Biochemical characterization of unusual meso-2,3-butanediol dehydrogenase from a strain of Bacillus subtilis

Abstract

meso-2,3-Butanediol dehydrogenase (BDH) catalyzes the redox reaction between (R)-acetoin and meso-2,3-butanediol (meso-BD). The BDHs isolated from bacteria are generally characterized as homo-tetramers. In the present work, an isolated and identified high acetoin (AC)-yielding (41.63g/L) bacteria strain belonging to Bacillus subtilis but without 2,3-butanediol (BD) byproduct during its fermentation was selected. To understand the characteristics of BDH activity from this high AC-yielding strain, we cloned, purified and compared the BDH from Enterobacter aerogenes (CICC10293) (E. a-BDH) with the BDH from this high AC-yielding strain B. subtilis. Sequence alignments indicate a non-conservative amino acid substitution from Asp to Gly at site 194 on the B. s-BDH compared to that of E. a-BDH. Enzymatic analysis of E. a-BDH and D194G B. s-BDH shows D194G B. s-BDH has almost lost its entire enzymatic activity. Moreover, Isothermal titration calorimetry (ITC) measurements detected the substrate binding for the D194G B. s-BDH but no reaction was detected. Circular dicroism (CD) spectroscopy characterization revealed an identical secondary structure of E. a-BDH and D194G B. s-BDH. Remarkably, D194G B. s-BDH is highly susceptible to protease digestion, suggesting that the aspartic acid to glycine substitution might cause the proteolytic susceptibility of D194G B. s-BDH. Furthermore, by homology modeling with meso-2,3-butanediol dehydrogenase from Klebsiella pneumoniae (K. p-BDH) as a template, Gly194 seems to lose the hydrogen bond interactions with the surrounding residues (Gly206, Gly207 and Thr209), resulting in a putative conformational changes of D194G B. s-BDH which might be responsible for the loss of activity.