Abstract
The enzyme ThiI 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 ThiI) 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, R., and Lauhon, C. T. (1999) Biochemistry 38, 16561-16568), we proposed two mechanisms for the sulfur transfer mediated by ThiI, and both suggested possible involvement of the thiol group of another cysteine residue in ThiI. 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 ThiI activity, and C207A ThiI was only mildly impaired. Substitution of Cys-344, the only cysteine residue conserved among all sequenced ThiI, resulted in the loss of function in vivo and a 2700-fold reduction in activity measured in vitro. We also examined the possibility that ThiI 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 ThiI and that disulfide bonds do form in ThiI during turnover. We also discuss the relevance of these findings to the biosynthesis of thiamin and iron-sulfur clusters.
Highlights
The metabolism of many sulfur-containing biomolecules remains incompletely understood
We earlier noted the presence of a motif shared with sulfurtransferases, and we reported that the cysteine residue (Cys-456 of Escherichia coli ThiI) found in this motif is essential for activity
The catalytically essential Cys-456 is found on a C-terminal extension of ϳ100 amino acids that was found only in ThiI from E. coli and its close relatives Salmonella typhimurium and Hemophilus influenzae among the organisms for which a ThiI sequence was known [31]
Summary
General—Unless otherwise stated, all materials were purchased from either Sigma or Fisher and used as provided. Generation, Overexpression, Purification, and Physical Characterization of Altered ThiI—To substitute cysteine residues in ThiI with alanine, the QuikChangeTM mutagenesis protocol was used with appropriate primers (Table I) as we described previously [34]. The isolated PCR product was ligated into pGEM-T using the pGEM-T Easy Vector System II (Promega) as specified by the manufacturer, and restriction analysis confirmed the generation of the target plasmid This plasmid was digested with NdeI and EcoRI (New England Biolabs, Beverly, MA), and the iscS fragment was isolated by agarose gel electrophoresis and ligated into pET29b (Novagen) that had been opened with the same enzymes. IscS bearing the C-terminal His tag will be denoted IscS1⁄7His6 Both alterations of iscS in pBH400 were corrected using the QuikChangeTM protocol (Stratagene) with appropriate primers (Table I) as we have described previously [34]. The plasmid encoding L244P IscS (with no C-terminal His tag) is pBH401; the plasmid encoding IscS1⁄7His (with Leu-244 restored) is pBH402; the plasmid encoding native IscS (with Leu-244 restored and no C-terminal His tag) is pBH403
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