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Significance Lytic polysaccharide monooxygenases (LPMOs) have unique catalytic centers, at which a single copper catalyzes the oxidative cleavage of a glycosidic bond. The mechanism by which LPMOs activate molecular oxygen is key to understanding copper (bio)catalysis but remains poorly understood, largely because the insoluble and heterogeneous nature of LPMO substrates precludes the use of usual laboratory techniques. Using an integrated NMR/EPR approach, we have unraveled structural and electronic details of the interactions of an LPMO from Bacillus licheniformis and β-chitin. EPR spectroscopy on uniformly isotope 15N-labeled 63Cu(II)-LPMO provided insight into substrate-driven rearrangement of the copper coordination sphere that predisposes the enzyme for O2 activation., Lytic polysaccharide monooxygenases (LPMOs) have a unique ability to activate molecular oxygen for subsequent oxidative cleavage of glycosidic bonds. To provide insight into the mode of action of these industrially important enzymes, we have performed an integrated NMR/electron paramagnetic resonance (EPR) study into the detailed aspects of an AA10 LPMO–substrate interaction. Using NMR spectroscopy, we have elucidated the solution-phase structure of apo-BlLPMO10A from Bacillus licheniformis, along with solution-phase structural characterization of the Cu(I)-LPMO, showing that the presence of the metal has minimal effects on the overall protein structure. We have, moreover, used paramagnetic relaxation enhancement (PRE) to characterize Cu(II)-LPMO by NMR spectroscopy. In addition, a multifrequency continuous-wave (CW)-EPR and 15N-HYSCORE spectroscopy study on the uniformly isotope-labeled 63Cu(II)-bound 15N-BlLPMO10A along with its natural abundance isotopologue determined copper spin-Hamiltonian parameters for LPMOs to markedly improved accuracy. The data demonstrate that large changes in the Cu(II) spin-Hamiltonian parameters are induced upon binding of the substrate. These changes arise from a rearrangement of the copper coordination sphere from a five-coordinate distorted square pyramid to one which is four-coordinate near-square planar. There is also a small reduction in metal–ligand covalency and an attendant increase in the d(x2−y2) character/energy of the singly occupied molecular orbital (SOMO), which we propose from density functional theory (DFT) calculations predisposes the copper active site for the formation of a stable Cu–O2 intermediate. This switch in orbital character upon addition of chitin provides a basis for understanding the coupling of substrate binding with O2 activation in chitin-active AA10 LPMOs.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides in the presence of an external electron donor (reductant). In the classical O2-driven monooxygenase reaction, the reductant is needed in stoichiometric amounts. In a recently discovered, more efficient H2O2-driven reaction, the reductant would be needed only for the initial reduction (priming) of the LPMO to its catalytically active Cu(I) form. However, the influence of the reductant on reducing the LPMO or on H2O2 production in the reaction remains undefined. Here, we conducted a detailed kinetic characterization to investigate how the reductant affects H2O2-driven degradation of 14C-labeled chitin by a bacterial LPMO, SmLPMO10A (formerly CBP21). Sensitive detection of 14C-labeled products and careful experimental set-ups enabled discrimination between the effects of the reductant on LPMO priming and other effects, in particular enzyme-independent production of H2O2 through reactions with O2 When supplied with H2O2, SmLPMO10A catalyzed 18 oxidative cleavages per molecule of ascorbic acid, suggesting a "priming reduction" reaction. The dependence of initial rates of chitin degradation on reductant concentration followed hyperbolic saturation kinetics, and differences between the reductants were manifested in large variations in their half-saturating concentrations (K mR app). Theoretical analyses revealed that K mR app decreases with a decreasing rate of polysaccharide-independent LPMO reoxidation (by either O2 or H2O2). We conclude that the efficiency of LPMO priming depends on the relative contributions of reductant reactivity, on the LPMO's polysaccharide monooxygenase/peroxygenase and reductant oxidase/peroxidase activities, and on reaction conditions, such as O2, H2O2, and polysaccharide concentrations.

Processive glycoside hydrolases associate with recalcitrant polysaccharides such as cellulose and chitin and repeatedly cleave glycosidic linkages without fully dissociating from the crystalline surface. The processive mechanism is efficient in the degradation of insoluble substrates, but comes at the cost of reduced enzyme speed. We show that less processive chitinase variants with reduced ability to degrade crystalline chitin, regain much of this ability when combined with a lytic polysaccharide monooxygenase (LPMO). When combined with an LPMO, several less processive chitinase mutants showed equal or even increased activity on chitin compared to the wild-type. Thus, LPMOs affect the need for processivity in polysaccharide degrading enzyme cocktails, which implies that the composition of such cocktails may need reconsideration.

Enzymes known as lytic polysaccharide monooxygenases (LPMOs) are recognized as important contributors to aerobic enzymatic degradation of recalcitrant polysaccharides such as chitin and cellulose. LPMOs are remarkably abundant in nature, with some fungal species possessing more than 50 LPMO genes, and the biological implications of this diversity remain enigmatic. For example, chitin-active LPMOs have been encountered in biological niches where chitin conversion does not seem to take place. We have carried out an in-depth kinetic characterization of a putatively chitin-active LPMO from Aspergillus fumigatus (AfAA11B), which, as we show here, has multiple unusual properties, such as a low redox potential and high oxidase activity. Furthermore, AfAA11B is hardly active on chitin, while being very active on soluble oligomers of N-acetylglucosamine. In the presence of chitotetraose, the enzyme can withstand considerable amounts of H2O2, which it uses to efficiently and stoichiometrically convert this substrate. The unique properties of AfAA11B allowed experiments showing that it is a strict peroxygenase and does not catalyze a monooxygenase reaction. This study shows that nature uses LPMOs for breaking glycosidic bonds in non-polymeric substrates in reactions that depend on H2O2. The quest for the true substrates of these enzymes, possibly carbohydrates in the cell wall of the fungus or its competitors, will be of major interest.

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.

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.

