Structural and Mechanistic Characterization of MoaA and MoaC in Molybdenum Cofactor Biosynthesis

Abstract

Molybdenum cofactor (Moco) is a redox enzyme cofactor found in all domains of life.Inactivation of Moco biosynthetic enzymes by genetic mutations or pharmacological inhibition can lead to the fatal Moco deficiency (MoCD) disease in humans or pathogenic defects in bacteria. In all organisms, the characteristic pyranopterin backbone of Moco is formed from GTP by the actions of two enzymes, MoaA and MoaC. MoaA, a member of the radical S-adenosyl-L-methionine (SAM) superfamily, catalyzes radical-mediated 3',8-cyclization of GTP into 3',8-cyclo-7,8- dihydro-GTP (3',8-cH2GTP). Subsequently, MoaC catalyzes the complex rearrangement of 3',8- cH₂GTP to cyclic pyranopterin monophosphate (cPMP). However, the detailed catalytic mechanisms of both enzymes remained unclear. In this dissertation, I report the first elucidation of the role of key structural components for MoaA catalysis. Using an NMR-guided structure of the previously disordered C-terminal tail and computational docking combined with functional validation, I demonstrate that MoaA uses the conformationally flexible C-terminal tail with two conserved Gly residues (GG motif) at the C- terminus as a sensor to detect GTP binding and trigger reductive SAM cleavage. For MoaC, I report the first crystal structure covalently linked with the reaction intermediate of mechanistic inhibitor 3',8-cH2GMP[CH2]PP through the conserved active site residue Lys131. By identifying four reaction intermediates, I prove that MoaC catalysis proceeds via a novel covalent carbon-carrying mechanism to navigate the complex multi-step rearrangement reaction. This reveals an enzyme- participating covalent mechanism distinct from other covalent catalysis enzymes. Through comprehensive mutagenesis studies, substrate analog analysis, computational modeling, and X-ray crystallography, these investigations of both MoaA and MoaC enable mechanistic model proposals for both enzymes. The elucidation of these mechanisms provides the complete picture of the first step in Moco biosynthesis and facilitates future development of antibiotic or anti-virulent agents targeting pathogenic bacterial Moco biosynthesis enzymes.