Characterization of a lytic polysaccharide monooxygenase from Aspergillus fumigatus shows functional variation among family AA11 fungal LPMOs.

The discovery of oxidative cleavage of recalcitrant polysaccharides by lytic polysaccharide monooxygenases (LPMOs) has affected the study and industrial application of enzymatic biomass processing. Despite being widespread in fungi, LPMOs belonging to the auxiliary activity (AA) family AA11 have been understudied. While these LPMOs are considered chitin active, some family members have little or no activity toward chitin, and the only available crystal structure of an AA11 LPMO lacks features found in bacterial chitin-active AA10 LPMOs. Here, we report structural and functional characteristics of a single-domain AA11 LPMO from Aspergillus fumigatus, AfAA11A. The crystal structure shows a substrate-binding surface with features resembling those of known chitin-active LPMOs. Indeed, despite the absence of a carbohydrate-binding module, AfAA11A has considerable affinity for α-chitin and, more so, ß-chitin. AfAA11A is active toward both these chitin allomorphs and enhances chitin degradation by an endoacting chitinase, in particular for α-chitin. The catalytic activity of AfAA11A on chitin increases when supplying reactions with hydrogen peroxide, showing that, like LPMOs from other families, AfAA11A has peroxygenase activity. These results show that, in stark contrast to the previously characterized AfAA11B from the same organism, AfAA11A likely plays a role in fungal chitin turnover. Thus, members of the hitherto rather enigmatic family of AA11 LPMOs show considerable structural and functional differences and may have multiple roles in fungal physiology.

The Fish Pathogen Aliivibrio salmonicida LFI1238 Can Degrade and Metabolize Chitin despite Gene Disruption in the Chitinolytic Pathway.

The fish pathogen Aliivibrio (Vibrio) salmonicida LFI1238 is thought to be incapable of utilizing chitin as a nutrient source, since approximately half of the genes representing the chitinolytic pathway are disrupted by insertion sequences. In the present study, we combined a broad set of analytical methods to investigate this hypothesis. Cultivation studies revealed that A. salmonicida grew efficiently on N-acetylglucosamine (GlcNAc) and chitobiose [(GlcNAc)2], the primary soluble products resulting from enzymatic chitin hydrolysis. The bacterium was also able to grow on chitin particles, albeit at a lower rate than on the soluble substrates. The genome of the bacterium contains five disrupted chitinase genes (pseudogenes) and three intact genes encoding a glycoside hydrolase family 18 (GH18) chitinase and two auxiliary activity family 10 (AA10) lytic polysaccharide monooxygenases (LPMOs). Biochemical characterization showed that the chitinase and LPMOs were able to depolymerize both α- and ß-chitin to (GlcNAc)2 and oxidized chitooligosaccharides, respectively. Notably, the chitinase displayed up to 50-fold lower activity than other well-studied chitinases. Deletion of the genes encoding the intact chitinolytic enzymes showed that the chitinase was important for growth on ß-chitin, whereas the LPMO gene deletion variants only showed minor growth defects on this substrate. Finally, proteomic analysis of A. salmonicida LFI1238 growth on ß-chitin showed expression of all three chitinolytic enzymes and, intriguingly, also three of the disrupted chitinases. In conclusion, our results show that A. salmonicida LFI1238 can utilize chitin as a nutrient source and that the GH18 chitinase and the two LPMOs are needed for this ability. IMPORTANCE The ability to utilize chitin as a source of nutrients is important for the survival and spread of marine microbial pathogens in the environment. One such pathogen is Aliivibrio (Vibrio) salmonicida, the causative agent of cold water vibriosis. Due to extensive gene decay, many key enzymes in the chitinolytic pathway have been disrupted, putatively rendering this bacterium incapable of chitin degradation and utilization. In the present study, we demonstrate that A. salmonicida can degrade and metabolize chitin, the most abundant biopolymer in the ocean. Our findings shed new light on the environmental adaption of this fish pathogen.

Chromatographic Assays for the Enzymatic Degradation of Chitin.

