Isolation and in vitro characterization of BchE, the cobalamin-dependent anaerobic magnesium protoporphyrin IX monomethylester cyclase.

The radical S-adenosylmethionine (SAM) superfamily comprises more than 800,000 enzymes that use [Fe4S4] clusters to initiate radical chemistry that mediates an exceptionally broad range of chemical transformations. Within this superfamily, cobalamin (Cbl)-dependent radical SAM enzymes constitute a major subclass predominantly associated with methylation reactions. However, several notable members catalyze nonmethylase reactions, for which the mechanistic role of Cbl is poorly understood. Bacteriochlorophyll biosynthesis enzyme BchE is a Cbl-dependent radical SAM enzyme that catalyzes a six-electron oxidation of Mg-protoporphyrin IX monomethylester to protochlorophyllide (PChlide), installing a ketone and forming the fifth ring of bacteriochlorophyll under anaerobic conditions. Although prior in vivo and in vitro studies have demonstrated a requirement for Cbl, SAM, and a low-potential reductant, detailed mechanistic analysis has been impeded by the inability to obtain soluble, catalytically active enzyme. Here, we report the successful isolation and spectroscopic characterization of BchE, enabling in vitro reconstitution of its enzymatic activity. Using both chemical and biological reducing systems, we observe the formation of PChlide along with proposed reaction intermediates and several off-pathway products. These results provide insight into the oxidative chemistry mediated by Cbl in nonmethylase radical SAM enzymes and establish BchE as a tractable model for elucidating how cobalamin is deployed in this understudied subclass.