Cleavage of cellulose by a CBM33 protein

Forsberg, Zarah; Vaaje‐Kolstad, Gustav; Westereng, Bjørge; Bunæs, Anne Cathrine; Stenstrøm, Yngve; Mackenzie, Alasdair; Sørlie, Morten; Horn, Svein Jarle; Eijsink, Vincent G. H.

doi:10.1002/pro.689

Cited by 338 publications

(407 citation statements)

References 28 publications

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“…Although CBM33-type and GH61-type LPMOs seem to catalyze essentially the same reaction (4,5,12,13,23) and have similar active sites ( Fig. 1 D and E), their metal binding sites do show some notable differences.…”

Section: Discussionmentioning

confidence: 98%

NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions

Aachmann

Sørlie

Skjåk‐Bræk

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Lytic polysaccharide monooxygenases currently classified as carbohydrate binding module family 33 (CBM33) and glycoside hydrolase family 61 (GH61) are likely to play important roles in future biorefining. However, the molecular basis of their unprecedented catalytic activity remains largely unknown. We have used NMR techniques and isothermal titration calorimetry to address structural and functional aspects of CBP21, a chitin-active CBM33. NMR structural and relaxation studies showed that CBP21 is a compact and rigid molecule, and the only exception is the catalytic metal binding site. NMR data further showed that His28 and His114 in the catalytic center bind a variety of divalent metal ions with a clear preference for Cu 2+ (K d = 55 nM; from isothermal titration calorimetry) and higher preference for Cu 1+ (K d ∼ 1 nM; from the experimentally determined redox potential for CBP21-Cu 2+ of 275 mV using a thermodynamic cycle). Strong binding of Cu 1+ was also reflected in a reduction in the pK a values of the histidines by 3.6 and 2.2 pH units, respectively. Cyanide, a mimic of molecular oxygen, was found to bind to the metal ion only. These data support a model where copper is reduced on the enzyme by an externally provided electron and followed by oxygen binding and activation by internal electron transfer. Interactions of CBP21 with a crystalline substrate were mapped in a 2 H/ 1 H exchange experiment, which showed that substrate binding involves an extended planar binding surface, including the metal binding site. Such a planar catalytic surface seems well-suited to interact with crystalline substrates.cellulose | biomass C hitin and cellulose represent some of nature's largest reservoirs of organic carbon in the form of monomeric hexose sugars (N-acetyl-glucosamine and glucose, respectively) linearly linked by β-1,4 glycosidic bonds. In their natural form, both polysaccharides are organized in crystalline arrangements that make up robust biological structures, like crustacean cuticles (chitin) or plant cell walls (cellulose). Although this crystalline nature is crucial for biological function, it provides a thorough challenge in industrial biorefining of biomass, where efficient enzymatic depolymerization of particularly cellulose is a critical step.Enzymatic degradation of recalcitrant polysaccharides has traditionally been thought to occur through the synergistic action of hydrolytic enzymes that have complementary activities (1, 2). Endo-acting hydrolases make random scissions on the polysaccharide chains, whereas exo-acting processive hydrolases mainly target chain ends. However, during the last 2 years, a new enzyme family targeting recalcitrant polysaccharides has been identified, namely the lytic polysaccharide monooxygenases [LPMOs; also referred to as lytic polysaccharide oxidases (3), polysaccharide monooxygenases (4), and oxidohydrolases (5)]. In contrast to the classic hydrolytic enzymes that comprise many enzyme families, LPMOs only group into two distinct families (6): carbohydrate binding modu...

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Section: Discussionmentioning

confidence: 98%

NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions

Aachmann

Sørlie

Skjåk‐Bræk

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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251

346

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“…Chitinases as virulence factors substrates such as cellulose have been observed (Forsberg et al, 2011). Host-related alternative binding substrates have also been described, with the virulence-related GbpA protein (Table 2) from V. cholerae as an example.…”

Section: Substrate Specificities Of Chitin-binding Domains Relevant Tmentioning

confidence: 99%

Bacterial chitinases and chitin-binding proteins as virulence factors

et al. 2013

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“…Recently, a class of oxygen activating enzymes with a single copper center has been identified, the polysaccharride monooxygenases [PMOs; often termed lytic polysaccharide monooxygenases (LPMOs), reflecting their ability to break polysaccharides chains and loosen crystalline structure] (6-8), or AA9 to 11 enzymes (AA = auxiliary activity) in the Carbohydrate-Active enZYmes (CAZy) database (9). AA9 enzymes [formerly glycoside hydrolases family 61 (GH61s)] are fungal enzymes that can enhance major cellulases' enzymatic degradation of cellulose (hence "auxiliary activity"), whereas AA10 enzymes [formerly carbohydrate binding module family 33 (CBM33)] are predominantly bacterial enzymes that can enhance major chitinases' degradation of chitin (6,7,(10)(11)(12)(13). Enzymes in the latest discovered subclass, AA11, are fungal enzymes, where the currently lone characterized example uses chitin as a substrate (8).…”

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confidence: 99%

Spectroscopic and computational insight into the activation of O ₂ by the mononuclear Cu center in polysaccharide monooxygenases

Kjaergaard

Qayyum

Wong

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Strategies for O 2 activation by copper enzymes were recently expanded to include mononuclear Cu sites, with the discovery of the copper-dependent polysaccharide monooxygenases, also classified as auxiliary-activity enzymes 9-11 (AA9-11). These enzymes are finding considerable use in industrial biofuel production. Crystal structures of polysaccharide monooxygenases have emerged, but experimental studies are yet to determine the solution structure of the Cu site and how this relates to reactivity. From X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopies, we observed a change from four-coordinate Cu(II) to three-coordinate Cu(I) of the active site in solution, where three protein-derived nitrogen ligands coordinate the Cu in both redox states, and a labile hydroxide ligand is lost upon reduction. The spectroscopic data allowed for density functional theory calculations of an enzyme active site model, where the optimized Cu(I) and (II) structures were consistent with the experimental data. The O 2 reactivity of the Cu(I) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O 2 and regeneration of the resting Cu(II) enzyme were observed. This reactivity was evaluated computationally, and by calibration to Cu-superoxide model complexes, formation of an end-on Cu-AA9-superoxide species was found to be thermodynamically favored. We discuss how this thermodynamically difficult one-electron reduction of O 2 is enabled by the unique protein structure where two nitrogen ligands from His1 dictate formation of a T-shaped Cu(I) site, which provides an open coordination position for strong O 2 binding with very little reorganization energy.X-ray absorption spectroscopy | DFT | dioxygen activation | biofuels

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Cleavage of cellulose by a CBM33 protein

Cited by 338 publications

References 28 publications

NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions

NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions

Bacterial chitinases and chitin-binding proteins as virulence factors

Spectroscopic and computational insight into the activation of O ₂ by the mononuclear Cu center in polysaccharide monooxygenases

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Cleavage of cellulose by a CBM33 protein

Cited by 338 publications

References 28 publications

NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions

NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions

Bacterial chitinases and chitin-binding proteins as virulence factors

Spectroscopic and computational insight into the activation of O 2 by the mononuclear Cu center in polysaccharide monooxygenases

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Spectroscopic and computational insight into the activation of O ₂ by the mononuclear Cu center in polysaccharide monooxygenases