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

publishedVersio

Hydrolys- och glykosyntasaktivitetsstudier av vildtyps-laminarinas, Lam16A, och dess katalytiskt inaktiva mutanter E115G, E115S och E120A från Phanerochaete chrysosporium

Laminarinase 16A from Phanerochaete chrysosporium is a 36 kDa enzyme with typical endo-β-1,3(4)-glucanase activity, belonging to family 16 of glycoside hydrolases. The catalytic amino acids in the active site are identified as nucleophile Glu 115 and acid/base Glu 120. Prior to this project, these residues had been mutated to make catalytic deficient mutants E115G, E115S and E120A in order to obtain structures in complex with natural substrates. In the present work, the nucleophile mutant E115G has been expressed and successfully purified and shown to possess glycosynthase activity when using an α-fluoride derivative of laminariheptaose as substrate by the detection of circular laminariheptaose using HPLC. Activity measurements revealed that the E115G mutant had substantial activity in presence of acetate and formiate, and the other nucleophile mutant, E115S, in the presence of formiate, which indicates that those molecules can act as external nucleophiles. Circular β-glucans from two different nitrogen-fixing bacterial species were tested as potential substrates for wildtype Lam16A and the E115G mutant in presence of acetate. HPLC analysis revealed that there was no effect on the preparation with circular β-1,2-glucan, whereas one component of the other β-1,3/1,6-glucan preparation was consumed and new peaks appeared in the chromatogram, although further studies are needed to identify these components. The findings from this project reveals that this enzyme has many interesting capacities that hopefully in the future may be used in the synthesis of interesting oligosaccharides that can be applied in the fields of environmental protection, pre-biotics and medicine

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

publishedVersio

Discovery and characterization of cellulose-active lytic polysaccharide monooxygenases

The efficient depolymerization of lignocellulosic biomass to fermentable sugars by enzymatic hydrolysis is a key step in the transition towards a more environmentally friendly and sustainable bio-economy. However, the complexity and recalcitrant nature of the substrate limit enzyme performance on lignocellulosic plant biomass, and at present the enzyme cocktails required for depolymerization represent a major cost in the production of biomass-based chemicals and fuels. The recent discovery of lytic polysaccharide monooxygenases (LPMOs) has changed our general understanding of polysaccharide deconstruction, and given rise to high expectations for further development of enzyme tools for biomass processing, since LPMOs enhance the activity of glycoside hydrolases. LPMOs are copper-dependent enzymes that oxidize recalcitrant polysaccharides such as chitin and cellulose in the presence of dioxygen, and an external electron donor. Before the discovery of their enzymatic function, in 2010, LPMOs were classified as either family 33 of carbohydrate-binding modules (now family 10 of auxiliary activities, AA10, LPMO10) or family 61 of glycoside hydrolases (now AA9, LPMO9). Prior to the studies presented here, catalytic activity had just been demonstrated for a chitin-active bacterial AA10-type of LPMO from Serratia marcescens called CBP21. The work on CBP21 formed the basis for the first goal of this study, namely finding or engineering an LPMO targeting cellulose substrates. Paper I describes CelS2, a naturally occurring AA10-type LPMO from the Gram-positive bacterium Streptomyces coelicolor that cleaves crystalline cellulose and produces C1-oxidized cello-oligosaccharides appearing in solution as aldonic acids. The generation of oxidized products was demonstrated using both mass spectrometry and chromatographic methods. CelS2, which comprises an N-terminal AA10 and a Cterminal cellulose-binding carbohydrate-binding module classified as CBM2, represents the first described LPMO that is active on cellulose. It was shown that CelS2 stimulates the release of soluble sugars from filter paper by Celluclast® (a commercial enzyme cocktail). Papers II and III of this study describe structure-function studies of cellulose-active AA10-type LPMOs with the purpose of unraveling the basic characteristics of these proteins and perhaps identify factors determining substrate specificity and the regioselectivity of hydroxylation (C1 versus C4 oxidation). Paper II describes a comparative study of four C1-oxidizing LPMOs, two of which are active on chitin and two on cellulose, and includes the description of one novel chitin-active LPMO10 (BlLPMO10A from Bacillus licheniformis) and one novel cellulose-active LPMO10 (E8 from Thermobifida fusca). Sequence analysis showed that all residues in the immediate copper coordination sphere were conserved in these four LPMOs. Conversely, electron paramagnetic resonance spectroscopy (EPR) analyses indicated that the electronic environments of the copper differed between the chitin- and cellulose-active LPMOs. The differences in the EPR spectra are thus likely to reflect variation in residues outside the direct copper coordination sphere, where the chitin-active and cellulose-active AA10-type of LPMOs indeed show considerable variation. Paper III presents the first crystal structures of cellulose-active AA10-type LPMOs, which allowed for the first time a structural comparison of LPMOs with different substrate specificities. The two S. coelicolor LPMO for which the structures were determined, CelS2 and ScLPMO10B, represent a conserved pair of LPMOs found in cellulolytic actinomycetes. The two enzymes are pregulated during growth on cellulose substrates and we show that they act synergistically when degrading cellulose. CelS2 shows strict C1- oxidation on cellulose substrates, whereas ScLPMO10B catalyzes oxidation of C1 and C4 in cellulose, as well as C1-oxidation on β-chitin. A structural comparison of the two cellulose-active LPMO10s revealed a difference in the copper oordination sphere that may relate to the (in)ability to oxidize C4. Structural comparisons of chitin-active and celluloseactive LPMO10s revealed a potential binding-pocket for a C2 acetamido group in chitinactive LPMO10s only. All LPMO10s had similar redox potentials and copper binding affinities, but showed a substrate-dependent difference in EPR spectra, as discussed in Paper II. Substrate-specificity thus seems to be determined by variation in substrate-binding and –positioning combined with variation in the electronic structure of the copper site. In conclusion, this study represents the discovery and first in-depth characterization of LPMOs from family 10 of auxiliary activities that are active on cellulose. The work presented here has provided fundamental insight into how these enzymes work and contributed to method development, thereby constituting an important basis for future LPMO research

