Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity

The recently discovered lytic polysaccharide monooxygenases (LPMOs) carry out oxidative cleavage of polysaccharides and are of major importance for efficient processing of biomass. NcLPMO9C from Neurospora crassa acts both on cellulose and on non-cellulose β-glucans, including cellodextrins and xyloglucan. The crystal structure of the catalytic domain of NcLPMO9C revealed an extended, highly polar substrate-binding surface well suited to interact with a variety of sugar substrates. The ability of NcLPMO9C to act on soluble substrates was exploited to study enzyme-substrate interactions. EPR studies demonstrated that the Cu2+ center environment is altered upon substrate binding, whereas isothermal titration calorimetry studies revealed binding affinities in the low micromolar range for polymeric substrates that are due in part to the presence of a carbohydrate-binding module (CBM1). Importantly, the novel structure of NcLPMO9C enabled a comparative study, revealing that the oxidative regioselectivity of LPMO9s (C1, C4, or both) correlates with distinct structural features of the copper coordination sphere. In strictly C1-oxidizing LPMO9s, access to the solvent-facing axial coordination position is restricted by a conserved tyrosine residue, whereas access to this same position seems unrestricted in C4-oxidizing LPMO9s. LPMO9s known to produce a mixture of C1- and C4-oxidized products show an intermediate situation

Strukturell og funksjonell karakterisering av NcLPMO9C : en lytisk polysakkarid monooksygenase med bred spesifisitet

Lignocellulose represents a major source of renewable organic matter and is of interest as feedstock for the biorefining industry, not only as a promising strategy for replacing fossil fuels in the transportation sector but also for production of higher value chemicals and animal feed. However, the complex and recalcitrant nature of lignocellulosic biomass puts major challenges to biorefining of this feedstock. Enzymatic saccharification of the polysaccharides in the feedstock, especially cellulose, is considered a crucial and challenging step in biorefining and this step is one of the limiting factors in the transition towards a sustainable bioeconomy. To overcome the recalictrant nature of lignocellulosic biomass, microorganisms have developed intricate enzyme systems, including lytic polysaccharide monooxygenases (LPMOs). LPMOs are copper-dependent enzymes that, in the presence of an electron donor, cleave the glycoside bonds of various polysaccharides using an oxidative mechanism. When working in synergy with cellulases, which are hydrolytic enzymes, LPMOs boost the enzymatic degradation of cellulose. Since their discovery in 2010, the novel catalytic abilities of LPMOs and their great potential in biomass degradation have attracted researchers in academia as well as industry. However, despite considerable research efforts, our knowledge of the action of LPMOs is still limited.Lignocellulose representerer en viktig kilde til fornybart organisk materiale og er av interesse som råmateriale for bioraffineringsindustrien, ikke bare som en lovende strategi for erstatning av fossile brensler i transportsektoren, men også for produksjon av kjemikalier og dyrefôr. Den komplekse og gjenstridige sammensetningen av lignocellulose gir store utfordringer i bioraffineringen av dette råmaterialet. Enzymatisk omdanning av polysakkaridene i lignocellulosen, spesielt cellulose, regnes som et avgjørende og utfordrende trinn i bioraffineringsprosessen. Dette trinnet representerer også en av hovedbegrensningene i overgangen til en bærekraftig bioøkonomi. For å overvinne den tungt nedbrytbare lignocellulosen har mikroorganismer utviklet intrikate enzymsystemer, som blant annet inneholder lytiske polysakkarid monooksygenaser (LPMOer). LPMOer er kobberavhengige enzymer som, i nærvær av en elektrondonor, bryter glykosidbindingene i forskjellige polysakkarider ved bruk av en oksidativ mekanisme. I synergi med cellulaser, som er hydrolytiske enzymer, øker LPMOer hastigheten til den enzymatiske nedbrytningen av cellulose. Siden oppdagelsen av LPMOer i 2010 har den katalytiske funksjonen til disse enzymene, samt deres potensiale innen nedbryting av biomasse, tiltrukket forskere fra både akademia og industri. Til tross for betydelig forskningsinnsats er kunnskapen om LPMOenes funksjon fortsatt begrenset.Norges forskningsråd ; Borregaard ; BioStruc

