Hayashi Maria, Kobayashi Kazuya, Esaki Hiroyoshi, Konno Hiroyuki, Akaji Kenichi, Tazuya Keiko, Yamada Kazuko, Nakabayashi Toshikatsu, Nosaka Kazuto
Department of Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Japan.
Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Kyoto, Japan.
Biochim Biophys Acta. 2014 Apr;1844(4):803-9. doi: 10.1016/j.bbapap.2014.02.017. Epub 2014 Feb 27.
Studies on thiamin biosynthesis have so far been achieved in eubacteria, yeast and plants, in which the thiamin structure is formed as thiamin phosphate from a thiazole and a pyrimidine moiety. This condensation reaction is catalyzed by thiamin phosphate synthase, which is encoded by the thiE gene or its orthologs. On the other hand, most archaea do not seem to have the thiE gene, but instead their thiD gene, coding for a 2-methyl-4-amino-5-hydroxymethylpyrimidine (HMP) kinase/HMP phosphate kinase, possesses an additional C-terminal domain designated thiN. These two proteins, ThiE and ThiN, do not share sequence similarity. In this study, using recombinant protein from the hyperthermophile archaea Pyrobaculum calidifontis, we demonstrated that the ThiN protein is an analog of the ThiE protein, catalyzing the formation of thiamin phosphate with the release of inorganic pyrophosphate from HMP pyrophosphate and 4-methyl-5-β-hydroxyethylthiazole phosphate (HET-P). In addition, we found that the ThiN protein can liberate an inorganic pyrophosphate from HMP pyrophosphate in the absence of HET-P. A structure model of the enzyme-product complex of P. calidifontis ThiN domain was proposed on the basis of the known three-dimensional structure of the ortholog of Pyrococcus furiosus. The significance of Arg320 and His341 residues for thiN-coded thiamin phosphate synthase activity was confirmed by site-directed mutagenesis. This is the first report of the experimental analysis of an archaeal thiamin synthesis enzyme.
迄今为止，关于硫胺素生物合成的研究已在真细菌、酵母和植物中完成，在这些生物中，硫胺素结构由噻唑和嘧啶部分形成硫胺素磷酸酯。这种缩合反应由硫胺素磷酸合酶催化，该酶由thiE基因或其直系同源基因编码。另一方面，大多数古细菌似乎没有thiE基因，但其编码2-甲基-4-氨基-5-羟甲基嘧啶（HMP）激酶/HMP磷酸激酶的thiD基因拥有一个额外的C末端结构域，称为thiN。这两种蛋白质，ThiE和ThiN，不具有序列相似性。在本研究中，我们使用嗜热古细菌嗜热栖热袍菌的重组蛋白，证明ThiN蛋白是ThiE蛋白的类似物，催化硫胺素磷酸酯的形成，并从HMP焦磷酸和4-甲基-5-β-羟乙基噻唑磷酸酯（HET-P）中释放无机焦磷酸。此外，我们发现ThiN蛋白在没有HET-P的情况下可以从HMP焦磷酸中释放无机焦磷酸。基于激烈火球菌直系同源物的已知三维结构，提出了嗜热栖热袍菌ThiN结构域的酶-产物复合物的结构模型。通过定点诱变证实了Arg320和His341残基对thiN编码的硫胺素磷酸合酶活性的重要性。这是关于古细菌硫胺素合成酶实验分析报道。
