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

Author(s)

Pereira, Caroline S. ; Silveira, Rodrigo L. ; Skaf, Munir S.

• Document type: Journal article
• Source: JOURNAL OF CHEMICAL INFORMATION AND MODELING; v. 61, n. 4, p. 11-pg., 2021-03-24.

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 (PcCe145A) defies the classical inverting mechanism as its crystal structure conspicuously lacks a general Asp or Glu base residue. Instead, PcCe145A 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 PcCe145A 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 PcCe145A 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 PcCe145A significantly restrains the sugar ring to the C-4(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 PcCe145A 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 PcCe145A suggest that carbohydrate hydrolysis catalyzed by GHs may take an alternative route from the classical mechanism. (AU)

• FAPESP's process: 13/08293-7 - CCES - Center for Computational Engineering and Sciences
• Grantee: Munir Salomao Skaf
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 14/10448-1 - Molecular aspects of plant cell wall architecture
• Grantee: Rodrigo Leandro Silveira
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 15/25031-1 - Molecular Dynamics of Carbohydrate Modifying Enzymes for Lignocellulosic Biomass Deconstruction and Valorization
• Grantee: Caroline Simões Pereira
• Support Opportunities: Scholarships in Brazil - Doctorate