Sequence Analysis, Expression, And The Relation Between Structure And Function Of Hydantoinase

This paper comprises the isolation and expression of D-hydantoinase from BT811 and the homology modeling of the spacial structure and the analysis of site-directed mutagenesis of L-hydantoin hydrolase from Arthrobacter BT801.BT811 is a D-hydantoinase producing strain isolated from soil. The genomic library of BT811 was constructed with a shuttle cosmid, pKC505, and a positive clone was selected by detection of biological activity of the transformants. A sequence of 4.5 kb encoding hydantoin utility operon was detected by subclone with pUC18. Two open reading frames was confirmed, an hyuH gene encoded a hydantoin hydrolase, a protein of 53 kD with isoelectric point at 4.82, an hyuC gene encoded N-carbamoylase, a protein of 45.2 kD with isoelectric point at 4.95. The BLAST search on database of GenBank revealed that hydantoinase gene is most homologous to a D-hydantoinase gene from Brucella suis, with a homology of 85% in N-carabmoyl amino acids hydrolase sequence and 84% in hydantoin hydrolyase sequence. The two genes were functionally expressed with pQE vectors. The activity of the enzyme increased by 1.8 times and 1.6 times respectively, compared to the parental strain. The protein bands were detected by SDS-PAGE, being identical to the result of the primary structure and sequence analysis.Arthrobacter BT801 is an L-hydantoinase producing bacterium, in which the hydantoin hydrolyase possesses a different substrate selectivity with other L-hydantoinas reported. Therefore its hydantoin hydrolase is a good material in the study of the relationship of the spatial stucture and the substrate specificity of the enzyme. A model of spatial structure of the L-hydantoinase from Arthrobacter BT801 was proposed, based on homology modeling with that of the L-hydantoinase from Arthobacter DSM3745 as a template. The model consists of two domains, the central is a (α/β)g domain, which is flanked by a p-sheet rich domain, comprised of both N- and the C-terminus. The strong homology to other related enzymes both in fold and in active site is exploited for identification ofsubstrate binding site of L-hydantoinase from Arthrobacter BT801. A preliminary analysis of the characteristic of the active sites for this enzyme was made and possible sites relating to substrate selectives of L-hydantoinase was proposed. The mutants of hydantoin hydrolyase were constructed by site-directed mutagenesis with overlap extentsion PCR technology. The carboxylated lysine residue 147 is confirmed to be necessary for the biological activity, while when the asparagine residue at 72 and serine residue at 97 were subsituted by tyrosine and phenylalanine (Asn72→Tyr72, Ser97→Phe97). no obvious change in the substrate selectivity was detected.