The effect of linker conformation on performance and stability of a two-domain lytic polysaccharide monooxygenase.

A considerable number of lytic polysaccharide monooxygenases (LPMOs) and other carbohydrate-active enzymes are modular, with catalytic domains being tethered to additional domains, such as carbohydrate-binding modules, by flexible linkers. While such linkers may affect the structure, function, and stability of the enzyme, their roles remain largely enigmatic, as do the reasons for natural variation in length and sequence. Here, we have explored linker functionality using the two-domain cellulose-active ScLPMO10C from Streptomyces coelicolor as a model system. In addition to investigating the WT enzyme, we engineered three linker variants to address the impact of both length and sequence and characterized these using small-angle X-ray scattering, NMR, molecular dynamics simulations, and functional assays. The resulting data revealed that, in the case of ScLPMO10C, linker length is the main determinant of linker conformation and enzyme performance. Both the WT and a serine-rich variant, which have the same linker length, demonstrated better performance compared with those with either a shorter linker or a longer linker. A highlight of our findings was the substantial thermostability observed in the serine-rich variant. Importantly, the linker affects thermal unfolding behavior and enzyme stability. In particular, unfolding studies show that the two domains unfold independently when mixed, whereas the full-length enzyme shows one cooperative unfolding transition, meaning that the impact of linkers in biomass-processing enzymes is more complex than mere structural tethering.

A comparative biochemical investigation of the impeding effect of C1-oxidizing LPMOs on cellobiohydrolases.

Lytic polysaccharide monooxygenases (LPMOs) are known to act synergistically with glycoside hydrolases in industrial cellulolytic cocktails. However, a few studies have reported severe impeding effects of C1-oxidizing LPMOs on the activity of reducing-end cellobiohydrolases. The mechanism for this effect remains unknown, but it may have important implications as reducing-end cellobiohydrolases make up a significant part of such cocktails. To elucidate whether the impeding effect is general for different reducing-end cellobiohydrolases and study the underlying mechanism, we conducted a comparative biochemical investigation of the cooperation between a C1-oxidizing LPMO from Thielavia terrestris and three reducing-end cellobiohydrolases; Trichoderma reesei (TrCel7A), T. terrestris (TtCel7A), and Myceliophthora heterothallica (MhCel7A). The enzymes were heterologously expressed in the same organism and thoroughly characterized biochemically. The data showed distinct differences in synergistic effects between the LPMO and the cellobiohydrolases; TrCel7A was severely impeded, TtCel7A was moderately impeded, while MhCel7A was slightly boosted by the LPMO. We investigated effects of C1-oxidations on cellulose chains on the activity of the cellobiohydrolases and found reduced activity against oxidized cellulose in steady-state and pre-steady-state experiments. The oxidations led to reduced maximal velocity of the cellobiohydrolases and reduced rates of substrate complexation. The extent of these effects differed for the cellobiohydrolases and scaled with the extent of the impeding effect observed in the synergy experiments. Based on these results, we suggest that C1-oxidized chain ends are poor attack sites for reducing-end cellobiohydrolases. The severity of the impeding effects varied considerably among the cellobiohydrolases, which may be relevant to consider for optimization of industrial cocktails.

Oxidative cleavage of cellulose in the horse gut.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) belonging to the auxiliary activity 9 family (AA9) are widely found in aerobic fungi. These enzymes are O2-dependent copper oxidoreductases that catalyze the oxidative cleavage of cellulose. However, studies that have investigated AA9 LPMOs of aerobic fungi in the herbivore gut are scare. To date, whether oxidative cleavage of cellulose occurs in the herbivore gut is unknown. RESULTS: We report for the first time experimental evidence that AA9 LPMOs from aerobic thermophilic fungi catalyze the oxidative cleavage of cellulose present in the horse gut to C1-oxidized cellulose and C1- and C4-oxidized cello-oligosaccharides. We isolated and identified three thermophilic fungi and measured their growth and AA9 LPMO expression at 37 °C in vitro. We also assessed the expression and the presence of AA9 LPMOs from thermophilic fungi in situ. Finally, we used two recombinant AA9 LPMOs and a native AA9 LPMO from thermophilic fungi to cleave cellulose to yield C1-oxidized products at 37 °C in vitro. CONCLUSIONS: The oxidative cleavage of cellulose occurs in the horse gut. This finding will broaden the known the biological functions of the ubiquitous LPMOs and aid in determining biological significance of aerobic thermophilic fungi.

