Endo/exo mechanism and processivity of family 18 chitinases produced by Serratia marcescens

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.     

Hallmarks of Processivity in Glycoside Hydrolases from Crystallographic and Computational Studies of the Serratia marcescens Chitinases

Degradation of recalcitrant polysaccharides in nature is typically accomplished by mixtures of processive and nonprocessive glycoside hydrolases (GHs), which exhibit synergistic activity wherein nonprocessive enzymes provide new sites for productive attachment of processive enzymes. GH processivity is typically attributed to active site geometry, but previous work has demonstrated that processivity can be tuned by point mutations or removal of single loops. To gain additional insights into the differences between processive and nonprocessive enzymes that give rise to their synergistic activities, this study reports the crystal structure of the catalytic domain of the GH family 18 nonprocessive endochitinase, ChiC, from Serratia marcescens. This completes the structural characterization of the co-evolved chitinolytic enzymes from this bacterium and enables structural analysis of their complementary functions. The ChiC catalytic module reveals a shallow substrate-binding cleft that lacks aromatic residues vital for processivity, a calcium-binding site not previously seen in GH18 chitinases, and, importantly, a displaced catalytic acid (Glu-141), suggesting flexibility in the catalytic center. Molecular dynamics simulations of two processive chitinases (ChiA and ChiB), the ChiC catalytic module, and an endochitinase from Lactococcus lactis show that the nonprocessive enzymes have more flexible catalytic machineries and that their bound ligands are more solvated and flexible. These three features, which relate to the more dynamic on-off ligand binding processes associated with nonprocessive action, correlate to experimentally measured differences in processivity of the S. marcescens chitinases. These newly defined hallmarks thus appear to be key dynamic metrics in determining processivity in GH enzymes complementing structural insights.     

Serratia marcescens chitinases with tunnel-shaped substrate-binding grooves show endo activity and different degrees of processivity during enzymatic hydrolysis of chitosan

The modes of action of three family 18 chitinases (ChiA, ChiB, and ChiC) from Serratia marcescens during the degradation of a water-soluble polymeric substrate, chitosan, were investigated using a combination of viscosity measurements, reducing end assays, and characterization of the size-distribution of the oligomeric products. All three enzymes yielded a fast reduction in molecular weight of the chitosan substrate at a very early stage of hydrolysis, which is typical for endo-acting enzymes. For ChiA and ChiB, this is inconsistent with the previously proposed exo-attack mode of action. The main difference between ChiA, ChiB, and ChiC is the degree of processivity. ChiC is an endo enzyme with no apparent processivity. ChiA and ChiB are processive enzymes in which the substrate remains bound to the active cleft after successful hydrolysis and is moved along for the next hydrolysis to occur. ChiA and ChiB perform on average 9.1 and 3.4 cleavages, respectively, for the formation of each enzyme-substrate complex. ChiA and ChiB have deep, tunnel-like substrate-binding grooves. The demonstration of endo activity shows that substrate binding must involve the temporary restructuring of the loops that make up the roofs of the substrate-binding grooves, similar to what has been proposed for cellobiohydrolase Cel6A. The data suggest that the exo-type of activity observed for ChiA and ChiB during the degradation of solid crystalline chitin is due to the better accessibility of chain ends, rather than intrinsic enzyme properties.     

Enzyme processivity changes with the extent of recalcitrant polysaccharide degradation

Glycoside hydrolases depolymerize polysaccharides. They can subtract single carbohydrate chains from polymer crystals and cleave glycosidic bonds without dissociating from the substrate after each catalytic event. This processivity is thought to conserve energy during polysaccharide degradation. Herein, we compare the processivity of components of the chitinolytic machinery of Serratia marcescens. The two processive chitinases ChiA and ChiB, the ChiB-W97A mutant, and the endochitinase ChiC were analyzed for the extent of degradation of three different chitin substrates. Moreover, enzyme processivity was assessed on the basis of the [(GlcNAc)2]/[GlcNAc] product ratio. 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.     

Potentiation of the synergistic activities of chitinases ChiA, ChiB and ChiC from Serratia marcescens CFFSUR-B2 by chitobiase (Chb) and chitin binding protein (CBP)

With the goal of understanding the chitinolytic mechanism of the potential biological control strain Serratia marcescens CFFSUR-B2, genes encoding chitinases ChiA, ChiB and ChiC, chitobiase (Chb) and chitin binding protein (CBP) were cloned, the protein products overexpressed in Escherichia coli as 6His-Sumo fusion proteins and purified by affinity chromatography. Following affinity tag removal, the chitinolytic activity of the recombinant proteins was evaluated individually and in combination using colloidal chitin as substrate. ChiB and ChiC were highly active while ChiA was inactive. Reactions containing both ChiB and ChiC showed significantly increased N-acetylglucosamine trimer and dimer formation, but decreased monomer formation, compared to reactions with either enzyme alone. This suggests that while both ChiB and ChiC have a general affinity for the same substrate, they attack different sites and together degrade chitin more efficiently than either enzyme separately. Chb and CBP in combination with ChiB and ChiC (individually or together) increased their chitinase activity. We report for the first time the potentiating effect of Chb on the activity of the chitinases and the synergistic activity of a mixture of all five proteins (the three chitinases, Chb and CBP). These results contribute to our understanding of the mechanism of action of the chitinases produced by strain CFFSUR-B2 and provide a molecular basis for its high potential as a biocontrol agent against fungal pathogens.     

