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.
Mueller
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Department of Chemistry & Biochemistry, University of Delaware, Newark 19716, USA. emueller@udel.edu
Palenchar
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D019732:Amide Synthases / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001124:Argininosuccinate Synthase / Q000737:chemistry / Q000378:metabolism; D001217:Aspartate-Ammonia Ligase / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013466:Sulfurtransferases; D013831:Thiamine / Q000096:biosynthesis; D013891:Thiouridine / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D013891:Thiouridine; D013466:Sulfurtransferases; C107110:ThiI protein, bacteria; C491479:thiI protein, E coli; D019731:Carbon-Nitrogen Ligases; D019732:Amide Synthases; D001217:Aspartate-Ammonia Ligase; C038479:NAD+ synthase; D001124:Argininosuccinate Synthase; C020828:GMP synthase (glutamine-hydrolyzing); D013831:Thiamine