Comparison of three seemingly similar lytic polysaccharide monooxygenases from Neurospora crassa suggests different roles in plant biomass degradation.

Many fungi produce multiple lytic polysaccharide monooxygenases (LPMOs) with seemingly similar functions, but the biological reason for this multiplicity remains unknown. To address this question, here we carried out comparative structural and functional characterizations of three cellulose-active C4-oxidizing family AA9 LPMOs from the fungus Neurospora crassa, NcLPMO9A (NCU02240), NcLPMO9C (NCU02916), and NcLPMO9D (NCU01050). We solved the three-dimensional structure of copper-bound NcLPMO9A at 1.6-Å resolution and found that NcLPMO9A and NcLPMO9C, containing a CBM1 carbohydrate-binding module, bind cellulose more strongly and were less susceptible to inactivation than NcLPMO9D, which lacks a CBM. All three LPMOs were active on tamarind xyloglucan and konjac glucomannan, generating similar products but clearly differing in activity levels. Importantly, in some cases, the addition of phosphoric acid-swollen cellulose (PASC) had a major effect on activity: NcLPMO9A was active on xyloglucan only in the presence of PASC, and PASC enhanced NcLPMO9D activity on glucomannan. Interestingly, the three enzymes also exhibited large differences in their interactions with enzymatic electron donors, which could reflect that they are optimized to act with different reducing partners. All three enzymes efficiently used H2O2 as a cosubstrate, yielding product profiles identical to those obtained in O2-driven reactions with PASC, xyloglucan, or glucomannan. Our results indicate that seemingly similar LPMOs act preferentially on different types of copolymeric substructures in the plant cell wall, possibly because these LPMOs are functionally adapted to distinct niches differing in the types of available reductants.

Engineering chitinolytic activity into a cellulose-active lytic polysaccharide monooxygenase provides insights into substrate specificity.

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of recalcitrant polysaccharides such as cellulose and chitin and play an important role in the enzymatic degradation of biomass. Although it is clear that these monocopper enzymes have extended substrate-binding surfaces for interacting with their fibrous substrates, the structural determinants of LPMO substrate specificity remain largely unknown. To gain additional insight into substrate specificity in LPMOs, here we generated a mutant library of a cellulose-active family AA10 LPMO from Streptomyces coelicolor A3(2) (ScLPMO10C, also known as CelS2) having multiple substitutions at five positions on the substrate-binding surface that we identified by sequence comparisons. Screening of this library using a newly-developed MS-based high-throughput assay helped identify multiple enzyme variants that contained four substitutions and exhibited significant chitinolytic activity and a concomitant decrease in cellulolytic activity. The chitin-active variants became more rapidly inactivated during catalysis than a natural chitin-active AA10 LPMO, an observation likely indicative of suboptimal substrate binding leading to autocatalytic oxidative damage of these variants. These results reveal several structural determinants of LPMO substrate specificity and underpin the notion that productive substrate binding by these enzymes is complex, depending on a multitude of amino acids located on the substrate-binding surface.

A simple enzymatic assay for the quantification of C1-specific cellulose oxidation by lytic polysaccharide monooxygenases.

OBJECTIVE: The development of an enzymatic assay for the specific quantification of the C1-oxidation product, i.e. gluconic acid of cellulose active lytic polysaccharide monooxygenases (LPMOs). RESULTS: In combination with a ß-glucosidase, the spectrophotometrical assay can reliably quantify the specific C1- oxidation product of LPMOs acting on cellulose. It is applicable for a pure cellulose model substrate as well as lignocellulosic biomass. The enzymatic assay compares well with the quantification performed by HPAEC-PAD. In addition, we show that simple boiling is not sufficient to inactivate LPMOs and we suggest to apply a metal chelator in addition to boiling or to drastically increase pH for proper inactivation. CONCLUSIONS: We conclude that the versatility of this simple enzymatic assay makes it useful in a wide range of experiments in basic and applied LPMO research and without the need for expensive instrumentation, e.g. HPAEC-PAD.

