The role of the cysteine residues of ThiI in the generation of 4-thiouridine in tRNA.

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.

Functional importance of motif I of pseudouridine synthases: mutagenesis of aligned lysine and proline residues.

On the basis of sequence alignments, the pseudouridine synthases were grouped into four families that share no statistically significant global sequence similarity, though some common sequence motifs were discovered [Koonin, E. V. (1996) Nucleic Acids. Res. 24, 2411-2415; Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762]. We have investigated the functional significance of these alignments by substituting the nearly invariant lysine and proline residues in Motif I of RluA and TruB, pseudouridine synthases belonging to different families. Contrary to our expectations, the altered enzymes display only very mild kinetic impairment. Substitution of the aligned lysine and proline residues does, however, reduce structural stability, consistent with a temperature sensitive phenotype that results from substitution of the cognate proline residue in Cbf5p, a yeast homologue of TruB [Zerbarjadian, Y., King, T., Fournier, M. J., Clarke, L., and Carbon, J. (1999) Mol. Cell. Biol. 19, 7461-7472]. Together, our data support a functional role for Motif I, as predicted by sequence alignments, though the effect of substituting the highly conserved residues was milder than we anticipated. By extrapolation, our findings also support the assignment of pseudouridine synthase function to certain physiologically important eukaryotic proteins that contain Motif I, including the human protein dyskerin, alteration of which leads to the disease dyskeratosis congenita.

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).

Using genomic information to investigate the function of ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis.

The gene thiI encodes a protein (ThiI) that plays a role in the transfer of sulfur from cysteine to both thiamin and 4-thiouridine, but the reaction catalyzed by ThiI remains undetermined. Based upon sequence alignments, ThiI shares a unique "P-loop" motif with the PPi synthetase family, four enzymes that catalyze adenylation and subsequent substitution of carbonyl oxygens. To test whether or not this motif is critical for ThiI function, the Asp in the motif was converted to Ala (D189A), and a screen for in vivo 4-thiouridine production revealed the altered enzyme to be inactive. Further scrutiny of sequence data and the crystal structures of two members of the PPi synthetase family implicated Lys321 in the proposed adenylation function of ThiI, and the critical nature of Lys321 has been demonstrated by site-directed mutagenesis and genetic screening. Our results, then, indicate that ThiI catalyzes the adenylation of a substrate at the expense of ATP, a narrowing of possible reactions that provides a strong new basis for deducing the early steps in the transfer of sulfur from cysteine to both thiamin and 4-thiouridine.

Role of cysteine residues in pseudouridine synthases of different families.

The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine in RNA molecules. An attractive mechanism was proposed based on that of thymidylate synthase, in which the thiol(ate) group of a cysteine side chain serves as the nucleophile in a Michael addition to C6 of the isomerized uridine. Such a role for cysteine in the pseudouridine synthase TruA (also named Psi synthase I) has been discredited by site-directed mutagenesis, but sequence alignments have led to the conclusion that there are four distinct "families" of pseudouridine synthases that share no statistically significant global sequence similarity. It was, therefore, necessary to probe the role of cysteine residues in pseudouridine synthases of the families that do not include TruA. We examined the enzymes RluA and TruB, which are members of different families than TruA and each other. Substitution of cysteine for amino acids with nonnucleophilic side chains did not significantly alter the catalytic activity of either pseudouridine synthase. We conclude, therefore, that neither TruB nor RluA require thiol(ate) groups to effect catalysis, excluding their participation in a Michael addition to C6 of uridine, although not eliminating that mechanism (with an alternate nucleophile) from future consideration.

Critical aspartic acid residues in pseudouridine synthases.

The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.

Identification of a gene involved in the generation of 4-thiouridine in tRNA.

All organisms modify the bases of their RNA after transcription. Relatively little is known about the functions that these chemical alterations serve and, with very few exceptions, even less has been established regarding the enzymology involved. One modified base of known function is 4-thiouridine at position 8 of certain bacterial tRNAs, which serves as a photosensor for near-UV light. A gene involved in the conversion of uridine at position 8 into 4-thiouridine has been identified by genetic screening and its role in 4-thiouridine generation has been confirmed biochemically. This same gene, thiI , has recently been shown to play a role in thiamin biosynthesis. The purification and characteristics of the purified protein are also reported.

Stereochemical course of the reactions catalyzed by the bacterial phosphoenolpyruvate:mannitol phosphotransferase system.

We have determined the overall stereochemical course of the reactions leading to the phosphorylation of D-mannitol by mannitol-specific enzyme II (EIIMtl) of the Escherichia coli phosphoenolpyruvate- (PEP) dependent phosphotransferase system (PTS). In the presence of enzyme I and HPr of the PTS, and of membranes containing EIIMtl, the phospho group from [(R)-16O,17O,18O]PEP was transferred to D-mannitol to form mannitol 1-phosphate with overall inversion of the configuration at phosphorus with respect to that of PEP. Since in the course of these reactions enzyme I and HPr are each covalently phosphorylated at a single site and inversion of the chiral phospho group from PEP indicates an odd number of transfer steps overall, transfer from phospho-HPr to mannitol via EIIMtl must also occur in an odd number of steps. Taken together with the fact that catalytically important phospho-EIIMtl intermediates have been demonstrated biochemically, our results imply that EIIMtl is sequentially phosphorylated at two different sites during phospho transfer from phospho-HPr to mannitol. This conclusion is consistent with the available evidence on phospho-EIIMtl intermediates and in particular with the recent report that two different phospho peptides can be isolated from the fully phosphorylated protein [Pas, H. H., & Robillard, G. T. (1988) Biochemistry 27, 5835-5839].