Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase

Abstract

Lytic polysaccharide monooxygenases have attracted vast attention due to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. We have determined high resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II)-dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed "pre-bound" molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygen activation. These results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme-substrate complex. Graphical abstractX-ray crystallography produces the first structure of an LPMO enzyme with an activated dioxo species bound in the plane of the histidine brace. A "pre-binding" site for molecular oxygen is also identified and characterized with neutron protein crystallography and DFT calculations.

Structural and molecular dynamics studies of a C1‐oxidizing lytic polysaccharide monooxygenase from Heterobasidion irregulare reveal amino acids important for substrate recognition

“…other C1-specific LPMOs and some C1/C4-active LPMOs [38][39][40], is shorter compared to those C4-specific AA9 LPMO structures solved to date [41][42][43] (Fig. 4).…”

“…The position of the LPMO over cellulose is thought to have a major impact on the selectivity of the oxidation site [34,49]. Several scenarios have emerged from the available LPMO structures determined in complex with ligands mimicking O 2 molecules or superoxide ions [5, 34,38,42]. In the C1/C4active NcLPMO9M structure, a peroxide ion was bound at the axial position of the copper coordination plane within 3.3…”

“…A recent study used crystallographic approaches to reveal the binding process of reactive oxygen species by C4-specific NcLPMO9D in the absence of substrate. It showed that the water at the equatorial corner of the copper coordination plane was replaced by molecular oxygen, leading to the formation of a Cu(II)-superoxide intermediate [42]. Similarly, a chloride ion, as a mimic of a negatively charged dioxygen species, was found to mediate substrate binding when the C4-specific LsLPMO9A was cocrystalized with a variety of oligosaccharides.…”

Polysaccharide oxidation by lytic polysaccharide monooxygenase is enhanced by engineered cellobiose dehydrogenase

“…The detailed catalytic mechanism of LPMOs and the nature of their cosubstrate are currently debated. Both O 2 [12][13][14] and H 2 O 2 [15,16] are reported to interact with reduced LPMO, but with different implications on the reaction pathway. The monooxygenase reaction using O 2 requires two sequential electron transfer steps and is likely to proceed via an intermittent dioxo [9,12] or oxyl [17] species.…”

Oxidoreductases and Reactive Oxygen Species in Conversion of Lignocellulosic Biomass

“…The gray dotted line shows the axis defined as axial, and the orange triangle represents the equatorial plane defined by the three copper-coordinating nitrogens in the histidine brace (best visible in panel C). The red star indicates the location of an oxygen species observed in the neutron structural studies by O'Dell et al (69), and the Glu/Gln potentially interacting with this oxygen species is underlined. Note that Phe187, Phe219, and Tyr175 are in equivalent positions in their respective proteins, namely, the proximal axial coordination position.…”