Chitin-Active Lytic Polysaccharide Monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze the cleavage of 1,4-glycosidic bonds various plant cell wall polysaccharides and chitin. In contrast to glycoside hydrolases, LPMOs are active on the crystalline regions of polysaccharides and thus synergize with hydrolytic enzymes. This synergism leads to an overall increase in the biomass-degradation activity of enzyme mixtures. Chitin-active LPMOs were discovered in 2010 and are currently classified in families AA10, AA11, and AA15 of the Carbohydrate-Active enZYmes database, which include LPMOs from bacteria, fungi, insects, and viruses. LPMOs have become important enzymes both industrially and scientifically and, in this chapter, we provide a brief introduction to chitin-active LPMOs including a summary of the 20+ chitin-active LPMOs that have been characterized so far. Then, we describe their structural features, catalytic mechanism, and appended carbohydrate modules. Finally, we show how chitin-active LPMOs can be used to perform chemo-enzymatic modification of chitin substrates.

High-resolution structure of a lytic polysaccharide monooxygenase from Hypocrea jecorina reveals a predicted linker as an integral part of the catalytic domain.

For decades, the enzymes of the fungus Hypocrea jecorina have served as a model system for the breakdown of cellulose. Three-dimensional structures for almost all H. jecorina cellulose-degrading enzymes are available, except for HjLPMO9A, belonging to the AA9 family of lytic polysaccharide monooxygenases (LPMOs). These enzymes enhance the hydrolytic activity of cellulases and are essential for cost-efficient conversion of lignocellulosic biomass. Here, using structural and spectroscopic analyses, we found that native HjLPMO9A contains a catalytic domain and a family-1 carbohydrate-binding module (CBM1) connected via a linker sequence. A C terminally truncated variant of HjLPMO9A containing 21 residues of the predicted linker was expressed at levels sufficient for analysis. Here, using structural, spectroscopic, and biochemical analyses, we found that this truncated variant exhibited reduced binding to and activity on cellulose compared with the full-length enzyme. Importantly, a 0.95-Å resolution X-ray structure of truncated HjLPMO9A revealed that the linker forms an integral part of the catalytic domain structure, covering a hydrophobic patch on the catalytic AA9 module. We noted that the oxidized catalytic center contains a Cu(II) coordinated by two His ligands, one of which has a His-brace in which the His-1 terminal amine group also coordinates to a copper. The final equatorial position of the Cu(II) is occupied by a water-derived ligand. The spectroscopic characteristics of the truncated variant were not measurably different from those of full-length HjLPMO9A, indicating that the presence of the CBM1 module increases the affinity of HjLPMO9A for cellulose binding, but does not affect the active site.

Structural and Functional Analysis of a Lytic Polysaccharide Monooxygenase Important for Efficient Utilization of Chitin in Cellvibrio japonicus.

Cellvibrio japonicusis a Gram-negative soil bacterium that is primarily known for its ability to degrade plant cell wall polysaccharides through utilization of an extensive repertoire of carbohydrate-active enzymes. Several putative chitin-degrading enzymes are also found among these carbohydrate-active enzymes, such as chitinases, chitobiases, and lytic polysaccharide monooxygenases (LPMOs). In this study, we have characterized the chitin-active LPMO,CjLPMO10A, a tri-modular enzyme containing a catalytic family AA10 LPMO module, a family 5 chitin-binding module, and a C-terminal unclassified module of unknown function. Characterization of the latter module revealed tight and specific binding to chitin, thereby unraveling a new family of chitin-binding modules (classified as CBM73). X-ray crystallographic elucidation of theCjLPMO10A catalytic module revealed that the active site of the enzyme combines structural features previously only observed in either cellulose or chitin-active LPMO10s. Analysis of the copper-binding site by EPR showed a signal signature more similar to those observed for cellulose-cleaving LPMOs. The full-length LPMO shows no activity toward cellulose but is able to bind and cleave both α- and ß-chitin. Removal of the chitin-binding modules reduced LPMO activity toward α-chitin compared with the full-length enzyme. Interestingly, the full-length enzyme and the individual catalytic LPMO module boosted the activity of an endochitinase equally well, also yielding similar amounts of oxidized products. Finally, gene deletion studies show thatCjLPMO10A is needed byC. japonicusto obtain efficient growth on both purified chitin and crab shell particles.

