The hydroxymethylpyrimidine phosphate kinases (HMPPK) encoded by the thiD gene are involved in the thiamine biosynthesis pathway, can perform two consecutive phosphorylations of 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) and are found in thermophilic and mesophilic bacteria, but only a few characterizations of mesophilic enzymes are available. The presence of another homolog enzyme (pyridoxal kinase) that can only catalyze the first phosphorylation of HMP and encoded by pdxK gene, has hampered a precise annotation in this enzyme family. Here we report the kinetic characterization of two HMPPK with structure available, the mesophilic and thermophilic enzyme from Salmonella typhimurium (StHMPPK) and Thermus thermophilus (TtHMPPK), respectively. Also, given their high structural similarity, we have analyzed the structural determinants of protein thermal stability in these enzymes by molecular dynamics simulation. The results show that pyridoxal kinases (PLK) from gram-positive bacteria (PLK/HMPPK-like enzymes) constitute a phylogenetically separate group from the canonical PLK, but closely related to the HMPPK, so the PLK/HMPPK-like and canonical PLK, both encoded by pdxK genes, are different and must be annotated distinctly. The kinetic characterization of StHMPPK and TtHMPPK, shows that they perform double phosphorylation on HMP, both enzymes are specific for HMP, not using pyridoxal-like molecules as substrates and their kinetic mechanism involves the formation of a ternary complex. Molecular dynamics simulation shows that StHMPPK and TtHMPPK have striking differences in their conformational flexibility, which can be correlated with the hydrophobic packing and electrostatic interaction network given mainly by salt bridge bonds, but interestingly not by the number of hydrogen bond interactions as reported for other thermophilic enzymes. ENZYMES: EC 2.7.1.49, EC 2.7.4.7, EC 2.7.1.35, EC 2.7.1.50.

中文翻译：

---

嗜温和嗜热细菌中羟甲基嘧啶磷酸激酶的表征及其对不同热稳定性的结构见解。

由thiD基因编码的羟甲基嘧啶磷酸激酶（HMPPK）参与硫胺素的生物合成途径，可以连续进行两个连续的4-氨基-5-羟甲基-2-甲基嘧啶（HMP）磷酸化反应，并存在于嗜热和嗜温细菌中，但只有一些嗜温酶的表征。只能催化HMP首次磷酸化并由pdxK基因编码的另一种同源酶（吡rid醛激酶）妨碍了该酶家族的精确注释。在这里，我们报告了两个具有可用结构的HMPPK的动力学表征，分别是鼠伤寒沙门氏菌（StHMPPK）和嗜热栖热菌（TtHMPPK）的嗜温和嗜热酶。而且，由于它们的结构相似性很高，我们已经通过分子动力学模拟分析了这些酶中蛋白质热稳定性的结构决定因素。结果表明，革兰氏阳性细菌的吡ido醛激酶（PLK / HMPPK-like酶）构成了与规范PLK的系统发育分离基团，但与HMPPK密切相关，因此PLK / HMPPK-like和规范PLK两者都是由pdxK基因编码的，并且是不同的，必须加以清楚的注释。StHMPPK和TtHMPPK的动力学特征表明，它们对HMP进行了双重磷酸化，这两种酶都是HMP特有的，没有使用像吡ido醛样分子作为底物，它们的动力学机制涉及三元复合物的形成。分子动力学模拟表明，StHMPPK和TtHMPPK在构象柔韧性上存在显着差异，其可以与主要由盐桥键给出的疏水性堆积和静电相互作用网络相关，但是有趣的是与其他嗜热酶报道的氢键相互作用的数量无关。酶：EC 2.7.1.49，EC 2.7.4.7，EC 2.7.1.35，EC 2.7.1.50。