Characterization of an Acinetobacter baumannii Monofunctional Phosphomethylpyrimidine Kinase That Is Inhibited by Pyridoxal Phosphate
Characterization of an Acinetobacter baumannii Monofunctional Phosphomethylpyrimidine Kinase That Is Inhibited by Pyridoxal Phosphate
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DOI：
10.1021/acs.biochem.3c00640
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发表时间：
2024-02-02
期刊：
影响因子：
2.9
通讯作者：
French，Jarrod B.
中科院分区：
文献类型：
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作者：
De Vitto，Humberto;Belfon，Kafi K. J.;French，Jarrod B.
硫胺素及其磷酸盐衍生物是普遍存在的分子，作为许多细胞过程中必需的辅助因子。硫胺素的从头生物合成采用平行合成 4-甲基-5-(2-羟乙基)噻唑 (THZ-P) 和 4-氨基-2-甲基-5(二磷氧基甲基)嘧啶 (HMP) 焦磷酸盐 (HMP-PP)，它们偶联生成磷酸硫胺素。大多数能够生物合成硫胺素的生物体利用激酶（HMPK 或 ThiD）来生成 HMP-PP。几乎在所有情况下，这种酶都是双功能的，还可以回收游离的 HMP，产生 HMP-P，即 HMP-PP 的单磷酸盐前体。在这里，我们展示了来自鲍曼不动杆菌 (AbHMPK) 的 HMPK 的高分辨率晶体结构，包括未配体的和与 5-磷酸吡哆醛 (PLP) 非共价结合的。尽管 HMPK 和吡哆醛激酶之间存在相似性，但我们的动力学分析表明 AbHMPK 只接受 HMP 作为底物，不能转化吡哆醛、吡哆胺或吡哆醇，也不显示磷酸酶活性。然而，PLP 确实是 AbHMPK 的弱抑制剂，IC50 为 768 μM。令人惊讶的是，与其他 HMPK 不同，AbHMPK 只催化 HMP 的磷酸化，不会生成二磷酸 HMP-PP。这表明 A 中存在额外的激酶。鲍曼不动杆菌或替代机制正在运行以完成硫胺素的生物合成。
Thiamin and its phosphate derivatives are ubiquitous molecules involved as essential cofactors in many cellular processes. Thede novobiosynthesis of thiamin employs the parallel synthesis of 4-methyl-5-(2-hydroxyethyl)thiazole (THZ-P) and 4-amino-2-methyl-5(diphosphooxymethyl) pyrimidine (HMP) pyrophosphate (HMP-PP), which are coupled to generate thiamin phosphate. Most organisms that can biosynthesize thiamin employ a kinase (HMPK or ThiD) to generate HMP-PP. In nearly all cases, this enzyme is bifunctional and can also salvage free HMP, producing HMP-P, the monophosphate precursor of HMP-PP. Here we present high-resolution crystal structures of an HMPK fromAcinetobacter baumannii(AbHMPK), both unliganded and with pyridoxal 5-phosphate (PLP) noncovalently bound. Despite the similarity between HMPK and pyridoxal kinase enzymes, our kinetics analysis indicates that AbHMPK accepts HMP exclusively as a substrate and cannot turn over pyridoxal, pyridoxamine, or pyridoxine nor does it display phosphatase activity. PLP does, however, act as a weak inhibitor of AbHMPK with an IC50of 768 μM. Surprisingly, unlike other HMPKs, AbHMPK catalyzes only the phosphorylation of HMP and does not generate the diphosphate HMP-PP. This suggests that an additional kinase is present inA. baumannii, or an alternative mechanism is in operation to complete the biosynthesis of thiamin.