Cellulose surface degradation by a lytic polysaccharide monooxygenase and its effect on cellulase hydrolytic efficiency.

Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization.

A C4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides.

Lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels, chemicals, and materials. Efficient saccharification of this biomass to fermentable sugars will be a key technology in future biorefineries. Traditionally, saccharification was thought to be accomplished by mixtures of hydrolytic enzymes. However, recently it has been shown that lytic polysaccharide monooxygenases (LPMOs) contribute to this process by catalyzing oxidative cleavage of insoluble polysaccharides utilizing a mechanism involving molecular oxygen and an electron donor. These enzymes thus represent novel tools for the saccharification of plant biomass. Most characterized LPMOs, including all reported bacterial LPMOs, form aldonic acids, i.e., products oxidized in the C1 position of the terminal sugar. Oxidation at other positions has been observed, and there has been some debate concerning the nature of this position (C4 or C6). In this study, we have characterized an LPMO from Neurospora crassa (NcLPMO9C; also known as NCU02916 and NcGH61-3). Remarkably, and in contrast to all previously characterized LPMOs, which are active only on polysaccharides, NcLPMO9C is able to cleave soluble cello-oligosaccharides as short as a tetramer, a property that allowed detailed product analysis. Using mass spectrometry and NMR, we show that the cello-oligosaccharide products released by this enzyme contain a C4 gemdiol/keto group at the nonreducing end.

On the impact of carbohydrate-binding modules (CBMs) in lytic polysaccharide monooxygenases (LPMOs).

Lytic polysaccharide monooxygenases (LPMOs) have revolutionized our understanding of how enzymes degrade insoluble polysaccharides. Compared with the substantial knowledge developed on the structure and mode of action of the catalytic LPMO domains, the (multi)modularity of LPMOs has received less attention. The presence of other domains, in particular carbohydrate-binding modules (CBMs), tethered to LPMOs has profound implications for the catalytic performance of the full-length enzymes. In the last few years, studies on LPMO modularity have led to advancements in elucidating how CBMs, other domains, and linker regions influence LPMO structure and function. This mini review summarizes recent literature, with particular focus on comparative truncation studies, to provide an overview of the diversity in LPMO modularity and the functional implications of this diversity.

Influences of the Carbohydrate-Binding Module on a Fungal Starch-Active Lytic Polysaccharide Monooxygenase.

Noncatalytic carbohydrate-binding modules (CBMs) play important roles in the function of lytic polysaccharide monooxygenases (LPMOs) but have not been well demonstrated for starch-active AA13 LPMO. In this study, four new CBMs were investigated systematically for their influence on MtLPMO toward starch in terms of substrate binding, H2O2 production activity, oxidative product yields, and the degradation effect with α-amylase and glucoamylase toward different starch substrates. Among the four MtLPMO-CBM chimeras, MtLPMO-CnCBM harboring the CBM fromColletotrichum nymphaeae showed the highest substrate binding toward different types of starch compared to MtLPMO without CBM. MtLPMO-PvCBM harboring the CBM from Pseudogymnoascus verrucosus and MtLPMO-CnCBM showed dramatically enhanced H2O2 production activity of 4.6-fold and 3.6-fold, respectively, than MtLPMO without CBM. More importantly, MtLPMO-CBM generated more oxidative products from starch polysaccharides degradation than MtLPMO alone, with 6.0-fold and 4.6-fold enhancement obtained from the oxidation of amylopectin and corn starch with MtLPMO-CnCBM, and a 5.2-fold improvement obtained with MtLPMO-AcCBM for amylose. MtLPMO-AcCBM significantly boosted the yields of reducing sugar with α-amylase upon degrading amylopectin and corn starch. These findings demonstrate that CBMs greatly influence the performance of starch-active AA13 LPMOs due to their enhanced binding and H2O2 production activity.

Lytic polysaccharide monooxygenase (LPMO)-derived saccharification of lignocellulosic biomass.

