title: Characterization of Thiamin Phosphate Kinase in the Hyperthermophilic Archaeon Pyrobaculum calidifontis.
abstract: Thiamin pyrophosphate is an essential cofactor in all living systems. In its biosynthesis, the thiamin structure is initially formed as thiamin phosphate from a thiazole and a pyrimidine moiety, and then thiamin pyrophosphate is synthesized from thiamin phosphate. Many eubacterial cells directly synthesize thiamin pyrophosphate by the phosphorylation of thiamin phosphate by thiamin phosphate kinase (ThiL), whereas this final step occurs in two stages in eukaryotic cells and some eubacterial cells: hydrolysis of thiamin phosphate to free thiamin and its pyrophosphorylation by thiamin pyrophosphokinase. In addition, some eubacteria have thiamin kinase, a salvage enzyme that converts the incorporated thiamin from the environment to thiamin phosphate. This final step in thiamin biosynthesis has never been experimentally investigated in archaea, although the putative thiL genes are found in their genome database. In this study, we observed thiamin phosphate kinase activity in the soluble fraction of the hyperthermophilic archaeon Pyrobaculum calidifontis. On the other hand, neither thiamin pyrophosphokinase nor thiamin kinase activity was detected, suggesting that in this archaeon the phosphorylation of thiamin phosphate is only way to synthesize thiamin pyrophosphate and it cannot use exogenous thiamin for the salvage synthesis of thiamin pyrophosphate. We also investigated the kinetic properties of thiamin phosphate kinase activity using the recombinant ThiL protein from P. calidifontis. Furthermore, the results obtained by site-directed mutagenesis suggest that the Ser196 of ThiL protein plays a pivotal role in the catalytic process.
authors.affiliation: 2nd Department of Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University.
authors.forename: Maria
authors.initials: M
authors.lastname: Hayashi
authors.forename: Kazuto
authors.initials: K
authors.lastname: Nosaka
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D041543:Pyrobaculum / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D013831:Thiamine / Q000096:biosynthesis*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D017856:Phosphotransferases (Phosphate Group Acceptor); C022735:thiamin-phosphate kinase; D013831:Thiamine