QM/MM Simulations of Enzymatic Hydrolysis of Cellulose: Probing the Viability of an Endocyclic Mechanism for an Inverting Cellulase

Abstract

Glycoside hydrolases (GH) cleave carbohydrate glycosidic bonds and play pivotal roles in living organisms and in many industrial processes. Unlike acid-catalyzed hydrolysis of carbohydrates in solution, which can occur either via cyclic or acyclic oxocarbenium-like transition states, it is widely accepted that GH-catalyzed hydrolysis proceeds via a general acid mechanism involving a cyclic oxocarbenium-like transition state with protonation of the glycosidic oxygen. The GH45 subfamily C inverting endoglucanase from Phanerochaete chrysosporium (PcCel45A) defies the classical inverting mechanism as its crystal structure conspicuously lacks a general Asp or Glu base residue. Instead, PcCel45A has an Asn residue, a notoriously weak base in solution, as one of its catalytic residues at position 92. Moreover, unlike other inverting GHs, the relative position of the catalytic residues in PcCel45A impairs the proton abstraction from the nucleophilic water that attacks the anomeric carbon, a key step in the classical mechanism. Here, we investigate the viability of an endocyclic mechanism for PcCel45A using hybrid quantum mechanics/molecular mechanics (QM/MM) simulations, with the QM region treated with the self-consistent-charge density-functional tight-binding level of theory. In this mechanism, an acyclic oxocarbenium-like transition state is stabilized leading to the opening of the glucopyranose ring and formation of an unstable acyclic hemiacetal that can be readily decomposed into hydrolysis product. In silico characterization of the Michaelis complex shows that PcCel45A significantly restrains the sugar ring to the 4 C 1 chair conformation at the −1 subsite of the substrate binding cleft, in contrast to the classical exocyclic mechanism in which ring puckering is critical. We also show that PcCel45A provides an environment where the catalytic Asn92 residue in its standard amide form participates in a cooperative hydrogen bond network resulting in its increased nucleophilicity due to an increased negative charge on the oxygen atom. Our results for PcCel45A suggest that carbohydrate hydrolysis catalyzed by GHs may take an alternative route from the classical mechanism.

Reconstructing reactivity in dynamic host–guest systems at atomistic resolution: amide hydrolysis under confinement in the cavity of a coordination cage

“…47 Recently shown to provide comparable accuracy to DFT with large basis sets in terms of prediction of barrier heights and reaction energies for organic molecules, [48][49][50] SCC-DFTB has indeed been already successfully used to investigate hydrolysis reactions in biological sys-tems. 51,52 Despite its approximations, this method guarantees satisfactory accuracy in our case at an affordable computational cost, 24 a point particularly relevant given that our method of choice, i.e. infrequent WT-MetaD, requires to run multiple replica simulations in parallel.…”

Revisiting Theoretical Tools and Approaches for the Valorization of Recalcitrant Lignocellulosic Biomass to Value-Added Chemicals

“…At the level of accuracy of the applied theory, detailed structural and energetic information revealed an S(N)2-type-like mechanism via loose transition state structures. In similar work using QM/MM, an endocyclic mechanism for PcCel45A was revealed in which an acyclic oxocarbenium-like transition state is stabilized, leading to the opening of the glucopyranose ring and the formation of an unstable acyclic hemiacetal that can be readily decomposed into hydrolysis products (Pereira et al, 2021).…”

“…Forty nanosecond MD simulations of Trichoderma reesei Cel6A and Cel7A showed the flexible glycosylated linkers of CBD bind nonspecifically to cellulose and can serve as the rate-limiting step in cellulose degradation (Payne et al, 2013;Knott et al, 2014). While MD simulations have been used to study the interaction between cellulases and cellulose and cellulase CBDs and cellulose, QM/MM simulations have been used to reveal the transition state of oligosaccharide hydrolysis (Liu J. et al, 2010;Li et al, 2010;Wang et al, 2011;Wang et al, 2016;Iglesias-Fernández et al, 2017;Zong et al, 2019;Bharadwaj et al, 2020;Pereira et al, 2021). Li et al elucidated the mechanism of enzymatic catalysis of cellulase Cel7A from Trichoderma reesei (Li et al, 2010).…”

Computer Simulation to Rationalize “Rational” Engineering of Glycoside Hydrolases and Glycosyltransferases

“…Families GH4 and GH109 employ an oxidative mechanism, while GH172 operate through a retaining condensation mechanism involving a glycosyl-enzyme intermediate . Other unusual mechanisms have been recently proposed by theoretical methods. , It is not obvious why numerous enzymes evolved to catalyze similar reactions with different mechanisms. In the case of the oxidative mechanisms, it seems that by using less efficient paths (multiple C–H bonds are formed and broken), the chemistry is shifted toward the glycoside rather than the aglycon departure.…”