Given that traditional biorefineries have been based on microbial fermentation to produce useful fuels, materials, and chemicals as metabolites, saccharification is an important step to obtain fermentable sugars from biomass. It is well-known that glycosidic hydrolases (GHs) are responsible for the saccharification of recalcitrant polysaccharides through hydrolysis, but the discovery of lytic polysaccharide monooxygenase (LPMO), which is a kind of oxidative enzyme involved in cleaving polysaccharides and boosting GH performance, has profoundly changed the understanding of enzyme-based saccharification. This review briefly introduces the classification, structural information, and catalytic mechanism of LPMOs. In addition to recombinant expression strategies, synergistic effects with GH are comprehensively discussed. Challenges and perspectives for LPMO-based saccharification on a large scale are also briefly mentioned. Ultimately, this review can provide insights for constructing an economically viable lignocellulose-based biorefinery system and a closed-carbon loop to cope with climate change.

Advances in lytic polysaccharide monooxygenases with the cellulose-degrading auxiliary activity family 9 to facilitate cellulose degradation for biorefinery.

One crucial step in processing the recalcitrant lignocellulosic biomass is the fast hydrolysis of natural cellulose to fermentable sugars that can be subsequently converted to biofuels and bio-based chemicals. Recent studies have shown that lytic polysaccharide monooxygenase (LPMOs) with auxiliary activity family 9 (AA9) are capable of efficiently depolymerizing the crystalline cellulose via regioselective oxidation reaction. Intriguingly, the catalysis by AA9 LPMOs requires reductant to provide electrons, and lignin and its phenolic derivatives can be oxidized, releasing reductant to activate the reaction. The activity of AA9 LPMOs can be enhanced by in-situ generation of H2O2 in the presence of O2. Although scientific understanding of these enzymes remains somewhat unknown or controversial, structure modifications on AA9 LPMOs through protein engineering have emerged in recent years, which are prerequisite for their extensive applications in the development of cellulase-mediated lignocellulosic biorefinery processes. In this review, we critically comment on advances in studies for AA9 LPMOs, i.e., characteristic of AA9 LPMOs catalysis, external electron donors to AA9 LPMOs, especially the role of the oxidization of lignin and its derivatives, and AA9 LPMOs protein engineering as well as their extensive applications in the bioprocessing of lignocellulosic biomass. Perspectives are also highlighted for addressing the challenges.

1H, 13C, 15N resonance assignment of the apo form of the small, chitin-active lytic polysaccharide monooxygenase JdLPMO10A from Jonesia denitrificans.

The lytic polysaccharide monooxygenase JdLPMO10A is the N-terminal domain of the multimodular protein Jd1381. The isolated JdLPMO10A domain is one of the smallest chitin-active lytic polysaccharide monooxygenases known to date with a size of only 15.5 kDa. JdLPMO10A is a copper-dependent oxidative enzyme that depolymerizes chitin by hydroxylating the C1 carbon in the glycosidic bond. JdLPMO10A has been isotopically labeled and recombinantly expressed. Here, we report the 1H, 13C, 15N resonance assignment of JdLPMO10A. Secondary structural elements predicted based on the NMR assignment are in excellent agreement with the crystal structure of JdLPMO10A.

New approach to the evaluation of lignocellulose derived by-products impact on lytic-polysaccharide monooxygenase activity by using molecular descriptor structural causality model.

Lytic-polysaccharide monooxygenase (LPMO) is one of the most important enzyme involved in biocatalytic lignocellulose degradation, and therefore inhibition of LPMO has significant effects on all related processes. Structural causality model (SCM) were established to evaluate impact of phenolic by-products in lignocellulose hydrolysates on LPMO activity. The molecular descriptors GATS4c, ATS2m, BIC3 and VR2_Dzs were found to be significant in describing inhibition. The causalities of the molecular descriptors and LPMO activity are determined by evaluating the directed acyclic graph (DAG) and the d-separation algorithm. The maximum causality for LPMO activation is ß = 0.79 by BIC3 and the maximum causality of inhibition is ß = -0.56 for the GATS4c descriptor. The model has the potential to predict the inhibition of LPMO and its application could be useful in selecting an appropriate lignocellulose pretreatment method to minimise the production of a potent inhibitor. This will subsequently lead to more efficient lignocellulose degradation process.