Chito-oligosaccharides (CHOS) are homo- or hetero-oligomers of

Microorganisms use a host of enzymes, including processive glycoside hydrolases, to deconstruct recalcitrant polysaccharides to sugars. Processive glycoside hydrolases closely associate with polymer chains and repeatedly cleave glycosidic linkages without dissociating from the crystalline surface after each hydrolytic step; they are typically the most abundant enzymes in both natural secretomes and industrial cocktails by virtue of their significant hydrolytic potential. The ubiquity of aromatic residues lining the enzyme catalytic tunnels and clefts is a notable feature of processive glycoside hydrolases. We hypothesized that these aromatic residues have uniquely defined roles, such as substrate chain acquisition and binding in the catalytic tunnel, that are defined by their local environment and position relative to the substrate and the catalytic center. Here, we investigated this hypothesis with variants of Serratia marcescens family 18 processive chitinases ChiA and ChiB. We applied molecular simulation and free energy calculations to assess active site dynamics and ligand binding free energies. Isothermal titration calorimetry provided further insight into enthalpic and entropic contributions to ligand binding free energy. Thus, the roles of six aromatic residues, Trp-167, Trp-275, and Phe-396 in ChiA, and Trp-97, Trp-220, and Phe-190 in ChiB, have been examined. We observed that point mutation of the tryptophan residues to alanine results in unfavorable changes in the free energy of binding relative to wild-type. The most drastic effects were observed for residues positioned at the "entrances" of the deep substrate-binding clefts and known to be important for processivity. Interestingly, phenylalanine mutations in ChiA and ChiB had little to no effect on chito-oligomer binding, in accordance with the limited effects of their removal on chitinase functionality.

The Gram-negative bacteria Serratia marcescens and Serratia proteamaculans have efficient chitinolytic machineries that degrade chitin into N-acetylglucosamine (GlcNAc), which is used as a carbon and energy source. The enzymatic degradation of chitin in these bacteria occurs through the synergistic action of glycoside hydrolases (GHs) that have complementary activities; an endo-acting GH (ChiC) making random scissions on the polysaccharide chains and two exo-acting GHs mainly targeting single reducing (ChiA) and nonreducing (ChiB) chain ends. Both bacteria produce low amounts of a fourth GH18 (ChiD) with an unclear role in chitin degradation. Here, we have determined the thermodynamic signatures for binding of (GlcNAc)6 and the inhibitor allosamidin to SpChiD as well as the crystal structure of SpChiD in complex with allosamidin. The binding free energies for the two ligands are similar (ΔGr° = −8.9 ± 0.1 and −8.4 ± 0.1 kcal/mol, respectively) with clear enthalpic penalties (ΔHr° = 3.2 ± 0.1 and 1.8 ± 0.1 kcal/mol, respectively). Binding of (GlcNAc)6 is dominated by solvation entropy change (−TΔSsolv° = −17.4 ± 0.4 kcal/mol) and the conformational entropy change dominates for allosamidin binding (−TΔSconf° = −9.0 ± 0.2 kcal/mol). These signatures as well as the interactions with allosamidin are very similar to those of SmChiB suggesting that both enzymes are nonreducing end-specific.

The discovery of oxidative cleavage of glycosidic bonds by enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) has had a major impact on our current understanding of the enzymatic conversion of recalcitrant polysaccharides such as chitin and cellulose. The number of LPMO sequence families keeps expanding and novel substrate specificities and biological functionalities are being discovered. The catalytic mechanism of these LPMOs remains somewhat enigmatic. Recently, novel insights have been obtained from studies of enzyme–substrate complexes by X-ray crystallography, EPR, NMR, and modeling. Furthermore, it has been shown that LPMOs may carry out peroxygenase reactions, at much higher rates than monooxygenase reactions, which affects our understanding and exploitation of these powerful enzymes. © 2019 The Authors. Published by Elsevier Ltd. This article is available under the Creative Commons CC-BY-NC-ND license and permits non-commercial use of the work as published, without adaptation or alteration provided the work is fully attributed.

Enzymatic depolymerization of abundant polysaccharides such as chitin and cellulose is hampered by the recalcitrant, crystalline nature of these materials. Nature musters a large variety of hydrolytic and oxidative enzymes with varying properties that, together, manage the task of saccharifying chitin and cellulose. Processivity, i.e. the ability to carry out multiple hydrolytic reactions while sliding along the polysaccharide chain, is considered a key property of hydrolytic enzymes acting on the most recalcitrant parts of the substrate. Due to the insoluble nature of the substrate, this phenomenon is difficult to study. For family GH18 chitinases, the combination of a catalytic mechanism depending on acetyl groups in the substrate and the possibility to produce partially deacetylated soluble single chitin chains (chitosan) provides a unique tool for studying processivity. Here, we review how the use of well-defined chitosans has helped unraveling crucial and otherwise difficult to study properties of multiple chitinases, including well studied chitinases from Serratia marcescens and human chitotriosidase (HCHT). These studies have yielded pioneering insights into the structural basis and functional implications of processivity that apply to both chitinases and cellulases. Recent studies of processive chitinases and cellulases confirm the insights originally derived from the work with chitosan and take this further, for example by providing kinetic and thermodynamic data for processive enzyme action.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides in the presence of an external electron donor (reductant). In the classical O(2)-driven monooxygenase reaction, the reductant is needed in stoichiometric amounts. In a recently discovered, more efficient H(2)O(2)-driven reaction, the reductant would be needed only for the initial reduction (priming) of the LPMO to its catalytically active Cu(I) form. However, the influence of the reductant on reducing the LPMO or on H(2)O(2) production in the reaction remains undefined. Here, we conducted a detailed kinetic characterization to investigate how the reductant affects H(2)O(2)-driven degradation of (14)C-labeled chitin by a bacterial LPMO, SmLPMO10A (formerly CBP21). Sensitive detection of (14)C-labeled products and careful experimental set-ups enabled discrimination between the effects of the reductant on LPMO priming and other effects, in particular enzyme-independent production of H(2)O(2) through reactions with O(2). When supplied with H(2)O(2), SmLPMO10A catalyzed 18 oxidative cleavages per molecule of ascorbic acid, suggesting a “priming reduction” reaction. The dependence of initial rates of chitin degradation on reductant concentration followed hyperbolic saturation kinetics, and differences between the reductants were manifested in large variations in their half-saturating concentrations (K(mR)(app)). Theoretical analyses revealed that K(mR)(app) decreases with a decreasing rate of polysaccharide-independent LPMO reoxidation (by either O(2) or H(2)O(2)). We conclude that the efficiency of LPMO priming depends on the relative contributions of reductant reactivity, on the LPMO's polysaccharide monooxygenase/peroxygenase and reductant oxidase/peroxidase activities, and on reaction conditions, such as O(2), H(2)O(2), and polysaccharide concentrations.

