Abstract
Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharide monooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases, CfLPMO10 and TbLPMO10 from Cellulomonas fimi and Thermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducing CtCBM3a, from the Clostridium thermocellum cellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact of CtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM from CfLPMO10 or the introduction of a family 10 CBM from Cellvibrio japonicus LPMO10B into TbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations.
Highlights
Cellulose, a polymer of -1,4-linked glucose, is the most abundant component of plant biomass
high performance anion exchange chromatography (HPAEC) and mass spectrometry showed that both enzymes generated a range of oxidized oligosaccharides with a degree of polymerization (DP) ranging primarily from 2 to 7 (Figs. 1, A and B, and 2)
The data presented here show that the CBMs of two LPMO10s contribute to the activity of the enzymes against different forms of cellulose
Summary
A polymer of -1,4-linked glucose, is the most abundant component of plant biomass. CBMs have been grouped into sequence-based families on the CAZy database and, based on ligand specificity, into three types (14, 15) dependent on whether they bind to crystalline ligands (type A), the internal regions of glycan chains (type B), or the termini of polysaccharides and oligosaccharides (type C). Three-dimensional structural data of crystalline cellulose-specific LPMOs indicate that both the ligand binding site of the type A CBMs (24 –27) and the substrate binding site of the catalytic domains display a planar surface (4, 13, 28), no ligand complexes are yet available. It is evident that a more detailed analysis of the role of CBMs in LPMO action is required
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