Abstract
ThiI is an enzyme common to the biosynthetic pathways leading to both thiamin and 4-thiouridine in tRNA. Comparison of the ThiI sequence with protein sequences in the data bases revealed that the Escherichia coli enzyme contains a C-terminal extension displaying sequence similarity to the sulfurtransferase rhodanese. Cys-456 of ThiI aligns with the active site cysteine residue of rhodanese that transiently forms a persulfide during catalysis. We investigated the functional importance of this sequence similarity and discovered that, like rhodanese, ThiI catalyzes the transfer of sulfur from thiosulfate to cyanide. Mutation of Cys-456 to alanine impairs this sulfurtransferase activity, and the C456A ThiI is incapable of supporting generation of 4-thiouridine in tRNA both in vitro and in vivo. We therefore conclude that Cys-456 of ThiI is critical for activity and propose that Cys-456 transiently forms a persulfide during catalysis. To accommodate this hypothesis, we propose a general mechanism for sulfur transfer in which the terminal sulfur of the persulfide first acts as a nucleophile and is then transferred as an equivalent of S(2-) rather than S(0).
Highlights
From the ‡Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716 and the ¶Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061
We investigated the functional importance of this sequence similarity and discovered that, like rhodanese, ThiI catalyzes the transfer of sulfur from thiosulfate to cyanide
Analysis was accomplished by rapid size exclusion chromatography over a spin column of Sephadex G-25 followed by the addition of carrier s4U and the total digestion/C18 HPLC analysis described previously [9], which resolves all of the bases in E. coli tRNA [24, 25]
Summary
This role of s4U as a photosensor provides for a remarkably powerful selection: mutant bacteria that do not make s4U in tRNA continue to grow upon exposure to near-UV light [15,16,17,18], and the near-UV resistant phenotype can be complemented by a plasmid-borne functional copy of the defective chromosomal gene Using this near-UV screen, the role of ThiI in s4U biosynthesis was demonstrated [9] as was the functional importance of a catalytically critical “P-loop” motif idiosyncratic to a family of enzymes that catalyze the adenylation of a carbonyl oxygen for subsequent nucleophilic substitution [19]. Further scrutiny of sequence data revealed a motif shared with rhodanese, an enzyme that catalyzes the transfer of sulfur from thiosulfate to cyanide, generating sulfite and thiocyanate. We report that the cysteine residue, Cys-456, in this rhodanese sulfurtransferase motif is critical for ThiI function both in vivo and in vitro
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