Expression and characterization of CBM33 proteins from Cellulomonas flavigena and Aspergillus terreus

An essential element of a modern biorefinery is the enzymatic conversion of biomass to soluble sugars. Although the optimization of this process has been pursued by both academia and industry for decades, it still represents a bottleneck in the biorefinery concept. Recent discoveries of a lytic polysaccharide monooxygenase (LPM) activity among members of family 33 of carbohydrate binding modules (CBM33), boosting the degradation of recalcitrant polysaccharides, have given more insight to the degradation of complex polysaccharides in nature that could be adapted for biorefining processes. These findings have also prompted an effort in cloning, expressing and characterizing a wide variety of CBM33s in order to get information on CBM33-family diversity in terms of function and mechanism. The genome of the Gram negative soil bacterium Cellulomonas flavigena encodes four CBM33s (CfCBM33s), all possessing C-terminal CBM2a domains, indicating substrate specificity towards either cellulose or chitin. The bacterium is previously known to metabolize cellulose and xylan. Conversely, results from binding and activity assays performed with two of the CfCBM33s (CfCBM33A-N and CfCBM33B-N) in this study show no specificity towards either of these substrates. However, both show affinity for chitin, which is intriguing as the genome of C. flavigena contains no identified chitinases. In addition to the unexpected binding affinity towards chitin, CfCBM33B-N shows lytic chitin monooxygenase activity and is also able to boost the degradation of β-chitin by chitinase A, B and C from Serratia marcescens. Moreover, intriguingly, CfCBM33B-N generates partly deacetylated products from the oxidation of β-chitin possibly showing a new function or binding specificity not previously reported for LPMs. Finally, a CBM33 containing a CBM20 (indicating binding affinity for starch) from the fungus Aspergillus terreus was successfully cloned and expressed in Pichia pastoris. Preliminary binding experiments using isothermal titration calorimetry indicate that AtCBM33A binds specifically to starch. Analysis of LPM activity was prevented by the time restrains of this study, but will be the focus of consecutive work. A starch active LPM would be completely new to the field and could be an important finding for the starch processing industry

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

193817, 203402, 214613, 216162 and 217708,publishedVersio

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

Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation

The recently discovered lytic polysaccharide monooxygenases (LPMOs) are known to carry out oxidative cleavage of glycoside bonds in chitin and cellulose, thus boosting the activity of well-known hydrolytic depolymerizing enzymes. Because biomass-degrading microorganisms tend to produce a plethora of LPMOs, and considering the complexity and copolymeric nature of the plant cell wall, it has been speculated that some LPMOs may act on other substrates, in particular the hemicelluloses that tether to cellulose microfibrils. We demonstrate that an LPMO from Neurospora crassa, NcLPMO9C, indeed degrades various hemicelluloses, in particular xyloglucan. This activity was discovered using a glycan microarray-based screening method for detection of substrate specificities of carbohydrate-active enzymes, and further explored using defined oligomeric hemicelluloses, isolated polymeric hemicelluloses and cell walls. Products generated by NcLPMO9C were analyzed using high performance anion exchange chromatography and multidimensional mass spectrometry. We show that NcLPMO9C generates oxidized products from a variety of substrates and that its product profile differs from those of hydrolytic enzymes acting on the same substrates. The enzyme particularly acts on the glucose backbone of xyloglucan, accepting various substitutions (xylose, galactose) in almost all positions. Because the attachment of xyloglucan to cellulose hampers depolymerization of the latter, it is possible that the beneficial effect of the LPMOs that are present in current commercial cellulase mixtures in part is due to hitherto undetected LPMO activities on recalcitrant hemicellulose structures