Human chitotriosidase (HCHT) is one of two active glycoside hydrolase family 18 chitinases produced by humans. The enzyme is associated with several diseases and is thought to play a role in the anti-parasite responses of the innate immune system. HCHT occurs in two isoforms, one 50 kDa (HCHT50) and one 39 kDa variant (HCHT39). Common for both isoforms is a catalytic domain with the (β/α)8 TIM barrel fold. HCHT50 has an additional linker-region, followed by a C-terminal carbohydrate-binding module (CBM) classified as CBM family 14 in the CAZy database. To gain further insight into enzyme functionality and especially the effect of the CBM, we expressed both isoforms and compared their catalytic properties on chitin and high molecular weight chitosans. HCHT50 degrades chitin faster than HCHT39 and much more efficiently. Interestingly, both HCHT50 and HCHT39 show biphasic kinetics on chitosan degradation where HCHT50 is faster initially and HCHT39 is faster in the second phase. Moreover, HCHT50 produces distinctly different oligomer distributions than HCHT39. This is likely due to increased transglycosylation activity for HCHT50 due the CBM extending the positive subsites binding surface and therefore promoting transglycosylation. Finally, studies with both chitin and chitosan showed that both isoforms have a similarly low degree of processivity. Combining functional and structural features of the two isoforms, it seems that HCHT combines features of exo-processive and endo-nonprocessive chitinases with the somewhat unusual CBM14 to reach a high degree of efficiency, in line with its alleged physiological task of being a “complete” chitinolytic machinery by itself.

The enzymatic degradation of recalcitrant polysaccharides is accomplished by synergistic enzyme cocktails of glycoside hydrolases (GHs) and accessory enzymes. Many GHs are processive which means that they remain attached to the substrate in between subsequent hydrolytic reactions. Chitinases are GHs that catalyze the hydrolysis of chitin (β-1,4-linked N-acetylglucosamine). Previously, a relationship between active site topology and processivity has been suggested while recent computational efforts have suggested a link between the degree of processivity and ligand binding free energy. We have investigated these relationships by employing computational (molecular dynamics (MD)) and experimental (isothermal titration calorimetry (ITC)) approaches to gain insight into the thermodynamics of substrate binding to Serratia marcescens chitinases ChiA, ChiB, and ChiC. We show that increased processive ability indeed corresponds to more favorable binding free energy and that this likely is a general feature of GHs. Moreover, ligand binding in ChiB is entropically driven; in ChiC it is enthalpically driven, and the enthalpic and entropic contributions to ligand binding in ChiA are equal. Furthermore, water is shown to be especially important in ChiA-binding. This work provides new insight into oligosaccharide binding, getting us one step closer to understand how GHs efficiently degrade recalcitrant polysaccharides.

Processive enzymes are major components of the efficient enzyme systems that are responsible for the degradation of the recalcitrant polysaccharides cellulose and chitin. Despite intensive research, there is no consensus on which step is rate-limiting for these enzymes. Here, we performed a comparative study of two well characterized enzymes, the cellobiohydrolase Cel7A from Hypocrea jecorina and the chitinase ChiA from Serratia marcescens. Both enzymes were inhibited by their disaccharide product, namely chitobiose for ChiA and cellobiose for Cel7A. The products behaved as noncompetitive inhibitors according to studies using the (14)C-labeled crystalline polymeric substrates (14)C chitin nanowhiskers and (14)C-labeled bacterial microcrystalline cellulose for ChiA and Cel7A, respectively. The resulting observed Ki (obs) values were 0.45 ± 0.08 mm for ChiA and 0.17 ± 0.02 mm for Cel7A. However, in contrast to ChiA, the Ki (obs) of Cel7A was an order of magnitude higher than the true Ki value governed by the thermodynamic stability of the enzyme-inhibitor complex. Theoretical analysis of product inhibition suggested that the inhibition strength and pattern can be accounted for by assuming different rate-limiting steps for ChiA and Cel7A. Measuring the population of enzymes whose active site was occupied by a polymer chain revealed that Cel7A was bound predominantly via its active site. Conversely, the active-site-mediated binding of ChiA was slow, and most ChiA exhibited a free active site, even when the substrate concentration was saturating for the activity. Collectively, our data suggest that complexation with the polymer chain is rate-limiting for ChiA, whereas Cel7A is limited by dissociation.

For decades, the enzymatic conversion of recalcitrant polysaccharides such as cellulose and chitin was thought to solely rely on the synergistic action of hydrolytic enzymes, but recent work has shown that lytic polysaccharide monooxygenases (LPMOs) are important contributors to this process. Here, we have examined the initial rate enhancement an LPMO (CBP21) has on the hydrolytic enzymes (ChiA, ChiB, and ChiC) of the chitinolytic machinery of Serratia marcescens through determinations of apparent k(cat) (k(cat)(app)) values on a β-chitin substrate. k(cat)(app) values were determined to be 1.7±0.1 s(-1) and 1.7±0.1 s(-1) for the exo-active ChiA and ChiB, respectively and 1.2±0.1 s(-1) for the endo-active ChiC. The addition of CBP21 boosted the k(cat)(app) values of ChiA and ChiB giving values of 11.1±1.5 s(-1) and 13.9±1.4 s(-1), while there was no effect on ChiC (0.9±0.1 s(-1)).

Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides, such as cellulose and chitin, and are of interest in biotechnological utilization of these abundant biomaterials. It has recently been shown that LPMOs can use H

We determined the crystal structure of a LysM module from Pteris ryukyuensis chitinase-A (PrLysM2) at a resolution of 1.8 A. Structural and binding analysis of PrLysM2 indicated that this module recognizes chitin oligosaccharides in a shallow groove comprised of five sugar-binding subsites on one side of the molecule. The free energy changes (ΔGr°) for binding of (GlcNAc)6, (GlcNAc)5, and (GlcNAc)4 to PrLysM2 were determined to be -5.4, -5,4 and -4.6 kcal mol-1, respectively, by ITC. Thermodynamic dissection of the binding energetics of (GlcNAc)6 revealed that the driving force is the enthalpy change (ΔHr° = -11.7 ± 0.2 kcal/mol) and the solvation entropy change (-TΔSsolv° = -5.9 ± 0.6 kcal/mol). This is the first description of thermodynamic signatures of a chitin oligosaccharide binding to a LysM module.

Polysaccharide depolymerization in nature is primarily accomplished by processive glycoside hydrolases which abstract single carbohydrate chains from polymer crystals and cleave glycosidic bonds without dissociating from the substrate after each catalytic event. Processivity is thought to conserve energy during enzymatic polysaccharide degradation. Herein, we compare two processive chitinases, ChiA and ChiB, one mutant, ChiB-W97A, and the endochitinase ChiC of the wellcharacterized chitinolytic machinery of Serratia marcescens by monitoring the extent of degradation on three different chitin substrates, and using the [(GlcNAc)2]/[GlcNAc] product ratio as a measure of processivity. The results show that the apparent processivity (Papp) greatly diminishes with the extent of degradation and confirm the hypothesis that Papp is limited by the length of obstacle free path on the substrate.

Chitosan, obtained from chitin by partial N-deacetylation, shows little or no toxicity towards mammalian cells, is biodegradable, and non-allergenic. It is known that chitosan may have antifungal properties, but the effect of defined chitosan or chito-oligosaccharides (CHOS) with different degree of polymerization is not well known. The objective of this study was to produce CHOS with different DPn (average degree of polymerization) and determine the most effective DPn of chitosan and CHOS against Botrytris cinerea Pers. Ex Fr. and Mucor piriformis Fischer. In vitro testing showed that CHOS of DPn 23 and 40 had the highest germination inhibition against the tested pathogens. The original chitosan (DPn 206) and a collection of short CHOS (degree of polymerization of 3–10) were significantly (P < 0.01) less effective than CHOS of DPn 23 and 40. M. piriformis M119J showed the most abnormal swelling in presence of CHOS DPn 40, but all abnormally swollen conidia showed further germ tube elongation. In vivo testing showed that CHOS DPn 23 was the most effective in reducing flower infection by two isolates of B. cinerea. Our results show that CHOS inhibit fungal germination and growth and that the effect depends highly on the level of polymerization of the oligomers.

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.

Human chitotriosidase (HCHT) is a family 18 chitinase that is an innate part of the immune system. We have mapped preferred productive binding modes of chito-oligosaccharide substrates to HCHT and the data show that HCHT has strong binding affinity in the +3 subsite. Moreover, HCHT shows anomer-specific binding affinities in subsites +2 and +3. These features could endorse HCHT with higher endo-activity and a higher transglycosylation potential.

Chitin is an important structural component of numerous fungal pathogens and parasitic nematodes. The human macrophage chitotriosidase (HCHT) is a chitinase that hydrolyses glycosidic bonds between the N-acetyl-D-glucosamine units of this biopolymer. HCHT belongs to the Glycoside Hydrolase (GH) superfamily and contains a well-characterized catalytic domain appended to a chitin-binding domain (ChBDCHIT1). Although its precise biological function remains unclear, HCHT has been described to be involved in innate immunity. In this study, the molecular basis for interaction with insoluble chitin as well as with soluble chito-oligosaccharides has been determined. The results suggest a new mechanism as a common binding mode for many Carbohydrate Binding Modules (CBMs). Furthermore, using a phylogenetic approach, we have analysed the modularity of HCHT and investigated the evolutionary paths of its catalytic and chitin binding domains. The phylogenetic analyses indicate that the ChBDCHIT1 domain dictates the biological function of HCHT and not its appended catalytic domain. This observation may also be a general feature of GHs. Altogether, our data have led us to postulate and discuss that HCHT acts as an immune catalyser. © 2017 The Authors. Published by Nature Publishing Group. This is an open access article licensed under a Creative Commons Attribution 4.0 International License

Substrate binding of a family GH19 chitinase from a moss species, Bryum coronatum (BcChi-A, 22 kDa), which is smaller than the 26 kDa family GH19 barley chitinase due to the lack of several loop regions (‘loopless’), was investigated by oligosaccharide digestion, thermal unfolding experiments and isothermal titration calorimetry (ITC). Chitin oligosaccharides [β-1,4-linked oligosaccharides of N-acetylglucosamine with a polymerization degree of n, (GlcNAc)n, n = 3–6] were hydrolyzed by BcChi-A at rates in the order (GlcNAc)6 > (GlcNAc)5 > (GlcNAc)4 >> (GlcNAc)3. From thermal unfolding experiments using the inactive BcChi-A mutant (BcChi-A-E61A), in which the catalytic residue Glu61 is mutated to Ala, we found that the transition temperature (Tm) was elevated upon addition of (GlcNAc)n (n = 2–6) and that the elevation (ΔTm) was almost proportional to the degree of polymerization of (GlcNAc)n. ITC experiments provided the thermodynamic parameters for binding of (GlcNAc)n (n = 3–6) to BcChi-A-E61A, and revealed that the binding was driven by favorable enthalpy changes with unfavorable entropy changes. The change in heat capacity (ΔCp°) for (GlcNAc)6 binding was found to be relatively small (−105 ± 8 cal·K−1·mol−1). The binding free energy changes for (GlcNAc)6, (GlcNAc)5, (GlcNAc)4 and (GlcNAc)3 were determined to be −8.5, −7.9, −6.6 and −5.0 kcal·mol−1, respectively. Taken together, the substrate binding cleft of BcChi-A consists of at least six subsites, in contrast to the four-subsites binding cleft of the ‘loopless’ family 19 chitinase from Streptomyces coelicolor. Database Chitinase, EC 3.2.1.14

Bacterial proteins categorized as family 33 carbohydrate-binding modules (CBM33) were recently shown to cleave crystalline chitin, using a mechanism that involves hydrolysis and oxidation. We show here that some members of the CBM33 family cleave crystalline cellulose as demonstrated by chromatographic and mass spectrometric analyses of soluble products released from Avicel or filter paper on incubation with CelS2, a CBM33-containing protein from Streptomyces coelicolor A3(2). These enzymes act synergistically with cellulases and may thus become important tools for efficient conversion of lignocellulosic biomass. Fungal proteins classified as glycoside hydrolase family 61 that are known to act synergistically with cellulases are likely to use a similar mechanism.

