Immunization with lytic polysaccharide monooxygenase CbpD induces protective immunity against Pseudomonas aeruginosa pneumonia.

Pseudomonas aeruginosa (PA) CbpD belongs to the lytic polysaccharide monooxygenases (LPMOs), a family of enzymes that cleave chitin or related polysaccharides. Here, we demonstrate a virulence role of CbpD in PA pneumonia linked to impairment of host complement function and opsonophagocytic clearance. Following intratracheal challenge, a PA ΔCbpD mutant was more easily cleared and produced less mortality than the wild-type parent strain. The x-ray crystal structure of the CbpD LPMO domain was solved to subatomic resolution (0.75Å) and its two additional domains modeled by small-angle X-ray scattering and Alphafold2 machine-learning algorithms, allowing structure-based immune epitope mapping. Immunization of naive mice with recombinant CbpD generated high IgG antibody titers that promoted human neutrophil opsonophagocytic killing, neutralized enzymatic activity, and protected against lethal PA pneumonia and sepsis. IgG antibodies generated against full-length CbpD or its noncatalytic M2+CBM73 domains were opsonic and protective, even in previously PA-exposed mice, while antibodies targeting the AA10 domain were not. Preexisting antibodies in PA-colonized cystic fibrosis patients primarily target the CbpD AA10 catalytic domain. Further exploration of LPMO family proteins, present across many clinically important and antibiotic-resistant human pathogens, may yield novel and effective vaccine antigens.

Isophthalate:coenzyme A ligase initiates anaerobic degradation of xenobiotic isophthalate.

BACKGROUND: Environmental contamination from synthetic plastics and their additives is a widespread problem. Phthalate esters are a class of refractory synthetic organic compounds which are widely used in plastics, coatings, and for several industrial applications such as packaging, pharmaceuticals, and/or paints. They are released into the environment during production, use and disposal, and some of them are potential mutagens and carcinogens. Isophthalate (1,3-benzenedicarboxylic acid) is a synthetic chemical that is globally produced at a million-ton scale for industrial applications and is considered a priority pollutant. Here we describe the biochemical characterization of an enzyme involved in anaerobic degradation of isophthalate by the syntrophically fermenting bacterium Syntrophorhabdus aromaticivorans strain UI that activate isophthalate to isophthalyl-CoA followed by its decarboxylation to benzoyl-CoA. RESULTS: Isophthalate:Coenzyme A ligase (IPCL, AMP-forming) that activates isophthalate to isophthalyl-CoA was heterologously expressed in E. coli (49.6 kDa) for biochemical characterization. IPCL is homologous to phenylacetate-CoA ligase that belongs to the family of ligases that form carbon-sulfur bonds. In the presence of coenzyme A, Mg2+ and ATP, IPCL converts isophthalate to isophthalyl-CoA, AMP and pyrophosphate (PPi). The enzyme was specifically induced after anaerobic growth of S. aromaticivorans in a medium containing isophthalate as the sole carbon source. Therefore, IPCL exhibited high substrate specificity and affinity towards isophthalate. Only substrates that are structurally related to isophthalate, such as glutarate and 3-hydroxybenzoate, could be partially converted to the respective coenzyme A esters. Notably, no activity could be measured with substrates such as phthalate, terephthalate and benzoate. Acetyl-CoA or succinyl-CoA did not serve as CoA donors. The enzyme has a theoretical pI of 6.8 and exhibited optimal activity between pH 7.0 to 7.5. The optimal temperature was between 25 °C and 37 °C. Denaturation temperature (Tm) of IPCL was found to be at about 63 °C. The apparent KM values for isophthalate, CoA, and ATP were 409 µM, 642 µM, and 3580 µM, respectively. Although S. aromaticivorans is a strictly anaerobic bacterium, the enzyme was found to be oxygen-insensitive and catalysed isophthalyl-CoA formation under both anoxic and oxic conditions. CONCLUSION: We have successfully cloned the ipcl gene, expressed and characterized the corresponding IPCL enzyme, which plays a key role in isophthalate activation that initiates its activation and further degradation by S. aromaticivorans. Its biochemical characterization represents an important step in the elucidation of the complete degradation pathway of isophthalate.

