Homology modelling and site-directed mutagenesis studies of the XXXX hydrolase from Phanerochaete chrysosporium.

@entity3261 @entity73358 @entity4299 @entity73357 @entity4022 @entity2734 @entity73359 @entity73360 @entity2574
Three-dimensional structural model of @entity4299 hydrolase (PchEHA) from Phanerochaete chrysosporium was constructed based on X-ray structure of @entity3261 AD1 sEH using SWISS-MODEL server. Conserved residues constituting the active site cavity were identified, of which the functional roles of 14 residues were determined by site-directed mutagenesis. In catalytic triad, @entity2574 105 and His308 play a leading role in alkylation and hydrolysis steps, respectively. Distance between @entity2574 105 and @entity4299 ring of substrate may determine the regiospecificity in the substrate docking model. @entity2574 277 located at the entrance of substrate tunnel is concerned with catalysis but not essential. @entity73357 had the highest activity and lower enantioselectivity among 14 mutants, suggesting @entity2574 307 may be involved in choice of substrate configuration. @entity73358 and @entity73359 almost exhibited no activity, indicating that they are essential to bind substrate and facilitate opening of @entity4299 ring. Besides, His35- @entity4022 36- @entity2734 37-Pro38, Trp106 and Trp309 surrounding @entity2574 105, may coordinate the integration of active site cavity and influence substrate binding. Especially, W106I reversed the enantioselectivity, perhaps due to more deteriorative impact on the preferred @entity73360 . @entity4022 65 and Gly67 occurring at b-turns and Gly36 are vital in holding protein conformation. Conclusively, single conserved residue around the active sites has an important impact on catalytic properties.
