constructed. The catalytic zinc atom is likely coordinated by Cys37, His70, and Glu71, while the structural zinc site is probably composed of Cys100, Cys103, Cys106, and Cys114. Residues determining NADP specificity were predicted as well. The physiological role of Cb-ACR in C. beijerinckii is discussed.
Laboratory of Microbiology, Wageningen University and Research Centre, Wageningen, The Netherlands.
John
J
Raedts
Marco A J
MA
Siemerink
Mark
M
Levisson
John
J
van der Oost
Servé W M
SW
Kengen
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D002072:Butylene Glycols / Q000378:metabolism*; D003001:Cloning, Molecular; D046970:Clostridium beijerinckii / Q000201:enzymology* / Q000235:genetics; D003067:Coenzymes / Q000032:analysis / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism*; D011487:Protein Conformation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature; D015032:Zinc / Q000032:analysis
D002072:Butylene Glycols; D003067:Coenzymes; D011994:Recombinant Proteins; C026978:2,3-butylene glycol; D009249:NADP; D000429:Alcohol Oxidoreductases; C019801:butanediol dehydrogenase; D015032:Zinc