Pyridoxal enzymes acting on D-amino acids

Abstract

We isolated a thermophile, Bacillus sp. YM-1 which abundantly produced the thermostable D-amino acid aminotransferase (EC 2.6.1.21; D-AAT), and cloned the enzyme gene into E. coli. The primary structure of D-AAT was found to be homologous with that of branched-chain L-amino acid aminotransferase (EC 2.6.1.42; BCAT) of E. coli. Both enzymes are unique in their stereospecificity of pro-R C-4′ hydrogen transfer through the coenzyme-substrate Schiff base intermediates in contrast to various L-amino acid aminotransferases catalyzing the pro-5 hydrogen transfer. Thus, D-AAT and BCAT are categorized into the same group on the basis of stereospecificity of the hydrogen transfer, but different from all other aminotransferases. The thermostable alanine racemase (EC 5.1.1.1) of B. stearothermophilus has a molecular weight of about 78,000, and consists of two identical subunits (Mr 39,000). We generated a mutant which was genetically engineered to produce two polypeptide fragments corresponding to the domains. The fragments associate with each other to form an active structure, which was termed “fragmentary form”, and shows about 50% of the specific activity of the wild-type enzyme. The CD study showed that the secondary structure of the fragmentary form is closely similar to that of the wild-type enzyme. We have developed a procedure for the synthesis of various D-amino acids by means of bacterial thermostable D-amino acid aminotransferase, alanine racemase and L-alanine dehydrogenase (EC 1.4.1.1), and yeast formate dehydrogenase (EC 1.2.1.2) with a high yield. © 1994, Walter de Gruyter. All rights reserved.