Polysaccharide Breakdown One of the current challenges in the biofuels industry is achieving efficient bioconversion of complex polysaccharides like cellulose and chitin. Recently, chitin-binding proteins (CBPs) have been identified that potentiate chitin hydrolysis. Now, Vaaje-Kolstad et al. (p. 219 ) show that a CBP from the chitinolytic bacterium Serratia marcescens appears to catalyze an oxygenase reaction on the surface of crystallized chitin, leading to chain breakage and generating oxidized ends that can be degraded by chitinases. A structurally similar enzyme, GH61, may play a similar role in the degradation of cellulose.

Many glycoside hydrolases, such as chitinases and cellulases, degrade polysaccharides in a processive manner. Inhibition of chitinases is of great interest, because chitin-metabolizing pathogenic organisms such as certain fungi, insects and nematodes need chitinase activity for survival. Here we show how the processivity and the directionality of two chitinases, chitinase A (ChiA) and B (ChiB) from Serratia marcescens, affects the practical inhibition efficacy (IC50) of allosamidin, a general competitive inhibitor of family 18 chitinases. The results show that there is a clear negative correlation between processivity and the efficiency of competitive inhibition, and that this effect of processivity (i.e. reducing inhibitor efficacy) is largest when allosamidin binds to those enzyme subsites that interact with the polymeric part of the substrate. Besides providing further insight into the processivity and directionality of the two Serratia enzymes, these results reveal important aspects of ligand binding that should be taken into account when designing inhibitors of processive enzymes.

Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides, such as cellulose and chitin, and are of interest in biotechnological utilization of these abundant biomaterials. It has recently been shown that LPMOs can use H2O2, instead of O2, as a cosubstrate. This peroxygenase-like reaction by a monocopper enzyme is unprecedented in nature and opens new avenues in chemistry and enzymology. Here, we provide the first detailed kinetic characterization of chitin degradation by the bacterial LPMO chitin-binding protein CBP21 using H2O2 as cosubstrate. The use of 14C-labeled chitin provided convenient and sensitive detection of the released soluble products, which enabled detailed kinetic measurements. The kcat for chitin oxidation found here (5.6 s−1) is more than an order of magnitude higher than previously reported (apparent) rate constants for reactions containing O2 but no added H2O2. The kcat/Km for H2O2-driven degradation of chitin was on the order of 106 m−1 s−1, indicating that LPMOs have catalytic efficiencies similar to those of peroxygenases. Of note, H2O2 also inactivated CBP21, but the second-order rate constant for inactivation was about 3 orders of magnitude lower than that for catalysis. In light of the observed CBP21 inactivation at higher H2O2 levels, we conclude that controlled generation of H2O2 in situ seems most optimal for fueling LPMO-catalyzed oxidation of polysaccharides.

Glycoside hydrolases play an important role in the degradation of biomass such as cellulose and chitin. Many of these enzymes act by a processive mechanism, which is generally considered favorable because it improves substrate-accessiblity. Recently we showed that this only applies to insoluble substrates. Towards more soluble and accessible substrates, processivity may in fact reduce the catalytic activity. Here, we describe kinetic studies showing how the type of substrate, insoluble or soluble, affects the activation parameters and rate determining steps for catalysis by the processive two-domain chitinase A (ChiA) from Serratia marcescens. The activation parameters show a large entropic activation barrier, indicative of a bimolecular (associative) rate determining step, for the degradation of insoluble crystalline β-chitin. For the water-soluble polymeric chitin-derivative chitosan, the rate determining step is associated with product-displacement and release. Furthermore, the degree of processivity is reflected in the activation parameters for chitosan hydrolysis; increase in processivity results in increase in activation enthalpy change and decrease in activation entropy change.

Chitooligosaccharides (CHOS) are homo- or heterooligomers of N-acetylglucosamine and D-glucosamine. CHOS can be produced using chitin or chitosan as a starting material, using enzymatic conversions, chemical methods or combinations thereof. Production of well-defined CHOS-mixtures, or even pure CHOS, is of great interest since these oligosaccharides are thought to have several interesting bioactivities. Understanding the mechanisms underlying these bioactivities is of major importance. However, so far in-depth knowledge on the mode-of-action of CHOS is scarce, one major reason being that most published studies are done with badly characterized heterogeneous mixtures of CHOS. Production of CHOS that are well-defined in terms of length, degree of N-acetylation, and sequence is not straightforward. Here we provide an overview of techniques that may be used to produce and characterize reasonably well-defined CHOS fractions. We also present possible medical applications of CHOS, including tumor growth inhibition and inhibition of T(H)2-induced inflammation in asthma, as well as use as a bone-strengthener in osteoporosis, a vector for gene delivery, an antibacterial agent, an antifungal agent, an anti-malaria agent, or a hemostatic agent in wound-dressings. By using well-defined CHOS-mixtures it will become possible to obtain a better understanding of the mechanisms underlying these bioactivities.