Chitin is an insoluble linear polymer of ß(1→4)-linked N-acetylglucosamine. Enzymatic cleavage of chitin chains can be achieved using hydrolytic enzymes, called chitinases, and/or oxidative enzymes, called lytic polysaccharide monooxygenases (LPMOs). These two groups of enzymes have different modes of action and yield different product types that require different analytical methods for detection and quantitation. While soluble chromogenic substrates are readily available for chitinases, proper insight into the activity of these enzymes can only be obtained by measuring activity toward their polymeric, insoluble substrate, chitin. For LPMOs, only assays using insoluble chitin are possible and relevant. Working with insoluble substrates complicates enzyme assays from substrate preparation to product analysis. Here, we describe typical set-ups for chitin degradation reactions and the chromatographic methods used for product analysis. Graphical abstract: Overview of chromatographic methods for assessing the enzymatic degradation of chitin.

The lytic polysaccharide monooxygenase CbpD promotes Pseudomonas aeruginosa virulence in systemic infection.

The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but supporting functional data is scarce. Here we show that CbpD, the LPMO of Pseudomonas aeruginosa, is a chitin-oxidizing virulence factor that promotes survival of the bacterium in human blood. The catalytic activity of CbpD was promoted by azurin and pyocyanin, two redox-active virulence factors also secreted by P. aeruginosa. Homology modeling, molecular dynamics simulations, and small angle X-ray scattering indicated that CbpD is a monomeric tri-modular enzyme with flexible linkers. Deletion of cbpD rendered P. aeruginosa unable to establish a lethal systemic infection, associated with enhanced bacterial clearance in vivo. CbpD-dependent survival of the wild-type bacterium was not attributable to dampening of pro-inflammatory responses by CbpD ex vivo or in vivo. Rather, we found that CbpD attenuates the terminal complement cascade in human serum. Studies with an active site mutant of CbpD indicated that catalytic activity is crucial for virulence function. Finally, profiling of the bacterial and splenic proteomes showed that the lack of this single enzyme resulted in substantial re-organization of the bacterial and host proteomes. LPMOs similar to CbpD occur in other pathogens and may have similar immune evasive functions.

Secretion Systems in Gram-Negative Bacterial Fish Pathogens.

Bacterial fish pathogens are one of the key challenges in the aquaculture industry, one of the fast-growing industries worldwide. These pathogens rely on arsenal of virulence factors such as toxins, adhesins, effectors and enzymes to promote colonization and infection. Translocation of virulence factors across the membrane to either the extracellular environment or directly into the host cells is performed by single or multiple dedicated secretion systems. These secretion systems are often key to the infection process. They can range from simple single-protein systems to complex injection needles made from dozens of subunits. Here, we review the different types of secretion systems in Gram-negative bacterial fish pathogens and describe their putative roles in pathogenicity. We find that the available information is fragmented and often descriptive, and hope that our overview will help researchers to more systematically learn from the similarities and differences between the virulence factors and secretion systems of the fish-pathogenic species described here.

A trimodular bacterial enzyme combining hydrolytic activity with oxidative glycosidic bond cleavage efficiently degrades chitin.

Findings from recent studies have indicated that enzymes containing more than one catalytic domain may be particularly powerful in the degradation of recalcitrant polysaccharides such as chitin and cellulose. Some known multicatalytic enzymes contain several glycoside hydrolase domains and one or more carbohydrate-binding modules (CBMs). Here, using bioinformatics and biochemical analyses, we identified an enzyme, Jd1381 from the actinobacterium Jonesia denitrificans, that uniquely combines two different polysaccharide-degrading activities. We found that Jd1381 contains an N-terminal family AA10 lytic polysaccharide monooxygenase (LPMO), a family 5 chitin-binding domain (CBM5), and a family 18 chitinase (Chi18) domain. The full-length enzyme, which seems to be the only chitinase produced by J. denitrificans, degraded both α- and ß-chitin. Both the chitinase and the LPMO activities of Jd1381 were similar to those of other individual chitinases and LPMOs, and the overall efficiency of chitin degradation by full-length Jd1381 depended on its chitinase and LPMO activities. Of note, the chitin-degrading activity of Jd1381 was comparable with or exceeded the activities of combinations of well-known chitinases and an LPMO from Serratia marcescens Importantly, comparison of the chitinolytic efficiency of Jd1381 with the efficiencies of combinations of truncated variants-JdLPMO10 and JdCBM5-Chi18 or JdLPMO10-CBM5 and JdChi18-indicated that optimal Jd1381 activity requires close spatial proximity of the LPMO10 and the Chi18 domains. The demonstration of intramolecular synergy between LPMOs and hydrolytic enzymes reported here opens new avenues toward the development of efficient catalysts for biomass conversion.

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.

Characterization and synergistic action of a tetra-modular lytic polysaccharide monooxygenase from Bacillus cereus.

