Tuesday, April 1, 2025
4/1/2025
Elucidating the Role of Cobalamin in BchE, a B12-Binding Radical SAM Enzyme - Project Summary Radical SAM (RS) enzymes have been greatly influential in the fields of biochemistry, microbiology, cancer, and infectious diseases. These enzymes are characterized by their [4Fe-4S] cluster which facilitates reductive cleavage of SAM to methionine and a 5’-deoxyadenosyl radical (5’-dA•). The latter of which is able to abstract a hydrogen (H•) from a wide range of possible substrates, affording exotic biological reactions. In the last decade, many studies have focused on the cobalamin (Cbl) dependent subclass of RS enzymes. Based on bioinformatics, this subclass of the RS superfamily currently accounts for over 50,000 possible enzymes, the vast majority of which are unannotated. Most of the annotated enzymes in this class are methylases which can act upon unactivated or inert carbons. However, there are examples of both non-radical and non-methylase reactivity from this class of enzymes which further complicate understanding the role Cbl plays in the reaction mechanism. This proposed work aims to determine the role of Cbl in BchE, a Cbl-dependent RS enzyme that does not catalyze methyl transfer. Instead, it catalyzes two oxidations and a ring closure of substrate, Mg-protoporphyrin- IX monomethylester. This reaction forms the fifth ring of bacteriochlorophyll a during its biosynthesis. Though a unique reaction among Cbl-dependent RS enzymes, in vitro studies of this enzyme have been impeded due to its notorious insolubility. Preliminary data shows the Booker lab has found a soluble construct of this enzyme which is able to convert substrate to a new species, though not the predicted final product. This proposal outlines the spectroscopic and electrochemical characterization of BchE as the role of each cofactor (cobalamin and Fe- S cluster) is not understood. Furthermore, experiments to fulfill complete reactivity to the final product are outlined. These include determining what protein binding partners and native reductants are necessary for the reaction. Alternative strategies for inducing full reactivity are also proposed. Finally, structural characterization of BchE is proposed by means of X-ray crystallography. There is much interest to determine what structural features influence the cobalamin cofactor to proceed in this unique reaction opposed to the more conventional methylase chemistry seen in Cbl-dependent RS enzymes.
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