Structural diversity of lytic polysaccharide monooxygenases

acceptedVersio

1H, 13C, 15N resonance assignment of the chitin-active lytic polysaccharide monooxygenase BlLPMO10A from Bacillus licheniformis

The chitin-active 19.2 kDa lytic polysaccharide monooxygenase BlLPMO10A from Bacillus licheniformis has been isotopically labeled and recombinantly expressed. In this paper, we report the 1H, 13C, 15N resonance assignment of BlLPMO10A.acceptedVersio

Chemical shift assignments for the apo-form of the catalytic domain, the linker region, and the carbohydrate-binding domain of the cellulose-active lytic polysaccharide monooxygenase ScLPMO10C

acceptedVersio

Modularity impacts cellulose surface oxidation by a lytic polysaccharide monooxygenase from Streptomyces coelicolor

Open access funding provided by Norwegian University of Life Sciences. Contributions from O.R. and E.R.M. were financially supported by the Genome Canada and Ontario Genomics project: Synbiomics (Project Number 10405), the Research Council of Norway through grant 262853 to V.G.H.E. and the Novo Nordisk Foundation through grant NNF18OC0055736 to Z.F. also supported this work. Funding Information: The authors would like to acknowledge the financial support from Genome Canada, the Ontario Genomics project, the Research Council of Norway, and the Novo Nordisk Foundation. Publisher Copyright: © 2023, The Author(s).Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidation of β-(1,4)-linked polysaccharides, such as cellulose, in a reaction that requires an electron donor and H2O2 as co-substrate. Several LPMOs include a carbohydrate-binding module (CBM), which promotes action on insoluble substrates. Herein, a fluorescent labeling technique was used to track LPMO action on microcrystalline cellulose and evaluate the impact of CBMs on the distribution of LPMO activity across the fiber surface. Confocal microscopic images revealed that the distribution of oxidized positions on the cellulose surface was CBM-dependent: fluorescent spots were concentrated in reactions with a CBM-containing LPMO whereas they were more dispersed for a CBM-deficient LPMO variant. The more dispersed oxidation pattern for the CBM-free LPMO coincided with the release of fewer soluble reaction products.Peer reviewe

On the functional characterization of lytic polysaccharide monooxygenases (LPMOs)

Lytic polysaccharide monooxygenases (LPMOs) are abundant in nature and best known for their role in the enzymatic conversion of recalcitrant polysaccharides such as chitin and cellulose. LPMO activity requires an oxygen co‑substrate, which was originally thought to be O2, but which may also be H2O2. Functional characterization of LPMOs is not straightforward because typical reaction mixtures will promote side reactions, including auto‑catalytic inactivation of the enzyme. For example, despite some recent progress, there is still limited insight into the kinetics of the LPMO reaction. Recent discoveries concerning the role of H2O2 in LPMO catalysis further complicate the picture. Here, we review commonly used methods for characterizing LPMOs, with focus on benefits and potential pitfalls, rather than on technical details. We conclude by pointing at a few key problems and potential misconceptions that should be taken into account when interpreting existing data and planning future experiments.publishedVersio

The carbohydrate-binding module and linker of a modular lytic polysaccharide monooxygenase promote localized cellulose oxidation

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze the oxidative cleavage of polysaccharides such as cellulose and chitin, a feature that makes them key tools in industrial biomass conversion processes. The catalytic domains of a considerable fraction of LPMOs and other carbohydrate-active enzymes (CAZymes) are tethered to carbohydrate-binding modules (CBMs) by flexible linkers. These linkers preclude X-ray crystallographic studies, and the functional implications of these modular assemblies remain partly unknown. Here, we used NMR spectroscopy to characterize structural and dynamic features of full-length modular ScLPMO10C from Streptomyces coelicolor. We observed that the linker is disordered and extended, creating distance between the CBM and the catalytic domain and allowing these domains to move independently of each other. Functional studies with cellulose nanofibrils revealed that most of the substrate-binding affinity of full-length ScLPMO10C resides in the CBM. Comparison of the catalytic performance of full-length ScLPMO10C and its isolated catalytic domain revealed that the CBM is beneficial for LPMO activity at lower substrate concentrations and promotes localized and repeated oxidation of the substrate. Taken together, these results provide a mechanistic basis for understanding the interplay between catalytic domains linked to CBMs in LPMOs and CAZymes in general.acceptedVersion© 2018 Courtade et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. This is the authors' accepted and refereed manuscript to the article. The final authenticated version is available online at: http://doi.org/10.1074/jbc.RA118.00426