Phosphorylation Mechanism of N-acetyl-L-Glutamate Kinase, a QM/MM Study

James McClory, Gui-Xiang Hu, Jian-Wei Zou, David J. Timson, Meilan Huang

Research output: Contribution to journalArticleResearchpeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)2844-2852
Number of pages9
JournalJournal of Physical Chemistry B
Volume123
Issue number13
DOIs
Publication statusPublished - 8 Mar 2019

Fingerprint

glutamate 5-kinase
acetylglutamate kinase
phosphorylation
glutamates
Phosphorylation
Glutamic Acid
Phosphates
Substrates
phosphates
Microorganisms
Phosphotransferases
Arginine
Molecular mechanics
Mammals
Quantum theory
microorganisms
Adenosine
Glycine
Catalysis
Density functional theory

Cite this

McClory, James ; Hu, Gui-Xiang ; Zou, Jian-Wei ; Timson, David J. ; Huang, Meilan. / Phosphorylation Mechanism of N-acetyl-L-Glutamate Kinase, a QM/MM Study. In: Journal of Physical Chemistry B. 2019 ; Vol. 123, No. 13. pp. 2844-2852.
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abstract = "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.",
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Phosphorylation Mechanism of N-acetyl-L-Glutamate Kinase, a QM/MM Study. / McClory, James; Hu, Gui-Xiang; Zou, Jian-Wei; Timson, David J.; Huang, Meilan.

In: Journal of Physical Chemistry B, Vol. 123, No. 13, 08.03.2019, p. 2844-2852.

Research output: Contribution to journalArticleResearchpeer-review

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