Structure-function Relationship Of A Novel Carboxylesterase RPPE And A Cofactor-free Haloperoxidases RhEst

Crystal structures of rPPEWT and rPPEw187H were determined, both by themselves and in complex with the substrate (5)-2-acetoxy-2-(2’-chlorophenyl) acetate ((S)-AcO-CPA), to elucidate the molecular mechanism of substrate binding and chiral recognition. The structural analysis reveals that the side chain of residue Asp287in rPPEwT might have a potential steric conflict with (S)-AcO-CPA when the substrate binds at the active site of the enzyme. However, the mutation W187H could facilitate the relocation of Asp287, while D287A directly eliminates the hindrance of Asp287, both of which offer sufficient space for the binding and hydrolysis of the substrate. Therefore, the variants rPPEw187H and rPPED287A showed101-fold and81-fold increases in activity toward rac-2-acetoxy-2-(2’-chlorophenyl) acetate (rac-AcO-CPA), respectively. Moreover, Asp287as a hydrogen-bond donor constitutes one substrate binding site of "three-point attachment model", which determines the excellent enantioselectivity of rPPE in chiral recognition. D287A on the other hand would disrupt the hydrogen bond and result in the low enantioselectivity of rPPED287A.The structure of a novel cofector-free haloperoxidase from Rhodococcus sp. ECU1013has been determined at a resolution of1.9A, and two conformation of RhEst were captured in this crystal structure. The comparison of the B-factors between these two states reveals that the N-terminal domain of RhEst is very flexible, and the conformational change of N-terminal domain will result in the change of the substrate binding pocket. This will offer important structural information for further research on substrate binding or product release mechanism of RhEst.