A mechanistic investigation of the microbial chiral inversion of 2-phenylpropionic acid by Verticillium lecanii

Previous investigations have described the development of nongrowing suspension of Verticillium lecanii as a microbial model of the mammalian chiral inversion of the 2‐arylpropionic acids (2‐APAs). Mechanistic studies in mammals have shown that inversion involves loss of the α‐methine proton but retention of the original atoms at the β‐methyl position, and a mechanism has been proposed involving enzymatic epimerisation of acyl‐CoA thioester derivatives of the substrate. Inversion of the 2‐APAs by V. lecanii exhibits extensive intersubstrate variation in the presence, rate, extent, and direction of inversion, which are different from those observed in mammalian systems, possibly indicating differences in the mechanism of inversion between mammalian and microbial cells. This study involved the investigation of proton/deuterium exchange by 1H‐nuclear magnetic resonance following incubation of deuterated derivatives of 2‐phenylpropionic acid (2‐PPA), a model compound, in cell suspensions of V. lecanii and incubation of undeuterated 2‐PPA in cell suspensions containing D2O. The results indicated that the inversion of 2‐PPA by V. lecanii also involved exchange of the α‐methine proton but complete retention on the original atoms at the β‐methyl position. No kinetic deuterium isotope effect was observed, indicating that loss of the α‐methine proton is not the rate‐limiting step of the inversion process. This suggests that the observed differences between microbial and mammalian systems probably involve the stereoselective acyl‐CoA thioester formation step and not the subsequent epimerisation of the resultant diastereomers. Chirality 9:254–260, 1997. © 1997 Wiley‐Liss, Inc.