Mechanistic Study of a Radical SAM GTP 3’,8-cyclase MoaA in the Molybdenum Cofactor Biosynthetic Pathway

Molybdenum cofactor (Moco) is a ubiquitous cofactor essential for lives of most organisms. In humans, Moco is essential for healthy development of brain, and inability to produce Moco causes a fatal Moco deficiency (MoCD) disease. Moco biosynthesis is initiated by a conserved and complicated conversion from GTP to cyclic pyranopterin monophosphate (cPMP). In bacteria, this transformation is catalyzed by two enzymes, MoaA and MoaC. Of these two enzymes, MoaA attracts research interests as it is one of the founding members of the radical S-adenosyl-L-methionine (SAM) superfamily, and its human homolog, MOCS1A, harbors over 50% of MoCD-causing mutations identified in patients. MoaA catalyzes an unprecedented and chemically challenging 3’,8-cyclization of GTP into 3’,8-cyclo-7,8-dihydro-GTP (3’,8-cH2GTP) using an active site containing two 4Fe-4S clusters, three conserved Arg residues near the GTP binding site (R17, 266, 268) and two strictly conserved Gly residues on a disordered C-terminal tail (G339, 340; GG-motif). These amino acid residues were reported to be mutated in human MoCD disease patients. However, the details of catalytic mechanism of MoaA or the mechanism by which these mutations inactivate MoaA remained elusive.In this dissertation, I aimed to elucidate the catalytic mechanism of MoaA and the catalytic roles of amino acid residues whose mutations in human cause the MoCD disease. In Chapter 2, I describe enzymological characterization of the MoaA-catalyzed reaction. In this study, I found a shunt pathway that accumulates 5’-deoxyadenos-4’-yl radical and yields (4’S)-5’-deoxyadenosine. Using this shunt pathway as a reference, I was able to determine the rate constant for the MoaA-catalyzed C3’-C8 cyclization and revealed that MoaA accelerates the rate of radical-mediated C3’-C8 bond formation of GTP by 6 ~ 9 orders of magnitude using R17 to stabilize the transition state. In Chapter 3, I describe characterization of the redox function of 4Fe-4S clusters using a combination of protein film voltammography (PFV), X-band and Q-band EPR, and DFT calculation. Based on the results from these studies, I proposed a proton-coupled electron transfer (PCET) mechanism for the radical quenching step, where the auxiliary 4Fe-4S cluster acts as an electron donor and R17 acts as a proton donor. In Chapter 4, I describe structural and functional characterization of a 11mer peptide that mimics the C-terminal tail and rescues the activity of GG-motif mutants. The structure of the peptide in complex with MoaA was solved by nuclear magnetic resonance (NMR), which suggests that the GG-motif inserts into the active site and likely interacts with active site Arg residues. In Chapter 5, I describe characterization of the MoaA catalytic mechanisms using GTP analogs as mechanistic probes. Overall, this work provides new insights into the mechanisms by which MoaA controls the reactivity of radical intermediates and quenches the radical intermediate at the specific timing. The results have significant implications in understanding the catalytic mechanisms of radical SAM enzymes in general. It also contributes to our enzymological understanding of molecular causes of MoCD and will form foundations for development of novel therapeutics for MoCD.