Chitinolytic enzymes contribute to the pathogenicity of Aliivibrio salmonicida LFI1238 in the invasive phase of cold-water vibriosis.

BACKGROUND: Aliivibrio salmonicida is the causative agent of cold-water vibriosis in salmonids (Oncorhynchus mykiss and Salmo salar L.) and gadidae (Gadus morhua L.). Virulence-associated factors that are essential for the full spectrum of A. salmonicida pathogenicity are largely unknown. Chitin-active lytic polysaccharide monooxygenases (LPMOs) have been indicated to play roles in both chitin degradation and virulence in a variety of pathogenic bacteria but are largely unexplored in this context. RESULTS: In the present study we investigated the role of LPMOs in the pathogenicity of A. salmonicida LFI238 in Atlantic salmon (Salmo salar L.). In vivo challenge experiments using isogenic deletion mutants of the two LPMOs encoding genes AsLPMO10A and AsLPMO10B, showed that both LPMOs, and in particular AsLPMO10B, were important in the invasive phase of cold-water vibriosis. Crystallographic analysis of the AsLPMO10B AA10 LPMO domain (to 1.4 Å resolution) revealed high structural similarity to viral fusolin, an LPMO known to enhance the virulence of insecticidal agents. Finally, exposure to Atlantic salmon serum resulted in substantial proteome re-organization of the A. salmonicida LPMO deletion variants compared to the wild type strain, indicating the struggle of the bacterium to adapt to the host immune components in the absence of the LPMOs. CONCLUSION: The present study consolidates the role of LPMOs in virulence and demonstrates that such enzymes may have more than one function.

Genomic and Proteomic Study of Andreprevotia ripae Isolated from an Anthill Reveals an Extensive Repertoire of Chitinolytic Enzymes.

Chitin is an abundant natural polysaccharide that is hard to degrade because of its crystalline nature and because it is embedded in robust co-polymeric materials containing other polysaccharides, proteins, and minerals. Thus, it is of interest to study the enzymatic machineries of specialized microbes found in chitin-rich environments. We describe a genomic and proteomic analysis of Andreprevotia ripae, a chitinolytic Gram-negative bacterium isolated from an anthill. The genome of A. ripae encodes four secreted family GH19 chitinases of which two were detected and upregulated during growth on chitin. In addition, the genome encodes as many as 25 secreted GH18 chitinases, of which 17 were detected and 12 were upregulated during growth on chitin. Finally, the single lytic polysaccharide monooxygenase (LPMO) was strongly upregulated during growth on chitin. Whereas 66% of the 29 secreted chitinases contained two carbohydrate-binding modules (CBMs), this fraction was 93% (13 out of 14) for the upregulated chitinases, suggesting an important role for these CBMs. Next to an unprecedented multiplicity of upregulated chitinases, this study reveals several chitin-induced proteins that contain chitin-binding CBMs but lack a known catalytic function. These proteins are interesting targets for discovery of enzymes used by nature to convert chitin-rich biomass. The MS proteomic data have been deposited in the PRIDE database with accession number PXD025087.

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.

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.

Engineering chitinolytic activity into a cellulose-active lytic polysaccharide monooxygenase provides insights into substrate specificity.

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of recalcitrant polysaccharides such as cellulose and chitin and play an important role in the enzymatic degradation of biomass. Although it is clear that these monocopper enzymes have extended substrate-binding surfaces for interacting with their fibrous substrates, the structural determinants of LPMO substrate specificity remain largely unknown. To gain additional insight into substrate specificity in LPMOs, here we generated a mutant library of a cellulose-active family AA10 LPMO from Streptomyces coelicolor A3(2) (ScLPMO10C, also known as CelS2) having multiple substitutions at five positions on the substrate-binding surface that we identified by sequence comparisons. Screening of this library using a newly-developed MS-based high-throughput assay helped identify multiple enzyme variants that contained four substitutions and exhibited significant chitinolytic activity and a concomitant decrease in cellulolytic activity. The chitin-active variants became more rapidly inactivated during catalysis than a natural chitin-active AA10 LPMO, an observation likely indicative of suboptimal substrate binding leading to autocatalytic oxidative damage of these variants. These results reveal several structural determinants of LPMO substrate specificity and underpin the notion that productive substrate binding by these enzymes is complex, depending on a multitude of amino acids located on the substrate-binding surface.

