UC San Diego

Threonylcarbamoyl adenosine (t6A) and Queuosine (Q) are two structurally complex modified nucleosides found at position 37 and 34, respectively, in the anticodon stem-loop of tRNA, and are required for accurate and efficient ribosomal translation. The goal of this dissertation is to understand the molecular mechanisms underlying t6A and Q biosynthesis and explore the potential of the biosynthesis enzymes for drug target development. t6A biosynthesis involves the transfer of a threonylcarbamoyl (TC) moiety from a pathway intermediate to tRNA by a multisubunit enzyme complex possessing an ATPase activity required for multi-turnover of the t6A cycle. In chapter 2, I used structure-guided mutagenesis and kinetic assays to investigate the mechanism of ATP hydrolysis-driven turnover of the t6A cycle in T. maritima, and show that residues in conserved “switch” loops of the TsaE subunit of the complex mediate this process. In chapter 3, I show that a stable trimeric form of TsaE possesses a kinase activity, autophosphorylating at conserved switch residues, suggesting a role for phosphorylation in cycle reset. In chapter 4, I present efforts toward structure determination of the tRNA bound biosynthesis complex, resulting in a 10.1 Å cryo-EM map showing the mode of tRNA binding to a pre-TC-transfer state of the complex. Q biosynthesis starts with the GTP cyclohydrolase-catalyzed conversion of GTP to H2NTP, a step shared with the folate pathway in bacteria and the biopterin pathway in humans. Many pathogen rely on a form of the enzyme (GCYH-IB) that is structurally distinct from the human GCYH-IA enzyme, positioning GCYH-IB as an attractive antimicrobial target. In chapter 5, I investigate the catalytic mechanism of Neisseria gonorrhoeae GCYH-IB and the role of active-site S-nitrosylation in enzyme function; and test enzyme inhibition by 8-oxo-guanine analogs. The results provide a broad map of the catalytic site and suggest a novel role of S-nitrosothiol in catalysis. The outcome of these studies is a proposal of the detailed catalytic mechanism of GCYH-IB, and identification of a selective inhibitor of GCYH-IB. Together, these studies contribute to a better mechanistic understanding of t6A and Q biosyntheses, and the utilization of enzymes in these pathways for drug discovery.