Radical SAM (RS) enzymes are able to perform a wide variety of reactions throughout nature by utilizing a 4Fe-4S cluster to reductively cleave a molecule of S-adenosylmethionine to a methionine and 5’-deoxyadenosyl radical. Among this superfamily, there is a subclass of cobalamin-dependent RS enzymes which typically employ methylcobalamin to perform methylation on unactivated or inert carbon centers. Notably, there are exceptions of cobalamin-dependent RS enzymes that do not perform methylation. Among these is BchE, which catalyzes two oxidations and a ring closure of substrate, Mg-protoporphyrin-IX monomethylester. This reaction forms the fifth ring of bacteriochlorophyll during its biosynthesis, allowing absorbance of light at higher wavelengths (>600 nm). The mechanism of BchE has been of great interest for over two decades, particularly regarding the role of cobalamin. However, in vitro studies of this enzyme have been hampered by its notorious insolubility.
In this work, we show the first characterization of purified BchE and the analysis of its catalytic activity. UV-vis and electron paramagnetic resonance (EPR) spectroscopy were used to characterize the metallocofactors of isolated BchE. Enzymatic assays were performed to investigate the active form of cobalamin. Biological reducing systems were discovered to be compatible with the reaction. This allowed confident detection of proposed intermediates by high-resolution LC-MS. In addition to the proposed intermediates, another species was shown to accumulate with the reaction. The relevance and structure of this species was investigated by LC-MS/MS, TD-DFT, and in vivo studies with R. capsulatus. Together, this work gives insight into the mechanism of BchE while establishing a new platform for investigation of non-methylase cobalamin-dependent RS enzymes.