The rotamer of the second-sphere histidine in AA9 lytic polysaccharide monooxygenase is pH dependent.

Lytic polysaccharide monooxygenases (LPMOs) catalyze a reaction that is crucial for the biological decomposition of various biopolymers and for the industrial conversion of plant biomass. Despite the importance of LPMOs, the exact molecular-level nature of the reaction mechanism is still debated today. Here, we investigated the pH-dependent conformation of a second-sphere histidine (His) that we call the stacking histidine, which is conserved in fungal AA9 LPMOs and is speculated to assist catalysis in several of the LPMO reaction pathways. Using constant-pH and accelerated molecular dynamics simulations, we monitored the dynamics of the stacking His in different protonation states for both the resting Cu(II) and active Cu(I) forms of two fungal LPMOs. Consistent with experimental crystallographic and neutron diffraction data, our calculations suggest that the side chain of the protonated and positively charged form is rotated out of the active site toward the solvent. Importantly, only one of the possible neutral states of histidine (HIE state) is observed in the stacking orientation at neutral pH or when bound to cellulose. Our data predict that, in solution, the stacking His may act as a stabilizer (via hydrogen bonding) of the Cu(II)-superoxo complex after the LPMO-Cu(I) has reacted with O2 in solution, which, in fine, leads to H2O2 formation. Also, our data indicate that the HIE-stacking His is a poor acid/base catalyst when bound to the substrate and, in agreement with the literature, may play an important stabilizing role (via hydrogen bonding) during the peroxygenase catalysis. Our study reveals the pH titration midpoint values of the pH-dependent orientation of the stacking His should be considered when modeling and interpreting LPMO reactions, whether it be for classical LPMO kinetics or in industry-oriented enzymatic cocktails, and for understanding LPMO behavior in slightly acidic natural processes such as fungal wood decay.

Molecular mechanism of the chitinolytic peroxygenase reaction.

Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of monocopper enzymes broadly distributed across the tree of life. Recent reports indicate that LPMOs can use H2O2 as an oxidant and thus carry out a novel type of peroxygenase reaction involving unprecedented copper chemistry. Here, we present a combined computational and experimental analysis of the H2O2-mediated reaction mechanism. In silico studies, based on a model of the enzyme in complex with a crystalline substrate, suggest that a network of hydrogen bonds, involving both the enzyme and the substrate, brings H2O2 into a strained reactive conformation and guides a derived hydroxyl radical toward formation of a copper-oxyl intermediate. The initial cleavage of H2O2 and subsequent hydrogen atom abstraction from chitin by the copper-oxyl intermediate are the main energy barriers. Stopped-flow fluorimetry experiments demonstrated that the priming reduction of LPMO-Cu(II) to LPMO-Cu(I) is a fast process compared to the reoxidation reactions. Using conditions resulting in single oxidative events, we found that reoxidation of LPMO-Cu(I) is 2,000-fold faster with H2O2 than with O2, the latter being several orders of magnitude slower than rates reported for other monooxygenases. The presence of substrate accelerated reoxidation by H2O2, whereas reoxidation by O2 became slower, supporting the peroxygenase paradigm. These insights into the peroxygenase nature of LPMOs will aid in the development and application of enzymatic and synthetic copper catalysts and contribute to a further understanding of the roles of LPMOs in nature, varying from biomass conversion to chitinolytic pathogenesis-defense mechanisms.

How a Lytic Polysaccharide Monooxygenase Binds Crystalline Chitin.

Lytic polysaccharide monooxygenases (LPMOs) are major players in biomass conversion, both in Nature and in the biorefining industry. How the monocopper LPMO active site is positioned relative to the crystalline substrate surface to catalyze powerful, but potentially self-destructive, oxidative chemistry is one of the major questions in the field. We have adopted a multidisciplinary approach, combining biochemical, spectroscopic, and molecular modeling methods to study chitin binding by the well-studied LPMO from Serratia marcescens SmAA10A (or CBP21). The orientation of the enzyme on a single-chain substrate was determined by analyzing enzyme cutting patterns. Building on this analysis, molecular dynamics (MD) simulations were performed to study interactions between the LPMO and three different surface topologies of crystalline chitin. The resulting atomistic models showed that most enzyme-substrate interactions involve the polysaccharide chain that is to be cleaved. The models also revealed a constrained active site geometry as well as a tunnel connecting the bulk solvent to the copper site, through which only small molecules such as H2O, O2, and H2O2 can diffuse. Furthermore, MD simulations, quantum mechanics/molecular mechanics calculations, and electron paramagnetic resonance spectroscopy demonstrate that rearrangement of Cu-coordinating water molecules is necessary when binding the substrate and also provide a rationale for the experimentally observed C1 oxidative regiospecificity of SmAA10A. This study provides a first, experimentally supported, atomistic view of the interactions between an LPMO and crystalline chitin. The confinement of the catalytic center is likely crucially important for controlling the oxidative chemistry performed by LPMOs and will help guide future mechanistic studies.