School of Chemistry and Chemical Engineering , Queen's University Belfast , David Keir Building, Stranmillis Road , Belfast BT9 5AG , Northern Ireland , U.K.
School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology , Zhejiang University , 1 Xuefu Rd , Yinzhou Dist. Ningbo 315100 , China.
J Phys Chem B. 2019 Apr 4;123(13):2844-2852. doi: 10.1021/acs.jpcb.9b00547. Epub 2019 Mar 21.
In microorganisms and plants, N-acetyl-l-glutamate kinase (NAGK) catalyzes the second step in l-arginine synthesis, the phosphorylation of N-Acetyl-l-glutamate (NAG) to give N-acetyl-l-glutamate-5-phosphate. NAGK is only present in microorganisms and plants but absent in mammals, which makes it an attractive target for antimicrobial or biocidal development. Understanding the substrate binding mode and reaction mechanism of NAGK is crucial for targeting the kinase to develop potential therapies. Here, the substrate binding mode was studied by comparing the conformational change of NAGK in the presence and in the absence of the NAG substrate based on molecular dynamics simulations. We revealed that with substrate binding, the catalytic site of the kinase involving three loops in NAGK exhibits a closed conformation, which is predominantly controlled by an interaction between Arg98 and the α-COO of NAG. Lys41 is found to guide phosphate transfer through the interactions with the β-,γ-, and γ-phosphate oxygen atoms of adenosine 5'-triphosphate surrounded by two highly conserved glycine residues (Gly44 and Gly76), while Arg98 helps to position the NAG substrate in the catalytic site, which facilitates the phosphate transfer. Furthermore, we elucidated phosphate-transfer reaction mechanism using hybrid density functional theory-based quantum mechanics/molecular mechanics calculations (B97D/AMBER99) and found that the catalysis follows a dissociative mechanism.
在微生物和植物中，N-乙酰-l-谷氨酸激酶（NAGK）催化 l-精氨酸合成的第二步，即 N-乙酰-l-谷氨酸（NAG）磷酸化生成 N-乙酰-l-谷氨酸-5-磷酸。NAGK 仅存在于微生物和植物中，而不存在于哺乳动物中，这使其成为抗菌或杀菌开发的有吸引力的目标。了解 NAGK 的底物结合模式和反应机制对于靶向激酶开发潜在疗法至关重要。在这里，通过基于分子动力学模拟比较 NAGK 在存在和不存在 NAG 底物的情况下的构象变化，研究了底物结合模式。我们揭示了与底物结合后，激酶的催化位点涉及 NAGK 中的三个环，呈现出封闭构象，主要由 Arg98 与 NAG 的α-COO 之间的相互作用控制。发现 Lys41 通过与周围两个高度保守的甘氨酸残基（Gly44 和 Gly76）的腺苷 5'-三磷酸的β-、γ-和γ-磷酸氧原子相互作用来引导磷酸转移，而 Arg98 有助于将 NAG 底物定位在催化位点，从而促进磷酸转移。此外，我们使用基于杂化密度泛函理论的量子力学/分子力学计算（B97D/AMBER99）阐明了磷酸转移反应机制，并发现该催化遵循离解机制。
