Characterization of a novel bifunctional dihydropteroate synthase/dihydropteroate reductase enzyme from Helicobacter pylori.
Characterization of a novel bifunctional dihydropteroate synthase/dihydropteroate reductase enzyme from Helicobacter pylori.
复制标题
来自幽门螺杆菌的新型双功能二氢蝶酸合酶/二氢蝶酸还原酶的表征。
DOI：
10.1128/jb.01878-06
复制
发表时间：
2007
影响因子：
3.2
通讯作者：
Palfey,BruceA
中科院分区：
文献类型：
--
作者：
Levin,Itay;Mevarech,Moshe;Palfey,BruceA
四氢叶酸是细菌许多生物合成途径中普遍存在的一种C1载体，在甲硫酰tRNAfMet的生物合成中尤为重要，而甲硫酰tRNAfMet是启动翻译的关键。生物合成四氢叶酸的最后一步是由二氢叶酸还原酶(DHFR)完成的。对幽门螺杆菌全基因组序列的搜索可能揭示编码dhfr的任何序列。以前的研究表明，这一点。幽门螺杆菌二氢蝶酸合成酶基因folP可以补充大肠杆菌，其中缺失了编码两个不同的DHFRs的folA和folM。它也被展示了。PyloriFolP具有一个额外的N-末端结构域，与黄素单核苷酸(FMN)结合。在其他不具有DHFR的微生物的FolP蛋白中也发现了同源结构域。在这项研究中，我们证明了。PyloriFolP也是一种二氢翼酸还原酶，其还原能力来自于可溶性黄素、还原的FMN和还原的黄素腺嘌呤二核苷酸。我们还测定了酶结合黄素的化学计量比，结果表明，结合黄素的一半可与可溶性黄素交换。最后，对N-末端结构域中最保守的氨基酸残基进行了定点突变，表明这些残基对该酶作为二氢翼酸还原酶的活性具有重要意义。
Tetrahydrofolate is a ubiquitous C1carrier in many biosynthetic pathways in bacteria, importantly, in the biosynthesis of formylmethionyl tRNAfMet, which is essential for the initiation of translation. The final step in the biosynthesis of tetrahydrofolate is carried out by the enzyme dihydrofolate reductase (DHFR). A search of the complete genome sequence ofHelicobacter pylorifailed to reveal any sequence that encodes DHFR. Previous studies demonstrated that theH. pyloridihydropteroate synthase genefolPcan complement anEscherichia colistrain in whichfolAandfolM, encoding two distinct DHFRs, are deleted. It was also shown thatH. pyloriFolP possesses an additional N-terminal domain that binds flavin mononucleotide (FMN). Homologous domains are found in FolP proteins of other microorganisms that do not possess DHFR. In this study, we demonstrated thatH. pyloriFolP is also a dihydropteroate reductase that derives its reducing power from soluble flavins, reduced FMN and reduced flavin adenine dinucleotide. We also determined the stoichiometry of the enzyme-bound flavin and showed that half of the bound flavin is exchangeable with the soluble flavins. Finally, site-directed mutagenesis of the most conserved amino acid residues in the N-terminal domain indicated the importance of these residues for the activity of the enzyme as a dihydropteroate reductase.