Structural determinants of bacterial lytic polysaccharide monooxygenase functionality.

Bacterial lytic polysaccharide monooxygenases (LPMO10s) use redox chemistry to cleave glycosidic bonds in the two foremost recalcitrant polysaccharides found in nature, namely cellulose and chitin. Analysis of correlated mutations revealed that the substrate-binding and copper-containing surface of LPMO10s composes a network of co-evolved residues and interactions, whose roles in LPMO functionality are unclear. Here, we mutated a subset of these correlated residues in a newly characterized C1/C4-oxidizing LPMO10 from Micromonospora aurantiaca (MaLPMO10B) to the corresponding residues in strictly C1-oxidizing LPMO10s. We found that surface properties near the catalytic copper, i.e. side chains likely to be involved in substrate positioning, are major determinants of the C1:C4 ratio. Several MaLPMO10B mutants almost completely lost C4-oxidizing activity while maintaining C1-oxidizing activity. These mutants also lost chitin-oxidizing activity, which is typically observed for C1/C4-oxidizing, but not for C1-oxidizing, cellulose-active LPMO10s. Selective loss in C1-oxidizing activity was not observed. Additional mutational experiments disclosed that neither truncation of the MaLPMO10B family 2 carbohydrate-binding module nor mutations altering access to the solvent-exposed axial copper coordination site significantly change the C1:C4 ratio. Importantly, several of the mutations that altered interactions with the substrate exhibited reduced stability. This effect could be explained by productive substrate binding that protects LPMOs from oxidative self-inactivation. We discuss these stability issues in view of recent findings on LPMO catalysis, such as the involvement of H2O2 Our results show that residues on the substrate-binding surface of LPMOs have co-evolved to optimize several of the interconnected properties: substrate binding and specificity, oxidative regioselectivity, catalytic efficiency, and stability.

Systems analysis of the glycoside hydrolase family 18 enzymes from Cellvibrio japonicus characterizes essential chitin degradation functions.

Understanding the strategies used by bacteria to degrade polysaccharides constitutes an invaluable tool for biotechnological applications. Bacteria are major mediators of polysaccharide degradation in nature; however, the complex mechanisms used to detect, degrade, and consume these substrates are not well-understood, especially for recalcitrant polysaccharides such as chitin. It has been previously shown that the model bacterial saprophyte Cellvibrio japonicus is able to catabolize chitin, but little is known about the enzymatic machinery underlying this capability. Previous analyses of the C. japonicus genome and proteome indicated the presence of four glycoside hydrolase family 18 (GH18) enzymes, and studies of the proteome indicated that all are involved in chitin utilization. Using a combination of in vitro and in vivo approaches, we have studied the roles of these four chitinases in chitin bioconversion. Genetic analyses showed that only the chi18D gene product is essential for the degradation of chitin substrates. Biochemical characterization of the four enzymes showed functional differences and synergistic effects during chitin degradation, indicating non-redundant roles in the cell. Transcriptomic studies revealed complex regulation of the chitin degradation machinery of C. japonicus and confirmed the importance of CjChi18D and CjLPMO10A, a previously characterized chitin-active enzyme. With this systems biology approach, we deciphered the physiological relevance of the glycoside hydrolase family 18 enzymes for chitin degradation in C. japonicus, and the combination of in vitro and in vivo approaches provided a comprehensive understanding of the initial stages of chitin degradation by this bacterium.

Oxidative cleavage of polysaccharides by monocopper enzymes depends on H2O2.

Enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) play an important role in the conversion of recalcitrant polysaccharides, but their mode of action has remained largely enigmatic. It is generally believed that catalysis by LPMOs requires molecular oxygen and a reductant that delivers two electrons per catalytic cycle. Using enzyme assays, mass spectrometry and experiments with labeled oxygen atoms, we show here that H2O2, rather than O2, is the preferred co-substrate of LPMOs. By controlling H2O2 supply, stable reaction kinetics are achieved, the LPMOs work in the absence of O2, and the reductant is consumed in priming rather than in stoichiometric amounts. The use of H2O2 by a monocopper enzyme that is otherwise cofactor-free offers new perspectives regarding the mode of action of copper enzymes. Furthermore, these findings have implications for the enzymatic conversion of biomass in Nature and in industrial biorefining.

