The Rhodanese Domain of ThiI Is Both Necessary and Sufficient for Synthesis of the Thiazole Moiety of Thiamine in Salmonella enterica

Abstract

In Salmonella enterica, ThiI is a bifunctional enzyme required for the synthesis of both the 4-thiouridine modification in tRNA and the thiazole moiety of thiamine. In 4-thiouridine biosynthesis, ThiI adenylates the tRNA uridine and transfers sulfur from a persulfide formed on the protein. The role of ThiI in thiazole synthesis is not yet well understood. Mutational analysis described here found that ThiI residues required for 4-thiouridine synthesis were not involved in thiazole biosynthesis. The data further showed that the C-terminal rhodanese domain of ThiI was sufficient for thiazole synthesis in vivo. Together, these data support the conclusion that sulfur mobilization in thiazole synthesis is mechanistically distinct from that in 4-thiouridine synthesis and suggest that functional annotation of ThiI in genome sequences should be readdressed. Nutritional studies described here identified an additional cysteine-dependent mechanism for sulfur mobilization to thiazole that did not require ThiI, IscS, SufS, or glutathione. The latter mechanism may provide insights into the chemistry used for sulfur mobilization to thiazole in organisms that do not utilize ThiI.

Biosynthesis of 4-Thiouridine in tRNA in the Methanogenic Archaeon Methanococcus maripaludis

“…Upon complementation, the strain expressing MMP1354 (JW0413-MMP1354) still required thiamine for growth (data not shown), suggesting that MMP1354 was unable to transfer sulfur for thiamine biosynthesis in E. coli. This observation agreed with the proposal that the RLD of ThiI is necessary and sufficient for thiamine biosynthesis (35). In contrast, strain JW0413-MMP1354 contained s 4 U in its tRNAs (Fig.…”

“…Addition of the reducing agent ␤-mercaptoethanol (1%, v/v) to the protein mixture removed the radiolabel (lane 10 of Fig. 4 to Ala abolished the 35 S labeling of MMP1354 (lanes 4 -9 of Fig. 4), which further confirmed that these three cysteines are essential for the generation or stabilization of the persulfide under in vitro conditions.…”

“…The C78A variant had similar amounts of persulfide as the wild-type protein, whereas the C265A, C268A, and C348A variants contained no detectable amount of persulfide ( Fig. 4), indicating that MMP1354 can accept 35 S from IscS. Addition of the reducing agent ␤-mercaptoethanol (1%, v/v) to the protein mixture removed the radiolabel (lane 10 of Fig.…”

Functional Analysis of Bacillus subtilis Genes Involved in the Biosynthesis of 4-Thiouridine in tRNA

“…For example, B. subtilis genome encodes for NifZ and three additional cysteine desulfurases (YrvO, NifS, and SufS) which could act as alternative sulfur donors in strains lacking NifZ, thus providing functional replacement in the initial sulfur transfer step in thiazole biosynthesis. The lack of a specific sulfur donor in thiazole biosynthesis is also manifested under certain growth conditions in S. enterica; the thiamine auxotrophy of a thiI mutant strain is suppressed in cysteine-enriched cultures (14).…”

“…In vivo and in vitro data points to the participation of all three domains in the formation of s 4 U8 (11,20,28). A recent report by the Downs' group has demonstrated that, in S. enterica, the Rhd domain alone is sufficient for the sulfur incorporation into the thiazole moiety of thiamine (14). This discovery led to the proposal that ThiI is a bifunctional enzyme carrying distinct tRNA adenylation and sulfurtransferase activities.…”