Minjárez-Sáenz Martha, Rivero Maribel, Correa-Pérez Víctor, Boneta Sergio, Suárez Paula, Polo Víctor, Sadeghi Sheila J, Yruela Inmaculada, Martínez-Júlvez Marta, Medina Milagros
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain; Instituto de Biocomputación y Física de Sistemas Complejos (BIFI) Universidad de Zaragoza, GBsC (Unizar) Join Unit to CSIC, Zaragoza, Spain.
Instituto de Biocomputación y Física de Sistemas Complejos (BIFI) Universidad de Zaragoza, GBsC (Unizar) Join Unit to CSIC, Zaragoza, Spain; Departamento de Química Física, Universidad de Zaragoza, Zaragoza, Spain.
Arch Biochem Biophys. 2025 Mar;765:110288. doi: 10.1016/j.abb.2025.110288. Epub 2025 Jan 4.
The peptidoglycan biosynthetic pathway involves a series of enzymatic reactions in which UDP-N-acetylglucosamine-enolpyruvate reductase (MurB) plays a crucial role in catalyzing the conversion of UDP-N-acetylglucosamine-enolpyruvate (UNAGEP) to UDP-N-acetylmuramic acid. This reaction relies on NADPH and FAD and, since MurB is not found in eukaryotes, it is an attractive target for the development of antimicrobials. MurB from Brucella ovis, the causative agent of brucellosis in sheep, is characterized here. The FAD cofactor in MurB of B. ovis is reduced to the hydroquinone state without semiquinone stabilization with an estimated E of -260 mV. MurB from B. ovis catalyzes the oxidation of NADPH in a slow process that is positively influenced by the presence of the second product, UNAGEP. The crystallographic structure of the MurB:UNAGEP complex confirms its folding into three domains and the binding of UNAGEP, positioning its enolpyruvyl group for hydride transfer from FAD. MurB shows a complex thermal unfolding pathway that is influenced by UNAGEP and NADP, confirming its ability to bind both molecules. Molecular dynamics (MD) simulations predict that the nicotinamide of NADP is more stable at the active site than the enolpyruvyl of UNAGEP, and suggests that MurB can simultaneously accommodate NADPH and UNAGEP in the substrate channel, increasing overall protein-ligand flexibility. Sequence and evolutionary analyses show that MurB from B. ovis conserves all motifs predicted to be involved in catalysis within the Type IIa family.
肽聚糖生物合成途径涉及一系列酶促反应，其中UDP-N-乙酰葡糖胺-烯醇丙酮酸还原酶（MurB）在催化UDP-N-乙酰葡糖胺-烯醇丙酮酸（UNAGEP）转化为UDP-N-乙酰胞壁酸的过程中起关键作用。该反应依赖于NADPH和FAD，由于真核生物中不存在MurB，因此它是抗菌药物开发的一个有吸引力的靶点。本文对绵羊布鲁氏菌病的病原体绵羊布鲁氏菌的MurB进行了表征。绵羊布鲁氏菌MurB中的FAD辅因子被还原为对苯二酚状态，没有半醌稳定，估计E为-260 mV。绵羊布鲁氏菌的MurB在一个缓慢的过程中催化NADPH的氧化，该过程受到第二种产物UNAGEP存在的正向影响。MurB:UNAGEP复合物的晶体结构证实其折叠成三个结构域以及UNAGEP的结合，将其烯醇丙酮酸基团定位用于从FAD进行氢化物转移。MurB显示出受UNAGEP和NADP影响的复杂热解折叠途径，证实了其结合这两种分子的能力。分子动力学（MD）模拟预测，NADP的烟酰胺在活性位点比UNAGEP的烯醇丙酮酸更稳定，并表明MurB可以在底物通道中同时容纳NADPH和UNAGEP，增加整体蛋白质-配体的灵活性。序列和进化分析表明，绵羊布鲁氏菌的MurB保留了IIa型家族中预测参与催化的所有基序。
