A microbial D-hydantoinase is stabilized and overexpressed as a catalytically active dimer by truncation and insertion of the C-terminal region

Abstract

Previously, it was reported that the nonhomologous C-terminal regions of the D-hydantoinases are nonessential for catalysis, but affect the oligomeric structure of the enzyme [3]. In an effort to further confirm the above observation, the C-terminal region-inserted enzyme was constructed by attaching a peptide (22 residues) at the C-terminal of the D-hydantoinase from Bacillus thermocatenulatus GH2, and its structural and biochemical properties were compared with both the wild-type and C-terminal region-truncated enzymes. As a result, native tetrameric D-hydantoinase was dimerized as the truncated enzyme, and the inserted mutant with a new sequence was expressed as a catalytically active form in E. coli. Expression level of the inserted and truncated enzymes were found to be significantly increased compared to the level of the wild-type enzyme, and this appears to be due to the reduced toxic effect of the mutant enzymes on host cells. Dimerized enzymes exhibited increased thermo- and pH stabilities considerably when compared with the corresponding wild-type enzyme. Comparison of the substrate specificity between the mutant and wildtype enzymes suggests that the substrate specificity of the D-hydantoinase is closely linked with the oligomeric structure.