Binding of allosamidin to the three family 18 chitinases of Serratia marcescens has been studied using isothermal titration calorimetry (ITC). Interestingly, the thermodynamic signatures of allosamidin binding were different for all three chitinases. At pH 6.0, chitinase A (ChiA) binds allosamidin with a K(d) value of 0.17 +/- 0.06 microM where the main part of the driving force is due to enthalpic change (DeltaH(r) degrees = -6.2 +/- 0.2 kcal/mol) and less to entropic change (-TDeltaS(r) degrees = -3.2 kcal/mol). A large part of DeltaH is due to allosamidin stacking with Trp(167) in the -3 subsite. Binding of allosamidin to both chitinase B (ChiB) (K(d) = 0.16 +/- 0.04 microM) and chitinase C (ChiC) (K(d) = 2.0 +/- 0.2 microM) is driven by entropy (DeltaH(r) degrees = 3.8 +/- 0.2 kcal/mol and -TDeltaS(r) degrees = -13.2 kcal/mol for ChiB and DeltaH(r) degrees = -0.6 +/- 0.1 and -TDeltaS(r) degrees = -7.3 kcal/mol for ChiC). For ChiC, the entropic term is dominated by changes in solvation entropy (DeltaS(conf) = 1 cal/K.mol and DeltaS(solv) = 31 cal/K.mol), while, for ChiB, changes in conformational entropy dominate (DeltaS(conf) = 37 cal/K x mol and DeltaS(solv) = 15 cal/K x mol). Corresponding values for ChiA are DeltaS(conf) = 4 cal/K x mol and DeltaS(solv) = 15 cal/K x mol. These remarkable differences in binding parameters reflect the different architectures of the catalytic centers in these enzymes that are adapted to different types of actions: ChiA and ChiB are processive enzymes that move in opposite directions, meaning that allosamidin binds in to "product" subsites in ChiB, while it binds to polymer-binding subsites in ChiA. The values for ChiC are compatible with this enzyme being a nonprocessive endochitinase with a much more open and solvated substrate-binding-site cleft.

The determination of kinetic parameters of chitinases using natural substrates is difficult due to low K(m) values, which require the use of low substrate concentrations that are hard to measure. Using the natural substrate (GlcNAc)(4), we have developed an assay for the determination of k(cat) and K(m)values of chitinases. Product concentrations as low as 0.5 microM were detected using normal-phase high-performance liquid chromatography (HPLC) with an amide 80 column (0.20 x 25 cm) using spectrophotometric detection at 210 nm. By means of this assay, k(cat) and K(m)values for chitinases A (ChiA) and B (ChiB) of Serratia marcescens were found to be 33+/-1s(-1) and 9+/-1 microM and 28+/-2s(-1) and 4+/-2 microM, respectively. For ChiB, these values were compared to those found with commonly used substrates where the leaving group is a (nonnatural) chromophore, revealing considerable differences. For example, assays with 4-methylumbelliferyl-(GlcNAc)(2) yielded a k(cat) value of 18+/-2s(-1) and a K(m) value of 30+/-6 microM. For two ChiB mutants containing a Trp --> Ala mutation in the +1 or +2 subsites, the natural substrate and the 4-methylumbelliferyl-(GlcNAc)(2) assays yielded rather similar K(m) values (5-fold difference at most) but showed dramatic differences in k(cat) values (up to 90-fold). These results illustrate the risk of using artificial substrates for characterization of chitinases and, thus, show that the new HPLC-based assay is a valuable tool for future chitinase research.

Human chitotriosidase (HCHT) is a glycoside hydrolase family 18 chitinase synthesized and secreted in human macrophages thought be an innate part of the human immune system. It consists of a catalytic domain with the (β/α)8 TIM barrel fold having a large area of solvent-exposed aromatic amino acids in the active site and an additional family 14 carbohydrate-binding module. To gain further insight into enzyme functionality, especially the effect of the active site aromatic residues, we expressed two variants with mutations in subsites on either side of the catalytic acid, subsite -3 (W31A) and +2 (W218A), and compared their catalytic properties on chitin and high molecular weight chitosans. Exchange of Trp to Ala in subsite -3 resulted in a 12-fold reduction in extent of degradation and a 20-fold reduction in kcat(app) on chitin, while the values are 5-fold and 10-fold for subsite +2. Moreover, aromatic residue mutation resulted in a decrease of the rate of chitosan degradation contrasting previous observations for bacterial family 18 chitinases. Interestingly, the presence of product polymers of 40 sugar moieties and higher starts to disappear already at 8% degradation for HCHT50-W31A. Such behavior contrast that of the wild type and HCHT-W218A and resembles the action of endo-nonprocessive chitinases.

Contains fulltext : 35967.pdf (Publisher’s version ) (Closed access) Many enzymes that hydrolyze insoluble crystalline polysaccharides such as cellulose and chitin guide detached single-polymer chains through long and deep active-site clefts, leading to processive (stepwise) degradation of the polysaccharide. We have studied the links between enzyme efficiency and processivity by analyzing the effects of mutating aromatic residues in the substrate-binding groove of a processive chitobiohydrolase, chitinase B from Serratia marcescens. Mutation of two tryptophan residues (Trp-97 and Trp-220) close to the catalytic center (subsites +1 and +2) led to reduced processivity and a reduced ability to degrade crystalline chitin, suggesting that these two properties are linked. Most remarkably, the loss of processivity in the W97A mutant was accompanied by a 29-fold increase in the degradation rate for single-polymer chains as present in the soluble chitin-derivative chitosan. The properties of the W220A mutant showed a similar trend, although mutational effects were less dramatic. Processivity is thought to contribute to the degradation of crystalline polysaccharides because detached single-polymer chains are kept from reassociating with the solid material. The present results show that this processivity comes at a large cost in terms of enzyme speed. Thus, in some cases, it might be better to focus strategies for enzymatic depolymerization of polysaccharide biomass on improving substrate accessibility for nonprocessive enzymes rather than on improving the properties of processive enzymes.

Serratia marcescens produces three chitinases, ChiA, ChiB and ChiC which together enable the bacterium to efficiently degrade the insoluble chitin polymer. We present an overview of the structural properties of these enzymes, as well as an analysis of their activities towards artificial chromogenic chito-oligosaccharide-based substrates, chito-oligosaccharides, chitin and chitosan. We also present comparative inhibition data for the pseudotrisaccharide allosamidin (an analogue of the reaction intermediate) and the cyclic pentapeptide argadin. The results show that the enzymes differ in terms of their subsite architecture and their efficiency towards chitinous substrates. The idea that the three chitinases play different roles during chitin degradation was confirmed by the synergistic effects that were observed for certain combinations of the enzymes. Studies of the degradation of the soluble heteropolymer chitosan provided insight into processivity. Taken together, the available data for Serratia chitinas...