Metaproteomics: Sample Preparation and Methodological Considerations.

Meta-omic techniques have progressed rapidly in the past decade and are frequently used in microbial ecology to study microorganisms in their natural ecosystems independent from culture restrictions. Metaproteomics, in combination with metagenomics, enables quantitative assessment of expressed proteins and pathways from individual members of the consortium. Together, metaproteomics and metagenomics can provide a detailed understanding of which organisms occupy specific metabolic niches, how they interact, and how they utilize nutrients, and these insights can be obtained directly from environmental samples. Here, we outline key aspects of sample preparation, database generation, and other methodological considerations that are required for successful quantitative metaproteomic analyses and we describe case studies on the integration with metagenomics for enhanced functional output.

Interactions of a fungal lytic polysaccharide monooxygenase with ß-glucan substrates and cellobiose dehydrogenase.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds using molecular oxygen and an external electron donor. We have used NMR and isothermal titration calorimetry (ITC) to study the interactions of a broad-specificity fungal LPMO, NcLPMO9C, with various substrates and with cellobiose dehydrogenase (CDH), a known natural supplier of electrons. The NMR studies revealed interactions with cellohexaose that center around the copper site. NMR studies with xyloglucans, i.e., branched ß-glucans, showed an extended binding surface compared with cellohexaose, whereas ITC experiments showed slightly higher affinity and a different thermodynamic signature of binding. The ITC data also showed that although the copper ion alone hardly contributes to affinity, substrate binding is enhanced for metal-loaded enzymes that are supplied with cyanide, a mimic of O2 (-) Studies with CDH and its isolated heme b cytochrome domain unambiguously showed that the cytochrome domain of CDH interacts with the copper site of the LPMO and that substrate binding precludes interaction with CDH. Apart from providing insights into enzyme-substrate interactions in LPMOs, the present observations shed new light on possible mechanisms for electron supply during LPMO action.

How a Lytic Polysaccharide Monooxygenase Binds Crystalline Chitin.

Lytic polysaccharide monooxygenases (LPMOs) are major players in biomass conversion, both in Nature and in the biorefining industry. How the monocopper LPMO active site is positioned relative to the crystalline substrate surface to catalyze powerful, but potentially self-destructive, oxidative chemistry is one of the major questions in the field. We have adopted a multidisciplinary approach, combining biochemical, spectroscopic, and molecular modeling methods to study chitin binding by the well-studied LPMO from Serratia marcescens SmAA10A (or CBP21). The orientation of the enzyme on a single-chain substrate was determined by analyzing enzyme cutting patterns. Building on this analysis, molecular dynamics (MD) simulations were performed to study interactions between the LPMO and three different surface topologies of crystalline chitin. The resulting atomistic models showed that most enzyme-substrate interactions involve the polysaccharide chain that is to be cleaved. The models also revealed a constrained active site geometry as well as a tunnel connecting the bulk solvent to the copper site, through which only small molecules such as H2O, O2, and H2O2 can diffuse. Furthermore, MD simulations, quantum mechanics/molecular mechanics calculations, and electron paramagnetic resonance spectroscopy demonstrate that rearrangement of Cu-coordinating water molecules is necessary when binding the substrate and also provide a rationale for the experimentally observed C1 oxidative regiospecificity of SmAA10A. This study provides a first, experimentally supported, atomistic view of the interactions between an LPMO and crystalline chitin. The confinement of the catalytic center is likely crucially important for controlling the oxidative chemistry performed by LPMOs and will help guide future mechanistic studies.

Multipoint Precision Binding of Substrate Protects Lytic Polysaccharide Monooxygenases from Self-Destructive Off-Pathway Processes.

