Quantum mechanical calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl, oxygen-rebound mechanism

Abstract

Lytic polysaccharide monooxygenases (LPMOs) exhibit a mononuclear copper-containing active site and use dioxygen and a reducing agent to oxidatively cleave glycosidic linkages in polysaccharides. LPMOs represent a unique paradigm in carbohydrate turnover and exhibit synergy with hydrolytic enzymes in biomass depolymerization. To date, several features of copper binding to LPMOs have been elucidated, but the identity of the reactive oxygen species and the key steps in the oxidative mechanism have not been elucidated. Here, density functional theory calculations are used with an enzyme active site model to identify the reactive oxygen species and compare two hypothesized reaction pathways in LPMOs for hydrogen abstraction and polysaccharide hydroxylation; namely, a mechanism that employs a η 1 -superoxo intermediate, which abstracts a substrate hydrogen and a hydroperoxo species is responsible for substrate hydroxylation, and a mechanism wherein a copper-oxyl radical abstracts a hydrogen and subsequently hydroxylates the substrate via an oxygen-rebound mechanism. The results predict that oxygen binds end-on (η 1 ) to copper, and that a copperoxyl-mediated, oxygen-rebound mechanism is energetically preferred. The N-terminal histidine methylation is also examined, which is thought to modify the structure and reactivity of the enzyme. Density functional theory calculations suggest that this posttranslational modification has only a minor effect on the LPMO active site structure or reactivity for the examined steps. Overall, this study suggests the steps in the LPMO mechanism for oxidative cleavage of glycosidic bonds.

Structural and Electronic Snapshots during the Transition from a Cu(II) to Cu(I) Metal Center of a Lytic Polysaccharide Monooxygenase by X-ray Photoreduction

“…This order of events is in accordance with the notion that molecular oxygen tends to bind copper proteins when the metal ion is in the reduced monovalent state (59). Subsequent to dioxygen binding, the catalytic cycle is initiated, which results in substrate hydroxylation, followed by elimination to cleave the glycosidic linkage (10,11). This general mechanism will incorporate a single oxygen atom from molecular oxygen into the products.…”

“…tively, in an AA9 LPMO with a similar ASM approach (11). Interestingly, the charge distribution of the coordinating histidine residues does not show a significant change, despite the substantial change in the copper ion oxidation state.…”

“…The catalytic mechanism of AA9 LPMOs, which to date only are found in fungi, was recently examined with density functional theory (DFT) calculations (11). Kim et al (11) predicted that AA9 LPMOs employ a Cu(II)-oxyl reactive oxygen species for hydrogen abstraction from the substrate, followed by an oxygen-rebound mechanism for substrate hydroxylation.…”

Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity

“…A similar difference has been observed when comparing a strictly C1-oxidizing LPMO10 with an LPMO10 that can oxidize both C1 and C4 (17). It has been shown that end-on binding of dioxygen is energetically favorable (12,49); however, although Kim et al (49) model O 2 binding at the exposed axial position, Kjaergaard et al (12) propose a model where the equatorial OH ligand is replaced by O 2 . This discrepancy is probably a result of different initial structural models and basis sets used in the density functional theory (DFT) calculations.…”

“…Although binding of multiple bivalent metal ions has been observed in the crystal structures for several LPMOs (5,17), available experimental and theoretical data consistently indicate that the active site copper centers are mononuclear (11,12,17,49). The additional metal ions observed in the zinc structure of NcLPMO9C are coordinated by amino acids belonging to a sequence insertion unique for strictly C4-oxidizing LPMO9s, but Fig.…”