The role of the cysteine residues of ThiI in the generation of 4-thiouridine in tRNA

The enzyme Thil is common to the biosynthetic pathways leading to both thiamin and 4-thiouridine in tRNA. We earlier noted the presence of a motif shared with sulfurtransferases, and we reported that the cysteine residue (Cys-456 of Escherichia coli Thil found in this motif is essential for activity (Palenchar, P. M., Buck, C. J., Cheng, H., Larson, T. J., and Mueller, E. G. (2000) J. Biol. Chem 275, 8283-8286). In light of that finding and the report of the involvement of the protein IscS in the reaction (Kambampati, M, and Lauhon, C. T. (1999) Biochemistry 38, 16561-16568), we proposed two mechanisms for the sulfur transfer mediated by Thil, and both suggested possible involvement of the thiol group of another cysteine residue in Thil. We have now substituted each of the cysteine residues with alanine and characterized the effect on activity in vivo and in vitro. Cys-108 and Cys-202 were converted to alanine with no significant effect on Thil activity, and C207A Thil was only mildly impaired. Substitution of Cys-344, the only cysteine residue conserved among all sequenced Thil, resulted in the loss of function in vivo and a 2700-fold reduction in activity measured in vitro. We also examined the possibility that Thil contains an iron-sulfur cluster or disulfide bonds in the resting state, and we found no evidence to support the presence of either species. We propose that Cys-344 forms a disulfide bond with Cys-456 during turnover, and we present evidence that a disulfide bond can form between these two residues in native Thil and that disulfide bonds do form in Thil during turnover. We also discuss the relevance of these findings to the biosynthesis of thiamin and iron-sulfur clusters.