Rational design and molecular evolution of D-hydantoinase based on the structure

Abstract

The D-hydantoinase from Bacillus stearothermophilus SD1 is a versatile industrial enzyme for commercial production of D-amino acids, but has relatively low activity for aromatic hydantoin derivatives used as raw materials for important pharmaceuticals, such as semi-synthetic antibiotics. Hydantoinase has been proposed to be a member of cyclic amidohydrolases, including dihydropyrimidinase, allantoinase, and dihydroorotase. Based on the rigidly conserved region of the related enzymes, a putative cyclic amidohydrolase gene from Escherichia coli was identified and characterized as phenylhydantoinase from its distinct substrate specificity for aromatic hydantoin derivatives. To elucidate the substrate specificity determinants and improve the catalytic properties of D-hydantoinase, we determined the three dimensional structure of D-hydantoinase and compared it with that of dihydroorotase. Major difference between their active sites is shown in the conformations of three loops, which are designated stereochemistry gate loops or SGLs. In addition, simulation of substrate binding in the active site of D-hydantoinase revealed that the hydrophobic and bulky residues of SGLs constitute a hydrophobic substrate binding pocket around the exocyclic substituent of substrate. Substitution of these residues, specifically Leu 65, Tyr 155, and Phe 159 near exocyclic group of substrate, to a charged amino aicd, Glu resulted in a significant decrease in the activity for hydantoin, but not for aromatic substrate such as D,L-5-hydroxyphenyl hydantoin (HPH). Based on the structures and these results, we compared the active sites of various D-hydantoinases with distinct substrate specificity, and designed new D-hydantoinase with desirable property. Met 63, Leu 65, Phe 152, and Phe 159 of SGLs were site-directed mutated to improve activity for aromatic substrates. The designed mutant enzymes show a remarkable change in substrate specificity. Particularly, the mutation F159A significantly...