Structure-based mutational analysis of the active site residues of d-hydantoinase

We previously proposed the hydrophobic and bulky residues of the three loops, designated stereochemistry gate loops (SGLs), to constitute a hydrophobic substrate binding pocket of d-hydantoinase from Bacillus stearothermophilus SD1. Simulation of substrate binding in the active site of d-hydantoinase and sequence alignment of various d-hydantoinases revealed the critical hydrophobic residues closely located around the exocyclic substituent of substrate. To evaluate the roles of these residues in substrate binding pocket, site-directed mutagenesis was performed specifically for Leu 65, Tyr 155, and Phe 159. When Tyr 155 was mutated to Phe and Glu, both mutants Y155F and Y155E were totally inactive for nonsubstituted hydantoin and d,l-5-hydroxyphenyl hydantoin (HPH), which indicates that Tyr 155 is involved in substrate binding via a hydrogen bond with the hydantoinic ring. Furthermore, replacement of the hydrophobic residues Leu 65 and Phe 159 with Glu, a charged amino acid, resulted in a significant decrease in activity for nonsubstituted hydantoin, but not for HPH. The Kcat values of both mutants for nonsubstituted hydantoin also severely decreased, but a slight change in the Kcat values was observed towards HPH. These results suggest that the hydrophobic residues in SGLs play an essential role in substrate binding, and differentially interact according to the property of the exocyclic substituent. [Copyright &y& Elsevier]