Evidence That ThiI, an Enzyme Shared between Thiamin and 4-Thiouridine Biosynthesis, May Be a Sulfurtransferase That Proceeds through a Persulfide Intermediate

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 .

Biosynthesis of the thiazole moiety of thiamin in Escherichia coli : Identification of an acyldisulfide-linked protein–protein conjugate that is functionally analogous to the ubiquitin/E1 complex

“…A protein ThiS (12), which has been posttranslationally modified by conversion of its carboxyl-terminal glycine to a thiocarboxylate (ThiS-COSH), has been identified as an intermediate sulfur carrier (5,6), and the structure of this protein has been determined (7). Two enzymes involved in the formation of ThiS-COSH also have been identified and characterized: ThiF (5) catalyzes the formation of ThiS-COAMP, and IscS (8,9) catalyzes the sulfur transfer from cysteine to the acyl adenylate of ThiS in a pyridoxal 5Ј-phosphate-dependent reaction.…”

3-Mercaptopyruvate Sulfurtransferase of LeishmaniaContains an Unusual C-terminal Extension and Is Involved in Thioredoxin and Antioxidant Metabolism

“…Bovine rhodanese has a 1000-fold higher affinity for the reduced form of thioredoxin than for cyanide and so may function in peroxide detoxification (4,10). A reaction analogous to that of sulfane-loaded sulfurtransferase with thioredoxin is also thought to be a critical step in the synthesis of thiouridine (7,11,12), and the formation of thiocarboxylate during thiamine biosynthesis by the multidomain protein ThiI of Escherichia coli (7,12). Sulfurtransferases have also been implicated in the synthesis of biotin (13) and molybdopterin (14).…”

Substrate Specificity for 4-Thiouridine Modification in Escherichia coli

“…We have shown through gel mobility shift assays that ThiI and not IscS binds to tRNA and that ThiI binds ATP, most probably for activation of the uridine via adenylation (22). The mechanism involves mobilization of sulfur from cysteine by IscS in the form of an enzyme-bound persulfide (23,24), which is then transferred to a cysteine residue on ThiI before final insertion into U8 of tRNA (25)(26)(27). The nature of the final sulfur transfer step is unclear, but it has been recently shown that ThiI is oxidized to a disulfide under single turnover conditions (28).…”