Lytic polysaccharide monooxygenases (LPMOs) play a crucial role in the degradation of polysaccharides in biomass by catalyzing powerful oxidative chemistry using only a single copper ion as a cofactor. Despite the natural abundance and importance of these powerful monocopper enzymes, the structural determinants of their functionality have remained largely unknown. We have used site-directed mutagenesis to probe the roles of 13 conserved amino acids located on the flat substrate-binding surface of CBP21, a chitin-active family AA10 LPMO from Serratia marcescens, also known as SmLPMO10A. Single mutations of residues that do not interact with the catalytic copper site, but rather are involved in substrate binding had remarkably strong effects on overall enzyme performance. Analysis of product formation over time showed that these mutations primarily affected enzyme stability. Investigation of protein integrity using proteomics technologies showed that loss of activity was caused by oxidation of essential residues in the enzyme active site. For most enzyme variants, reduced enzyme stability correlated with a reduced level of binding to chitin, suggesting that adhesion to the substrate prevents oxidative off-pathway processes that lead to enzyme inactivation. Thus, the extended and highly evolvable surfaces of LPMOs are tailored for precise multipoint substrate binding, which provides the confinement that is needed to harness and control the remarkable oxidative power of these enzymes. These findings are important for the optimized industrial use of LPMOs as well as the design of LPMO-inspired catalysts.

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.

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.

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.

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.

Structural and functional characterization of a conserved pair of bacterial cellulose-oxidizing lytic polysaccharide monooxygenases.

For decades, the enzymatic conversion of cellulose was thought to rely on the synergistic action of hydrolytic enzymes, but recent work has shown that lytic polysaccharide monooxygenases (LPMOs) are important contributors to this process. We describe the structural and functional characterization of two functionally coupled cellulose-active LPMOs belonging to auxiliary activity family 10 (AA10) that commonly occur in cellulolytic bacteria. One of these LPMOs cleaves glycosidic bonds by oxidation of the C1 carbon, whereas the other can oxidize both C1 and C4. We thus demonstrate that C4 oxidation is not confined to fungal AA9-type LPMOs. X-ray crystallographic structures were obtained for the enzyme pair from Streptomyces coelicolor, solved at 1.3 Å (ScLPMO10B) and 1.5 Å (CelS2 or ScLPMO10C) resolution. Structural comparisons revealed differences in active site architecture that could relate to the ability to oxidize C4 (and that also seem to apply to AA9-type LPMOs). Despite variation in active site architecture, the two enzymes exhibited similar affinities for Cu(2+) (12-31 nM), redox potentials (242 and 251 mV), and electron paramagnetic resonance spectra, with only the latter clearly different from those of chitin-active AA10-type LPMOs. We conclude that substrate specificity depends not on copper site architecture, but rather on variation in substrate binding and orientation. During cellulose degradation, the members of this LPMO pair act in synergy, indicating different functional roles and providing a rationale for the abundance of these enzymes in biomass-degrading organisms.

Proteomic investigation of the secretome of Cellvibrio japonicus during growth on chitin.

Studies of the secretomes of microbes grown on insoluble substrates are important for the discovery of novel proteins involved in biomass conversion. However, data in literature and this study indicate that secretome samples tend to be contaminated with cytoplasmic proteins. We have examined the secretome of the Gram-negative soil bacterium Cellvibrio japonicus using a simple plate-based culturing technique that yields samples with high fractions (60-75%) of proteins that are predicted to be secreted. By combining this approach with label-free quantification using the MaxLFQ algorithm, we have mapped and quantified proteins secreted by C. japonicus during growth on α- and ß-chitin. Hierarchical clustering of the detected protein quantities revealed groups of up-regulated proteins that include all five putative C. japonicus chitinases as well as a chitin-specific lytic polysaccharide monooxygenase (CjLPMO10A). A small set of secreted proteins were co-regulated with known chitin-specific enzymes, including several with unknown catalytic functions. These proteins provide interesting targets for further studies aimed at unraveling the enzymatic machineries used by C. japonicus for recalcitrant polysaccharide degradation. Studies of chitin degradation indicated that C. japonicus indeed produces an efficient chitinolytic enzyme cocktail. All MS data have been deposited in the ProteomeXchange with the dataset identifier PXD002843 (http://proteomecentral.proteomexchange.org/dataset/PXD002843).