D-Amino Acid Aminotransferase from a Thermophile, Bacillus SP. YM-1: Enzymological Properties, Cloning of the Gene, and the Amino Acid Sequence

We have isolated a thermophile which grows in a medium containing D-amino acids as a nitrogen source from soil and identified as a new Bacillus species. The bacterium (Bacillus sp. YM-1) showed a very high activity of D-amino acid aminotransferase. The enzyme purified to homogeneity from cell extracts of YM-1 has a molecular weight of about 62,000, and is composed of two subunits identical in molecular weight (30,000). The D-amino acid aminotransferase gene was cloned into Escherichia coli with the vector plasmid pBR322. The clone cells carrying the plasmid of 4.3 kb DNA (pICT113) produced the enzyme as high as 10% of the total cellular proteins. The enzyme overproduced by the clone was purified from cell extracts about 10-fold to homogeneity in about 60% yield by four steps including heat treatment. The complete primary structure of the enzyme involving the position of the active site lysyl residue that binds pyridoxal 5′-phosphate was determined from the nucleotide sequence of the gene and the amino acid sequences of tryptic peptides. The primary structure of D- amino acid aminotransferase shows high sequence homology with that of branched-chain amino acid aminotransferase of E. coli (the ilvE gene product).