Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
Centro de Bioinformática, Simulación y Modelado, Facultad de Ingeniería, Universidad de Talca, Talca, Chile.
Arch Biochem Biophys. 2020 Jul 30;688:108389. doi: 10.1016/j.abb.2020.108389. Epub 2020 May 5.
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.
羟甲基嘧啶磷酸激酶（HMPPK）由 thiD 基因编码，参与硫胺素生物合成途径，能够对 4-氨基-5-羟甲基-2-甲基嘧啶（HMP）进行两次连续磷酸化，存在于嗜热菌和中温菌中，但只有少数中温酶的特性得到了描述。另一种同系酶（吡哆醛激酶）的存在，该酶只能催化 HMP 的第一次磷酸化，由 pdxK 基因编码，这阻碍了该酶家族的精确注释。在这里，我们报告了两种具有结构可利用的 HMPPK 的动力学特征，即来自鼠伤寒沙门氏菌（StHMPPK）和嗜热栖热菌（TtHMPPK）的中温和嗜热酶。此外，鉴于它们具有很高的结构相似性，我们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，革兰氏阳性菌的吡哆醛激酶（PLK）（PLK/HMPPK 样酶）构成与典型 PLK 分开的进化分支，但与 HMPPK 密切相关，因此由 pdxK 基因编码的 PLK/HMPPK 样和典型 PLK 是不同的，必须明确注释。StHMPPK 和 TtHMPPK 的动力学特征表明，它们对 HMP 进行双重磷酸化，两种酶均特异性地作用于 HMP，不使用吡哆醛样分子作为底物，其动力学机制涉及形成三元复合物。分子动力学模拟表明，StHMPPK 和 TtHMPPK 在构象灵活性上存在显著差异，这可以与疏水性堆积和静电相互作用网络相关联，主要由盐桥键提供，但有趣的是，与其他嗜热酶报道的氢键相互作用数量无关。酶：EC 2.7.1.49、EC 2.7.4.7、EC 2.7.1.35、EC 2.7.1.50。
