QM-Cluster Model Study of the Guaiacol Hydrogen Atom Transfer and Oxygen Rebound with Cytochrome P450 Enzyme GcoA

Abstract

The key step of the O-demethylation of guaiacol by GcoA of the cytochrome P450-reductase pair was studied with DFT using two 10-residue and three 15-residue QM-cluster models. For each model, two reaction pathways were examined, beginning with a different guaiacol orientation. Based on this study, His354, Phe349, Glu249, and Pro250 residues were found to be important for keeping the heme in a planar geometry throughout the reaction. Val241 and Gly245 residues were needed in the QM-cluster models to provide the hydrophobic pocket for an appropriate guaiacol pose in the reaction. The aromatic triad Phe75, Phe169, and Phe395 may be necessary to facilitate guaiacol migrating into the enzyme active site, but it does not qualitatively affect kinetics and thermodynamics of the proposed mechanism. All QM-cluster models created by RINRUS agree very well with previous experimental work. This study provides details for better understanding enzymatic O-demethylation of lignins to form catechol derivatives by GcoA.

Single-Site Mutation Induces Water-Mediated Promiscuity in Lignin Breaking Cytochrome P450GcoA

“…The previous gas-phase DFT calculations with implicit solvent corrections have identified the hydrogen atom abstraction (HAA) as the rate-limiting step and a barrierless rebound step for both the guaiacol and syringol substrates. 19 , 20 , 27 , 28 …”

“… 19 , 20 However, F169A in complex with guaiacol and syringol showed an increased probability of an open substrate access channel and exposes the active site of P450 GcoA to the bulk solvent. 19 Previous DFT calculations 19 , 20 , 27 , 28 on the HAA and the rebound step of guaiacol or syringol in complex P450 GcoA and its variant F169A neglected the effect of the open substrate access channel, the role of solvent in catalysis and the demethylation of the second methoxy group of syringol.…”

“…The previous gas-phase DFT calculations with implicit solvent corrections have identified the hydrogen atom abstraction (HAA) as the rate-limiting step and a barrierless rebound step for both the guaiacol and syringol substrates. 19,20,27,28 Unfortunately, the native P450 GcoA enzyme shows aryl-Odemethylase activity toward guaiacol only but not against syringol. 20 This creates an initial roadblock to achieving greener and sustainable downstream lignin biodegradation through a natural enzymatic process.…”

A Case Study of the Glycoside Hydrolase Enzyme Mechanism Using an Automated QM-Cluster Model Building Toolkit

“…Ranking of residue importance can be obtained through distance metrics, but also more importantly via qualitative topological features of the residue interaction network ( Shannon et al, 2003 ; del Sol et al, 2006 ; Csermely, 2008 ; Vishveshwara et al, 2009 ; Doncheva et al, 2011 ; Di Paola et al, 2013 ) or from first-principles interaction energies computed via Symmetry Adapted Perturbation Theory ( Summers et al, 2019 ). RINRUS has been applied to many other enzyme case studies which provided valuable results ( DeYonker and Webster, 2013 ; DeYonker and Webster, 2015 ; Summers et al, 2018 ; Cheng and DeYonker, 2020 ; Summers et al, 2021 ; Cheng and DeYonker, 2021 ). It can currently interface with several quantum chemistry packages (such as Gaussian, Q-Chem, PSI4, and xtb).…”

A Single Site Mutation Induces a Water-Mediated Promiscuity in Lignin Breaking Cytochrome P450GcoA

“…19,20 However, F169A in complex with guaiacol and syringol showed an increased probability of an open substrate access channel and exposes the active site of P450 GcoA to the bulk solvent. 19 Previous DFT calculations 19,20,27,28 on the HAA and the rebound step of guaiacol or syringol in complex P450 GcoA and its variant F169A neglected the effect of the open substrate access channel, the role of solvent in catalysis and the demethylation of the second methoxy group of syringol.…”