Characterization of the NADH-Linked acetylacetoin reductase/2,3-butanedioldehydrogenase gene from Bacillus cereus YUF-4

A 1.4-kbp DNA fragment, including the NADH-linked acetylacetoin reductase/2 ,3-butanediol dehydrogenase (AACRII/BDH) gene from the chromosomal DNA of B acillus cereus YUF-4, was cloned in Escherichia coli DH5 alpha after its in sertion into pUC119, and the resulting plasmid was named pAACRII119. The AA CRII/BDH gene had an open reading frame consisting of 1047 bp encoding 349 amino acids. The enzyme exhibited not only AACR activity, but also BDH acti vity. However, the gene was not located in a 2,3-butanediol (BD) operon, as is the case in the BDH gene of Klebsiella pneumoniae and that of K. terrig ena. In addition, there was no BD-cycle-related enzyme gene in the region s urrounding the AACRII/BDH gene. The AACR and BDH activities in E. coli DH5 alpha /pAACRII119 were 200-fold higher than those in the original B. cereus YUF-4. The characteristics of the AACRII/BDH from E. coli DH 5 alpha /pAAC RII119 are similar to those of the AACRH/BDH from B. cereus YUF-4. The AACR H/BDH was considered to belong to the NAD(P)- and zinc-dependent long-chain alcohol dehydrogenase (group I ADH) family on the basis of the following d istinctive characteristics: it possessed 14 strictly conserved residues of microbial group I ADH and consisted of about 350 amino acids. The enzymatic and genetic characteristics of AACRII/BDH were completely different from t hose of BDHs belonging to the short-chain dehydrogenase/reductase family. T hese findings indicated that the AACRII/BDH could be considered a new type of BDH.