Evidence that ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis, may be a sulfurtransferase that proceeds through a persulfide intermediate.
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).
Palenchar
P M
PM
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Buck
C J
CJ
Cheng
H
H
Larson
T J
TJ
Mueller
E G
EG
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D003486:Cyanides / Q000378:metabolism; D029968:Escherichia coli Proteins; D008956:Models, Chemical; D008969:Molecular Sequence Data; D012343:RNA, Transfer / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013440:Sulfides / Q000378:metabolism*; D013466:Sulfurtransferases / Q000378:metabolism*; D013831:Thiamine / Q000096:biosynthesis*; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism; D013885:Thiosulfates / Q000378:metabolism; D013891:Thiouridine / Q000378:metabolism*
D001426:Bacterial Proteins; D003486:Cyanides; D029968:Escherichia coli Proteins; D013440:Sulfides; D013885:Thiosulfates; C051552:persulfides; D013891:Thiouridine; D012343:RNA, Transfer; D013466:Sulfurtransferases; C107110:ThiI protein, bacteria; C491479:thiI protein, E coli; D013884:Thiosulfate Sulfurtransferase; D013831:Thiamine