Lytic polysaccharide monooxygenases (LPMOs) contribute to enzymatic conversion of recalcitrant polysaccharides such as chitin and cellulose and may also play a role in bacterial infections. Some LPMOs are multimodular, the implications of which remain only partly understood. We have studied the properties of a tetra-modular LPMO from the food poisoning bacterium Bacillus cereus (named BcLPMO10A). We show that BcLPMO10A, comprising an LPMO domain, two fibronectin-type III (FnIII)-like domains, and a carbohydrate-binding module (CBM5), is a powerful chitin-active LPMO. While the role of the FnIII domains remains unclear, we show that enzyme functionality strongly depends on the CBM5, which, by promoting substrate binding, protects the enzyme from inactivation. BcLPMO10A enhances the activity of chitinases during the degradation of α-chitin.

Neutron and Atomic Resolution X-ray Structures of a Lytic Polysaccharide Monooxygenase Reveal Copper-Mediated Dioxygen Binding and Evidence for N-Terminal Deprotonation.

A 1.1 Å resolution, room-temperature X-ray structure and a 2.1 Å resolution neutron structure of a chitin-degrading lytic polysaccharide monooxygenase domain from the bacterium Jonesia denitrificans (JdLPMO10A) show a putative dioxygen species equatorially bound to the active site copper. Both structures show an elongated density for the dioxygen, most consistent with a Cu(II)-bound peroxide. The coordination environment is consistent with Cu(II). In the neutron and X-ray structures, difference maps reveal the N-terminal amino group, involved in copper coordination, is present as a mixed ND2 and ND-, suggesting a role for the copper ion in shifting the pKa of the amino terminus.

Genomic, proteomic and biochemical analysis of the chitinolytic machinery of Serratia marcescens BJL200.

The chitinolytic machinery of Serratia marcescens BJL200 has been studied in detail over the last couple of decades, however, the proteome secreted by this Gram-negative bacterium during growth on chitin has not been studied in depth. In addition, the genome of this most studied chitinolytic Serratia strain has until now, not been sequenced. We report a draft genome sequence for S. marcescens BJL200. Using label-free quantification (LFQ) proteomics and a recently developed plate-method for assessing secretomes during growth on solid substrates, we find that, as expected, the chitin-active enzymes (ChiA, B, C, and CBP21) are produced in high amounts when the bacterium grows on chitin. Other proteins produced in high amounts after bacterial growth on chitin provide interesting targets for further exploration of the proteins involved in degradation of chitin-rich biomasses. The genome encodes a fourth chitinase (ChiD), which is produced in low amounts during growth on chitin. Studies of chitin degradation with mixtures of recombinantly produced chitin-degrading enzymes showed that ChiD does not contribute to the overall efficiency of the process. ChiD is capable of converting N,N'-diacetyl chitobiose to N-acetyl glucosamine, but is less efficient than another enzyme produced for this purpose, the Chitobiase. Thus, the role of ChiD in chitin degradation, if any, remains unclear.

A novel analytical method for d-glucosamine quantification and its application in the analysis of chitosan degradation by a minimal enzyme cocktail.

Enzymatic depolymerization of chitosan, a ß-(1,4)-linked polycationic polysaccharide composed of d-glucosamine (GlcN) and N-acetyl-d-glucosamine (GlcNAc) provides a possible route to the exploitation of chitin-rich biomass. Complete conversion of chitosan to mono-sugars requires the synergistic action of endo- and exo- chitosanases. In the present study we have developed an efficient and cost-effective chitosan-degrading enzyme cocktail containing only two enzymes, an endo-attacking bacterial chitosanase, ScCsn46A, from Streptomyces coelicolor, and an exo-attacking glucosamine specific ß-glucosaminidase, Tk-Glm, from the archaeon Thermococcus kodakarensis KOD1. Moreover, we developed a fast, reliable quantitative method for analysis of GlcN using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The sensitivity of this method is high and less than 50 pmol was easily detected, which is about 1000-fold better than the sensitivity of more commonly used detection methods based on refractive index. We also obtained qualitative insight into product development during the enzymatic degradation reaction by means of ElectroSpray Ionization-Mass Spectrometry (ESI-MS).

Characterization of a chitinase from the cellulolytic actinomycete Thermobifida fusca.

