Mechanistic Studies of Radical SAM Enzymes in Cystobactamid, Anaerobilin and Thiamin Biosynthesis
Abstract
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.
Citation
Wang, Yuanyou (2019). Mechanistic Studies of Radical SAM Enzymes in Cystobactamid, Anaerobilin and Thiamin Biosynthesis. Doctoral dissertation, Texas A & M University. Available electronically from https : / /hdl .handle .net /1969 .1 /184924.