Inversion of enantioselectivity of arylmalonate decarboxylase via site-directed mutation based on the proposed reaction mechanism

Abstract

Arylmalonate decarboxylase (AMDase, EC. 4.1.1.76) catalyzes asymmetric decarboxylation of α-arylmalonic acid to give optically pure ( R)-α-arylpropionic acid. This enzyme has four cysteine residues, one of which, Cys188, is estimated to be located in the active site. It is believed that it delivers a proton from the si-face of the intermediate enolate to give ( R)-product. Based on database searches, it has been revealed that AMDase has some homology with glutamate racemase and other isomerases. Glutamate racemase has a pair of Cys residues in the active site, at positions 73 and 184, which are believed to abstract and deliver a proton from both sides of the substrate. On the other hand, AMDase has only one Cys at 188, and this is considered to be the reason why this enzyme gives optically pure products. The estimated 3D-structure of AMDase suggests that Gly74 is located in the opposite side of Cys188. Then, it was expected that introduction of one Cys around the region of Gly74, i.e., from 68 to 77 in addition to the replacement of Cys188 with less acidic Ser would invert the enantioselectivity of the enzyme. Thus, 10 mutants were prepared and their activities as well as their enantioselectivities were examined. As expected, two of them, S71C/C188S and G74C/C188S, exhibited decarboxylation activity and gave the opposite enantiomer to that formed by the wild type enzyme.