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. 6A 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.