Cloning, sequencing, and expression in Escherichia coli of the D- hydantoinase gene from Pseudomonas putida and distribution of homologous genes in other microorganisms

Abstract

Pseudomonas putida DSM 84 produces N-carbamyl-D-amino acids from the corresponding D-5-monosubstituted hydantoins. The gene encoding this D- hydantoinase enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the 1.8-kb insert of subclone pGES19 was determined. One open reading frame of 1,104 bp was found and was predicted to encode a polypeptide with a molecular size of 40.5 kDa. Local regions of identity between the predicted amino acid sequence and that of other known amidohydrolases (two other D-hydantoinases, allantoinase and dihydroorotase) were found. The D-hydantoinase gene was used as a probe to screen DNA isolated from diverse organisms. Within Pseudomonas strains of rRNA group I, the probe was specific. The probe did not detect D-hydantoinase genes in pseudomonads not in rRNA group I, other bacteria, or plants known to express D-hydantoinase activity.