| In some bacterial tRNA, U8 is modified to 4-thiouridine (s4U), which acts as a near-UV sensor by undergoing photochemistry upon absorbance of light at 335 rim. ThiI is an enzyme named for its role in thiamin biosynthesis. Experiments with Eschericia coli ThiI presented in this thesis demonstrate the involvement of ThiI in s4U biosynthesis and elucidate its mechanism. ThiI shares two motifs with enzymes that catalyze adenylation reactions, and amino acids conserved in those motifs are essential for ThiI function. E. coli ThiI has a C-terminal extension of ∼100 amino acids that is absent in most ThiI orthologs. This extension has sequence similarity to sulfurtransferases, and modeling studies indicate that it may adopt a sulfurtransferase-like fold. Cys-456 in E. coli ThiI aligns with cysteine residues in sulfurtransferases that are converted to persulfide groups during catalysis. Cys-456 is essential for s4U generation, leading to the proposal of two mechanisms that postulate conversion of Cys-456 into a persulfide group in the catalytic cycle.; In both mechanisms, Cys-456 and another enzymic Cys residue form a disulfide bond. Cys-344 is the only completely conserved Cys in ThiI, and C344A ThiI has greatly reduced activity. Both mechanisms, therefore, propose the formation of a transient, but essential, disulfide bond between Cys-344 and Cys-456 during catalysis. ThiI undergoes one turnover in the absence of an added reductant, and ThiI contained fewer free thiol groups after that turnover. All of these results support the formation of a disulfide bond between Cys-344 and Cys-456 during the catalytic cycle. Since only one of the mechanisms postulates a role for HS−, its role in s4U generation was probed. The experiments do not support HS− as an intermediate of s4U generation but do not definitively exclude such a role.; While several issues remain unresolved, the work presented here establishes the general features of s4U generation. Uridine is activated by adenylation of O4, and sulfur is transferred using a thiol to persulfide to disulfide cycle involving Cys-456 and Cys 344. This work has implications to the biosynthesis of other sulfur containing molecules including thiamin, 2-thiouridine, and molybdopterin. |