Processive glycoside hydrolases are the key components of enzymatic machineries that decompose recalcitrant polysaccharides, such as chitin and cellulose. The intrinsic processivity (P(Intr)) of cellulases has been shown to be governed by the rate constant of dissociation from polymer chain (koff). However, the reported koff values of cellulases are strongly dependent on the method used for their measurement. Here, we developed a new method for determining koff, based on measuring the exchange rate of the enzyme between a non-labeled and a (14)C-labeled polymeric substrate. The method was applied to the study of the processive chitinase ChiA from Serratia marcescens. In parallel, ChiA variants with weaker binding of the N-acetylglucosamine unit either in substrate-binding site -3 (ChiA-W167A) or the product-binding site +1 (ChiA-W275A) were studied. Both ChiA variants showed increased off-rates and lower apparent processivity on α-chitin. The rate of the production of insoluble reducing groups on the reduced α-chitin was an order of magnitude higher than koff, suggesting that the enzyme can initiate several processive runs without leaving the substrate. On crystalline chitin, the general activity of the wild type enzyme was higher, and the difference was magnifying with hydrolysis time. On amorphous chitin, the variants clearly outperformed the wild type. A model is proposed whereby strong interactions with polymer in the substrate-binding sites (low off-rates) and strong binding of the product in the product-binding sites (high pushing potential) are required for the removal of obstacles, like disintegration of chitin microfibrils.

The enzymatic degradation of the closely related insoluble polysaccharides; cellulose (β(1–4)-linked glucose) by cellulases and chitin (β(1–4)-linked N-acetylglucosamine) by chitinases, is of large biological and economical importance. Processive enzymes with different inherent directionalities, i.e. attacking the polysaccharide chains from opposite ends, are crucial for the efficiency of this degradation process. While processive cellulases with complementary functions differ in structure and catalytic mechanism, processive chitinases belong to one single protein family with similar active site architectures. Using the unique model system of Serratia marcescens with two processive chitinases attacking opposite ends of the substrate, we here show that different directionalities of processivity are correlated to distinct differences in the kinetic signatures for hydrolysis of oligomeric tetra-N-acetyl chitotetraose.

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 A (ScLPMO10B) and 1.5 A (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.

Polysaccharide depolymerization in nature is primarily accomplished by processive glycoside hydrolases (GHs), which abstract single carbohydrate chains from polymer crystals and cleave glycosidic linkages without dissociating after each catalytic event. Understanding the molecular-level features and structural aspects of processivity is of importance due to the prevalence of processive GHs in biomass-degrading enzyme cocktails. Here, we describe recent advances towards the development of a molecular-level theory of processivity for cellulolytic and chitinolytic enzymes, including the development of novel methods for measuring rates of key steps in processive action and insights gained from structural and computational studies. Overall, we present a framework for developing structure-function relationships in processive GHs and outline additional progress towards developing a fundamental understanding of these industrially important enzymes.

Chitosan is a linear heteropolymer consisting of β 1,4-linked N-acetyl-D-glucosamine (GlcNAc) and D-glucosamine (GlcN). We have compared the antifungal activity of chitosan with DPn (average degree of polymerization) 206 and F A (fraction of acetylation) 0.15 and of enzymatically produced chito-oligosaccharides (CHOS) of different DPn alone and in combination with commercially available synthetic fungicides, against Botrytis cinerea, the causative agent of gray mold in numerous fruit and vegetable crops. CHOS with DPn in the range of 15–40 had the greatest anti-fungal activity. The combination of CHOS and low dosages of synthetic fungicides showed synergistic effects on antifungal activity in both in vitro and in vivo assays. Our study shows that CHOS enhance the activity of commercially available fungicides. Thus, addition of CHOS, available as a nontoxic byproduct of the shellfish industry, may reduce the amounts of fungicides that are needed to control plant diseases.

Human chitotriosidase (HCHT) is involved in immune response to chitin-containing pathogens in humans. The enzyme is able to degrade chitooligosaccharides as well as crystalline chitin. The catalytic domain of HCHT is connected to the carbohydrate binding module (CBM) through a flexible hinge region. In humans, two active isoforms of HCHT are found-the full length enzyme and its truncated version lacking CBM and the hinge region. The active site architecture of HCHT is reminiscent to that of the reducing-end exo-acting processive chitinase ChiA from bacterium Serratia marcescens (SmChiA). However, the presence of flexible hinge region and occurrence of two active isoforms are reminiscent to that of non-processive endo-chitinase from S. marcescens, SmChiC. Although the studies on soluble chitin derivatives suggest the endo-character of HCHT, the mode of action of the enzyme on crystalline chitin is not known. Here, we made a thorough characterization of HCHT in terms of the mode of action, processivity, binding, and rate constants for the catalysis and dissociation using α-chitin as substrate. HCHT efficiently released the end-label from reducing-end labelled chitin and had also high probability (95%) of endo-mode initiation of processive run. These results qualify HCHT as an endo-processive enzyme. Processivity and the rate constant of dissociation of HCHT were found to be in-between those, characteristic to processive exo-enzymes, like SmChiA and randomly acting non-processive endo-enzymes, like SmChiC. Apart from increasing the affinity for chitin, CBM had no major effect on kinetic properties of HCHT.

The chitinolytic machinery of Serratia marcescens is one of the best known enzyme systems for the conversion of insoluble polysaccharides. This machinery includes four chitin-active enzymes: ChiC, an endo-acting non-processive chitinase; ChiA and ChiB, two processive chitinases moving along chitin chains in opposite directions; and CBP21, a surface-active CBM33-type lytic polysaccharide monooxygenase that introduces chain breaks by oxidative cleavage. Furthermore, an N-acetylhexosaminidase or chitobiase converts the oligomeric products from the other enzymes to monomeric N-acetylglucosamine. Here we discuss the catalytic mechanisms of these enzymes as well as the structural basis of each enzyme's specific role in the chitin degradation process. We also discuss how knowledge of this enzyme system may be extrapolated to other enzyme systems for conversion of insoluble polysaccharides, in particular conversion of cellulose by cellulases and GH61-type lytic polysaccharide monooxygenases.

