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 ⁴C₁ 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.

Scheme 1. Classical Inverting Mechanism

Geometric parameters (distances and angles) and substrate conformation (²S₀ and ^2,5B) required for the single-displacement exocyclic mechanism are shown in blue.

Figure 1

Free energy surface along puckering coordinates of a glycosidic ring of (A) cellobiose in solution and (B) PcCel45A substrate at −1 subsite. (C) Cremer–Pople puckering coordinate (θ) of PcCel45A substrate at −1 subsite along an unrestrained QM/MM simulation.

Figure 2

O_w–C1_glc–O5_glc angle versus (A) O_Asn92 and (B) O_Asp85 distances for structures having a potential nucleophilic water and with the substrate at −1 subsite in the ²S₀ metastable conformation. Representative snapshots where (C) the O_w–C1_glc–O5_glc angle is close to 90° (condition 1), but there is no hydrogen bond between the base and the water molecule (condition 2), and where (D) there is a hydrogen bond between the water molecule and the Asn92 base (condition 2), but the O_w–C1_glc–O5_glc angle of 150° is much greater than that required for the single-displacement nucleophilic attack (condition 1). (E and F) Superposition of simulated PcCel45A–substrate complex (enzyme in yellow and substrate in cyan) with crystal structures (in green) with the Asn92 in the tautomer imidic acid in two conformations. The dotted lines indicate the distances between the Asn92 N atom and the potential nucleophilic water that assumes the ideal the O_w–C1_glc–O5_glc angle of 90°.

Figure 3

(A) Representative snapshot of restrained QM/MM MD simulations of PcCel45A with Asn92 in imidic acid form, showing the restrained water molecule (highlighted in red shadow) at the optimal position to perform a nucleophilic attack on the anomeric carbon. (B) Scatter plot of the O_w–C1_glc distance versus the O_W–O_Asn92 distance during the restrained QM/MM MD simulations carrying a water molecule to the imminence of nucleophilic attack.

Figure 4

Molecular surface showing the active site architecture of (A) GH6 (a tunnel) and (B) PcCel45A (an open groove) and a representative snapshot showing the potential nucleophilic water surrounded by many water molecules connected to the bulk.

Scheme 2. Proposed Endocyclic Mechanism

Geometric parameters (distances and angle) and substrate conformation (⁴C₁) required for the endocyclic mechanism are shown in blue.

Figure 5

(A) O_w–C1_glc–O4_glc angle versus O_w–O_Asn92 distance. The red oval indicates structures visited in the simulation which satisfy the conditions for a single-displacement nucleophilic reaction. (B) Free energy surface of the reaction. Reactants (R) and products (P) are connected by a curved arrow passing through the transition state (TS). (C) Details of the ring-opening step showing the reactants (R), transition state (TS), and products (P).

Figure 6

(A) Resonance forms of amides in hydrogen bonding networks. (B) Gln39–Val89–Asn92 hydrogen bonding network. (C) Distribution of the distance (D1) between Asn92 hydrogen and Val89 oxygen. Distribution of Val89 (D) nitrogen and (E) oxygen Mulliken charges, considering both reactant (neutral Asn92) and product (protonated Asn92) states.