Wang, Yuanyou (2019-03). Mechanistic Studies of Radical SAM Enzymes in Cystobactamid, Anaerobilin and Thiamin Biosynthesis. Doctoral Dissertation.
The radical S-adenosylmethionine (SAM) enzymes represent a large class enzyme that catalyzes a wide range of reactions, including chemically challenging reactions in critical pathways across all domains of life. Work here describes the detailed mechanistic studies of three of these enzymes in unusual methylation, ring cleavage, and rearrangement reactions. CysS is a cobalamin-dependent radical SAM methyltransferase that catalyzes the iterative methylations on the 3-methoxy-4-aminobenzoic acid moieties of the antibiotic cystobactamids. We were able to reconstitute the activity of CysS in vitro and demonstrate that it can catalyze sequential methylations from a methyl group to form not only ethyl, isopropyl groups but also sec-butyl, t-butyl groups by a radical mechanism for the first time. To further elucidate the mechanism, different strategic substrate analogs were designed to trap the organic radical species, cobalamin intermediates and probe the radical substitution step. Based on all the isotope labeling studies and substrate analogs experiments, we were able to build a detailed mechanistic model to reveal how CysS uses cobalamin, SAM, [4Fe- 4S] cluster to perform the unusual iterative methylations. ChuW can catalyze a radical-mediated reaction to methylate and break the protoporphyrin ring of heme to release iron in an anaerobic heme degradation pathway. We were able to design a cyclopropyl heme analog to trap radical generated on the porphyrin ring by forming a porphyrin compound with cyclopropyl ring cleaved. Several tetrapyrrole ring and SAM adducts were also observed, which gives us further insight into the mechanism of the enzymatic porphyrin cleavage. ThiC is a non-canonical radical enzyme that catalyzes one of the most complicated rearrangements in all of the mechanistic enzymology, which is the conversion of the 5-aminoimidazole ribonucleotide (AIR) to form 4-amino-5 hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) in thiamin biosynthesis. Here, we were able to trap a four-carbon fragment from the ribose part of AIR by methionine during the enzymatic reaction. Using isotope labeling substrates and cofactors, we were able to show the intermediate leaks out after the first hydrogen atom abstraction but before the loss of C3? of AIR.