Natural cellulolytic enzyme systems as well as leading commercial cellulase cocktails are dominated by enzymes that degrade cellulose chains in a processive manner. Despite the abundance of processivity among natural cellulases, the molecular basis as well as the biotechnological implications of this mechanism are only partly understood. One of the major limitations lies in the fact that it is not straightforward to measure and quantify processivity in what essentially are biphasic experimental systems. Here, we describe and discuss both well-established methods and newer methods for measuring cellulase processivity. In addition, we discuss recent insights from studies on chitinases that may help direct further studies on processivity in cellulases.

Chitooligosaccharides (CHOS) inhibit angiogenesis and may be used in the treatment of cancer tumors. We have studied the effect of the fraction of acetylation (FA) and the degree of polymerization (DP) on CHOS anti-angiogenic activity. We tested enzymatically produced CHOS-mixtures with FA0.15, FA0.3 and FA0.6, and DP≤12 in initial experiments with chorioallantoic membranes. All of the samples reduced the formation of new blood vessels, CHOS with FA0.3 giving the best effect. Single-DP fractions from the FA0.3 sample purified by size-exclusion chromatography (DP3-DP12) were then tested for inhibition of migration of human endothelial cells, which is an important element of the angiogenesis process. All of the fractions inhibited migration, meaning that, within the DP area tested in this study, FA is more important than DP for the effect. Generally, the results reveal that DP3-DP12 CHOS have considerable potential as anti-angiogenic compounds.

Enzymatic depolymerisation of polysaccharides is a key technology in the biorefining of biomass. The enzymatic conversion of the abundant insoluble polysaccharides cellulose and chitin is of particular interest and complexity, because of the bi-phasic nature of the process, the seemingly complicated tasks faced by the enzymes, and the importance of these conversions for the future biorefinery. Here we review recent work on family 18 chitinases that sheds light on important aspects of the catalytic action of these depolymerising enzymes, including the structural basis of processivity and its direction, the energies involved in substrate-binding and displacement.

Chitotriosidase (HCHT) is one of two family 18 chitinases produced by humans, the other being acidic mammalian chitinase (AMCase). The enzyme is thought to be part of the human defense mechanism against fungal parasites, but its precise role and the details of its enzymatic properties have not yet been fully unraveled. We have studied the properties of HCHT by analyzing how the enzyme acts on high-molecular weight chitosans, soluble copolymers of β-1,4-linked N-acetylglucosamine (GlcNAc, A), and glucosamine (GlcN, D). Using methods for in-depth studies of the chitinolytic machinery of bacterial family 18 enzymes, we show that HCHT degrades chitosan primarily via an endoprocessive mechanism, as would be expected on the basis of the structural features of its substrate-binding cleft. The preferences of HCHT subsites for acetylated versus nonacetylated sugars were assessed by sequence analysis of obtained oligomeric products showing a very strong, absolute, and a relative weak preference for an acetylated unit in the −2, −1, and +1 subsites, respectively. The latter information is important for the design of inhibitors that are specific for the human chitinases and also provides insight into what kind of products may be formed in vivo upon administration of chitosan-containing medicines or food products. Copyright © 2012 American Chemical Society. This is the authors’ accepted and refereed manuscript to the article.

The processive Serratia marcescens chitinases A (ChiA) and B (ChiB) are thought to degrade chitin in the opposite directions. A recent study of ChiB suggested that processivity is governed by aromatic residues in the +1 and +2 (aglycon) subsites close to the catalytic center. To further investigate the roles of aromatic residues in processivity and to gain insight into the structural basis of directionality, we have mutated Trp167, Trp275, and Phe396 in the -3, +1, and +2 subsites of ChiA, respectively, and characterized the hydrolytic activities of the mutants toward β-chitin and the soluble chitin-derivative chitosan. Although the W275A and F396A mutants showed only modest reductions in processivity, it was almost abolished by the W167A mutation. Thus, although aglycon subsites seem to steer processivity in ChiB, a glycon (-3) subsite seems to be adapted to do so in ChiA, in line with the notion that the two enzymes have different directionalities. Remarkably, whereas all three single mutants and the W167A/W275A double mutant showed reduced efficiency toward chitin, they showed up to 20-fold higher activities toward chitosan. These results show that the processive mechanism is essential for an efficient conversion of crystalline substrates but comes at a large cost in terms of intrinsic enzyme speed. This needs to be taken into account when devising enzymatic strategies for biomass turnover.

We present a comparative study of ChiA, ChiB, and ChiC, the three family 18 chitinases produced by Serratia marcescens. All three enzymes eventually converted chitin to N-acetylglucosamine dimers (GlcNAc2) and a minor fraction of monomers. ChiC differed from ChiA and ChiB in that it initially produced longer oligosaccharides from chitin and had lower activity towards an oligomeric substrate, GlcNAc6. ChiA and ChiB could convert GlcNAc6 directly to three dimers, whereas ChiC produced equal amounts of tetramers and dimers, suggesting that the former two enzymes can act processively. Further insight was obtained by studying degradation of the soluble, partly deacetylated chitin-derivative chitosan. Because there exist nonproductive binding modes for this substrate, it was possible to discriminate between independent binding events and processive binding events. In reactions with ChiA and ChiB the polymer disappeared very slowly, while the initially produced oligomers almost exclusively had even-numbered chain lengths in the 2-12 range. This demonstrates a processive mode of action in which the substrate chain moves by two sugar units at a time, regardless of whether complexes formed along the way are productive. In contrast, reactions with ChiC showed rapid disappearance of the polymer and production of a continuum of odd- and even-numbered oligomers. These results are discussed in the light of recent literature data on directionality and synergistic effects of ChiA, ChiB and ChiC, leading to the conclusion that ChiA and ChiB are processive chitinases that degrade chitin chains in opposite directions, while ChiC is a nonprocessive endochitinase.