(A) comparative study on the structure-function relationship of microbial D-hydantoinases

Abstract

The gene encoding a microbial D-hydantoinase from thermophilic Bacillus thermocatenultus GH2 was cloned and expressed in Escherichia coli by its own promoter, and its nucleotide sequence was determined. The structural gene of D-hydantoinase consists of 1,416 nucleotides and encodes 471 amino acid residues with a calculated molecular weight of 51,8 kDa. The N- and C-terminal amino acid sequences deduced from the nucleotide sequence were coincident with those determined by Edman degradation and carboxypeptidase digestion of the purified enzyme from B. thermocatenultus GH2. To get insight the structure-function relationship and active domain, its tetrameric enzyme property was compared with those of dimeric enzyme from Bacillus stearothermophilus SD1 based on the primary sequences. The homology of amino acid sequences between two enzymes was shown as high as 92%, and the secondary structures were determined to possess a very similar content. Despite the extreme homology of the amino acid sequences, the oligomeric structure and substrate specificity were quite different. The native gel electrophoresis patterns of the D-hydantoinases in the presence of b-mercaptoethanol revealed a different oligomeric feature, showing that its monomeric state is still active but quietly unstable. Moreover, we found that the oligomeric structure of the enzyme affected on their substrate specificities. From the comparison of the enzyme with those of previously reported D-hydantoinases and dihydropyrimidinases in a dihydropyrimidinase family, the primary sequence among the enzymes showed a considerable homology with about 24 %, and the similar ORF sizes were revealed. If the highly mismatching C-terminal regions were not considered, the overall homology was increased up to about 35 %. With the considerable homology, the rigidly conserved histidine residues and highly conserved four regions were founded, but the C-terminal regions were completely mismatched. From the analyses of the ne...