Engineered LPMO Significantly Boosting Cellulase-Catalyzed Depolymerization of Cellulose.

Lytic polysaccharide monooxygenases (LPMOs) play a crucial role in the enzymatic depolymerization of cellulose through oxidative cleavage of the glycosidic bond in the highly recalcitrant crystalline cellulose region. Improving the activity of LPMOs is of considerable importance for second-generation biorefinery. In this study, we identified a beneficial amino acid substitution (N526S) located in the cellulose binding module (CBM) of HcLPMO10 (LPMO of Hahella chejuensis) using directed evolution. The improved variant HcLPMO10 M1 (N526S) exhibits 2.1-fold higher activity for the H2O2 production, 2.7-fold higher oxidation activity, and 1.9-fold higher binding capacity toward cellulose compared with those of the wild type (WT). Furthermore, M1 shows 2.1-fold higher activity for degradation of crystalline cellulose in synergy with cellulase, compared to the WT. Structural analysis through molecular modeling and molecular dynamics (MD) simulation revealed that the substitution N526S located in the CBM likely stabilizes the cellulose binding surface and enhances the binding capacity of HcLPMO10 to cellulose, thereby enhancing enzyme activity. These findings demonstrate the important role of the CBM in the catalytic function of LPMO.

Lytic polysaccharide monooxygenases (LPMOs) facilitate cellulose nanofibrils production.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that cleave polysaccharides through an oxidative mechanism. These enzymes are major contributors to the recycling of carbon in nature and are currently used in the biorefinery industry. LPMOs are commonly used in synergy with cellulases to enhance biomass deconstruction. However, there are few examples of the use of monocomponent LPMOs as a tool for cellulose fibrillation. In this work, we took advantage of the LPMO action to facilitate disruption of wood cellulose fibers as a strategy to produce nanofibrillated cellulose (NFC). RESULTS: The fungal LPMO from AA9 family (PaLPMO9E) was used in this study as it displays high specificity toward cellulose and its recombinant production in bioreactor is easily upscalable. The treatment of birchwood fibers with PaLPMO9E resulted in the release of a mixture of C1-oxidized oligosaccharides without any apparent modification in fiber morphology and dimensions. The subsequent mechanical shearing disintegrated the LPMO-pretreated samples yielding nanoscale cellulose elements. Their gel-like aspect and nanometric dimensions demonstrated that LPMOs disrupt the cellulose structure and facilitate the production of NFC. CONCLUSIONS: This study demonstrates the potential use of LPMOs as a pretreatment in the NFC production process. LPMOs weaken fiber cohesion and facilitate fiber disruption while maintaining the crystallinity of cellulose.

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

Lytic polysaccharide monooxygenases (LPMOs) are a group of recently discovered enzymes that play important roles in the decomposition of recalcitrant polysaccharides. Here, we report the biochemical, structural, and computational characterization of an LPMO from the white-rot fungus Heterobasidion irregulare (HiLPMO9B). This enzyme oxidizes cellulose at the C1 carbon of glycosidic linkages. The crystal structure of HiLPMO9B was determined at 2.1 Å resolution using X-ray crystallography. Unlike the majority of the currently available C1-specific LPMO structures, the HiLPMO9B structure contains an extended L2 loop, connecting ß-strands ß2 and ß3 of the ß-sandwich structure. Molecular dynamics (MD) simulations suggest roles for both aromatic and acidic residues in the substrate binding of HiLPMO9B, with the main contribution from the residues located on the extended region of the L2 loop (Tyr20) and the LC loop (Asp205, Tyr207, and Glu210). Asp205 and Glu210 were found to be involved in the hydrogen bonding with the hydroxyl group of the C6 carbon of glucose moieties directly or via a water molecule. Two different binding orientations were observed over the course of the MD simulations. In each orientation, the active-site copper of this LPMO preferentially skewed toward the pyranose C1 of the glycosidic linkage over the targeted glycosidic bond. This study provides additional insight into cellulose binding by C1-specific LPMOs, giving a molecular-level picture of active site substrate interactions. DATABASE: The atomic coordinates and structure factors for HiLPMO9B have been deposited in the Protein Data Bank with accession code 5NNS.