9MXP

Pseudomonas fluorescens isocyanide hydratase D17N mutant, pH=8.8

9MXP の概要

• エントリーDOI: 10.2210/pdb9mxp/pdb
• 関連するPDBエントリー: 9MXN
• 分子名称: Cyclohexyl-isocyanide hydratase, MAGNESIUM ION, CHLORIDE ION, ... (4 entities in total)
• 機能のキーワード: isonitrile, ph, cysteine, lyase
• 由来する生物種: Pseudomonas fluorescens
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 48489.99

構造登録者

Smith, N.,Prososki, K.,Wilson, M.A. (登録日: 2025-01-20, 公開日: 2025-10-29)

主引用文献

Corrigan Grove, R.A.,Moxley, M.A.,Negre, C.F.A.,Cawkwell, M.J.,Niklasson, A.M.N.,Mniszewski, S.M.,Smith, N.,Prososki, K.,Wilson, M.A.,Wall, M.E.
Combining Reactive Quantum-Mechanical Molecular-Dynamics Simulations with Mutagenesis, Crystallography, and Enzyme Kinetics to Reveal Plausible Steps of Isocyanide Hydratase Catalysis.
J.Chem.Inf.Model., 2025

PubMed Abstract: A complete understanding of enzyme mechanisms requires atomistic details of chemical reactions. Quantum-based molecular dynamics simulations (QMD) are a potential source of this information, but trade-offs between accuracy and computational cost have limited their use. We previously developed extended Lagrangian Born-Oppenheimer molecular dynamics (XL-BOMD) methods that leverage a negligible compromise in accuracy to substantially decrease the cost of QMD simulations. Here, we develop a reactive QMD approach using the latest XL-BOMD formulation, which enables efficient simulations of highly reactive systems, and use it to investigate mechanisms of intermediate formation in isocyanide hydratase (ICH) catalysis. In QMD simulations, molecular analogs of ICH active site residues reacted with para-nitrophenyl isocyanide, forming a thioimidate. Analysis of simulated atomic configurational and charge dynamics revealed a pathway where protonation of the isocyanide carbon occurs prior to thioimidate formation and suggested a possible role of Asp17 as a proton donor in the early phase of ICH catalysis. To test whether the pathway seen using the reactive QMD approach might be relevant to ICH catalysis, we performed X-ray crystallography and pre-steady-state enzyme kinetics studies of wild-type and D17N mutant ICH. Both the structure and kinetics are sensitive to the D17N mutation in a manner that is consistent with the order of the reaction steps seen in the simulations. Mobile protons play essential roles in many enzymes, yet they are difficult to observe experimentally, making the ordering of proton-dependent steps ambiguous in many enzyme mechanisms. The ability to directly simulate model reactions for the design of experiments that provide information about enzyme mechanisms involving mobile protons demonstrates the significance of our reactive QMD approach and motivates further biological applications.

PubMed: 41051317
DOI: 10.1021/acs.jcim.5c01152

実験手法

X-RAY DIFFRACTION (1.2 Å)