Thermobifida fusca is a well-known cellulose-degrading actinomycete, which produces various glycoside hydrolases for this purpose. However, despite the presence of putative chitinase genes in its genome, T. fusca has not been reported to grow on chitin as sole carbon source. In this study, a gene encoding a putative membrane-anchored GH18 chitinase (Tfu0868) from T. fusca has been cloned and overexpressed in Escherichia coli. The protein was produced as SUMO fusion protein and, upon removal of the SUMO domain, soluble pure TfChi18A was obtained with yields typically amounting to 150mg per litre of culture. The enzyme was found to be relatively thermostable (apparent Tm=57.5°C) but not particularly thermoactive, the optimum temperature being 40-45°C. TfChi18A bound to α- and ß-chitin and degraded both these substrates. Interestingly, activity towards colloidal chitin was minimal and in this case, substrate inhibition was observed. TfChi18A also cleaved soluble chito-oligosaccharides and showed a clear preference for substrates having five sugars or more. While these results show that TfChi18A is a catalytically competent GH18 chitinase, the observed catalytic rates were low compared to those of well-studied GH18 chitinases. This suggests that TfChi18A is not a true chitinase and not likely to endow T. fusca with the ability to grow on chitin.

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.

Structural and functional characterization of a small chitin-active lytic polysaccharide monooxygenase domain of a multi-modular chitinase from Jonesia denitrificans.

Lytic polysaccharide monooxygenases (LPMOs) boost enzymatic depolymerization of recalcitrant polysaccharides, such as chitin and cellulose. We have studied a chitin-active LPMO domain (JdLPMO10A) that is considerably smaller (15.5 kDa) than all structurally characterized LPMOs so far and that is part of a modular protein containing a GH18 chitinase. The 1.55 Å resolution structure revealed deletions of interacting loops that protrude from the core ß-sandwich scaffold in larger LPMO10s. Despite these deletions, the enzyme is active on alpha- and beta-chitin, and the chitin-binding surface previously described for larger LPMOs is fully conserved. JdLPMO10A may represent a minimal scaffold needed to catalyse the powerful LPMO reaction.

Neutron and high-resolution room-temperature X-ray data collection from crystallized lytic polysaccharide monooxygenase.

Bacteria and fungi express lytic polysaccharide monooxgyenase (LPMO) enzymes that act in conjunction with canonical hydrolytic sugar-processing enzymes to rapidly convert polysaccharides such as chitin, cellulose and starch to single monosaccharide products. In order to gain a better understanding of the structure and oxidative mechanism of these enzymes, large crystals (1-3 mm(3)) of a chitin-processing LPMO from the Gram-positive soil bacterium Jonesia denitrificans were grown and screened for their ability to diffract neutrons. In addition to the collection of neutron diffraction data, which were processed to 2.1 Å resolution, a high-resolution room-temperature X-ray diffraction data set was collected and processed to 1.1 Å resolution in space group P212121. To our knowledge, this work marks the first successful neutron crystallographic experiment on an LPMO. Joint X-ray/neutron refinement of the resulting data will reveal new details of the structure and mechanism of this recently discovered class of enzymes.

Comparative study of two chitin-active and two cellulose-active AA10-type lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs), found in family 9 (previously GH61), family 10 (previously CBM33), and the newly discovered family 11 of auxiliary activities (AA) in the carbohydrate-active enzyme classification system, are copper-dependent enzymes that oxidize sp(3)-carbons in recalcitrant polysaccharides such as chitin and cellulose in the presence of an external electron donor. In this study, we describe the activity of two AA10-type LPMOs whose activities have not been described before and we compare in total four different AA10-type LPMOs with the aim of finding possible correlations between their substrate specificities, sequences, and EPR signals. EPR spectra indicate that the electronic environment of the copper varies within the AA10 family even though amino acids directly interacting with the copper atom are identical in all four enzymes. This variation seems to be correlated to substrate specificity and is likely caused by sequence variation in areas that affect substrate binding geometry and/or by variation in a cluster of conserved aromatic residues likely involved in electron transfer. Interestingly, EPR signals for cellulose-active AA10 enzymes were similar to those previously observed for cellulose-active AA9 enzymes. Mutation of the conserved phenylalanine positioned in close proximity to the copper center in AA10-type LPMOs to Tyr (the corresponding residue in most AA9-type LPMOs) or Ala, led to complete or partial inactivation, respectively, while in both cases the ability to bind copper was maintained. Moreover, substrate binding affinity and degradation ability seemed hardly correlated, further emphasizing the crucial role of the active site configuration in determining LPMO functionality.