d-Amino acid aminotransferase: fragmentation at a flexible loop is an efficient method to generate mutant enzymes with new substrate specificities and elevated activities

d-Amino acid aminotransferase (d-AAT) (EC 2.6.1.21) catalyzes the interconversion between various d-amino acids and α-keto acids. A subunit of the homodimeric enzyme from a thermophile, Bacillus sp. YM-1, consists of two distinct structural domains connected by one loop. We previously constructed an active fragmentary enzyme whose backbone was cut at the interdomain loop [J. Biochem. 124 (1998) 905]. In this work, we constructed 13 fragmentary d-amino acid aminotransferase genes by inserting a termination codon, an SD sequence, and an initiation codon into the specific positions of the gene corresponding to various loop regions and expressed in Escherichia coli cells. We have obtained six genes producing active fragmentary enzymes, one producing an inactive fragmentary enzyme, four producing only large peptide fragment, and another two that gave no products. The six active fragmentary enzymes purified to near-homogeneity showed various substrate specificities and thermostabilities distinct from each other and also from the wild-type enzyme: two exhibited higher catalytic activity towards d-alanine, the most efficient substrate, than the wild-type enzyme. These results suggest that cleavage at a loop region is an efficient method for the alteration of enzyme properties.