Chitinases A,B, and C1 of Serratia marcescens 2170 produced by recombinant Escherichia coli: enzymatic properties and synergism on chitin degradation

To discover the individual roles of the chitinases from Serratia marcescens 2170, chitinases A, B, and C1 (ChiA, ChiB, and ChiC1) were produced by Escherichia coli and their enzymatic properties as well as synergistic effect on chitin degradation were studied. All three chitinases showed a broad pH optimum and maintained significant chitinolytic activity between pH 4 and 10. ChiA was the most active enzyme toward insoluble chitins, but ChiC1 was the most active toward soluble chitin derivatives among the three chitinases. Although all three chitinases released (GlcNAc)2 almost exclusively from colloidal chitin, ChiB and ChiC1 split (GlcNAc)6 to (GlcNAc)3, while ChiA exclusively generated (GlcNAc)2 and (GlcNAc)4. Clear synergism on the hydrolysis of powdered chitin was observed in the combination between ChiA and either ChiB or ChiC, and the sites attacked by ChiA on the substrate are suggested to be different from those by either ChiB or ChiC1.     

Comparative studies of chitinases A,B and C from Serratia marcescens

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 chitinases show that the chitinolytic machinery of this bacterium consists of two processive exo-enzymes that degrade the chitin chains in opposite directions (ChiA and ChiB) and a non-processive endo-enzyme, ChiC.     

Characterization of a cold-adapted and salt-tolerant exo-chitinase (ChiC) from <Emphasis Type="Italic">Pseudoalteromonas</Emphasis> sp. DL-6

We have previously reported a non-processive endo-type chitinase, ChiA, from a newly isolated marine psychrophilic bacterium, Pseudoalteromonas sp. DL-6. In this study, a processive exo-type chitinase, ChiC, was cloned from the same bacterium and characterized in detail. ChiC could hydrolyze crystalline chitin into (GlcNAc)₂ as the only observed product. It exhibited high catalytic activity even at low temperatures, e.g. close to 0 °C, or in the presence of 5 M NaCl, suggesting that ChiC was a cold-adapted and highly salt-tolerant chitinase. ChiC could also hydrolyze other substrates, including chitosan and Avicel, indicating its broad substrate specificity. Sequence features indicated that ChiC was a multi-domain protein having a deep substrate-binding groove that was regarded as characteristic of processive exo-chitinases. Enzymatic hydrolysis of chitin by ChiC could be remarkably boosted in the presence of ChiA, suggesting the synergy of ChiC and ChiA. This work provided a new evidence to prove that marine psychrophilic bacteria utilized a synergistic enzyme system to degrade recalcitrant chitin.     

Comparative studies of chitinases A, B and C from Serratia marcescens

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 chitinases show that the chitinolytic machinery of this bacterium consists of two processive exo-enzymes that degrade the chitin chains in opposite directions (ChiA and ChiB) and a non-processive endo-enzyme, ChiC.     

Extracellular chitinases of mutant superproducing strain <Emphasis Type="Italic">Serratia marcescens</Emphasis> M-1

Four extracellular proteins with chitinase activity capable of binding chitin substrates have been revealed in the culture liquid of chitinase superproducing mutant strain M-1 of Serratia marcescens. Proteins were analyzed by SDS-PAGE and MALDI-TOF mass spectrometry. Based on the data obtained, the proteins were identified as typical chitinases of S. marcescens: ChiA, ChiB, ChiC, and CBP21.     

Expression and characterization of the chitinases from Serratia marcescens GEI strain for the control of Varroa destructor, a honey bee parasite

Serratia marcescens GEI strain was isolated from the gut of the workers of Chinese honey bee Apis cerana and evaluated in the laboratory for the control of Varroa destructor, a parasite of western honey bee A. mellifera. The supernatant and the collected proteins by ammonium sulfate from the bacterial cultures showed a strong miticidal effect on the female mites, with 100% mite mortality in 5 days. Heat (100 °C for 10 min) and proteinase K treatment of the collected proteins destroyed the miticidal activity. The improved miticial activity of this bacterial strain on chitin medium indicated the involvement of chitinases. The expressed chitinases ChiA, ChiB and ChiC1 from S. marcescens GEI by recombinant Escherichia coli showed pathogenicity against the mites in the laboratory. These chitinases were active in a broad pH range (5-9) and the optimum temperatures were between 60 and 75 °C. Synergistic effects of ChiA and ChiB on the miticidal activity against V. destructor were observed. The workers of both honey bee species were not sensitive to the spraying and feeding chitinases. These results provided alternative control strategies for Varroa mites, by formulating chitinase agents and by constructing transgenetic honey bees.     

Characterization of Chitinase Genes from an Alkaliphilic Actinomycete, Nocardiopsis prasina OPC-131

An alkaliphilic actinomycete, Nocardiopsis prasina OPC-131, secretes chitinases, ChiA, ChiB, and ChiBΔ, in the presence of chitin. The genes encoding ChiA and ChiB were cloned and sequenced. The open reading frame (ORF) of chiA encoded a protein of 336 amino acids with a calculated molecular mass of 35,257 Da. ChiA consisted of only a catalytic domain and showed a significant homology with family 18 chitinases. The chiB ORF encoded a protein of 296 amino acids with a calculated molecular mass of 31,500 Da. ChiB is a modular enzyme consisting of a chitin-binding domain type 3 (ChtBD type 3) and a catalytic domain. The catalytic domain of ChiB showed significant similarity to Streptomyces family 19 chitinases. ChiBΔ was the truncated form of ChiB lacking ChtBD type 3. Expression plasmids coding for ChiA, ChiB, and ChiBΔ were constructed to investigate the biochemical properties of these recombinant proteins. These enzymes showed pHs and temperature optima similar to those of native enzymes. ChiB showed more efficient hydrolysis of chitin and stronger antifungal activity than ChiBΔ, indicating that the ChtBD type 3 of ChiB plays an important role in the efficient hydrolysis of chitin and in antifungal activity. Furthermore, the finding of family 19 chitinase in N. prasina OPC-131 suggests that family 19 chitinases are distributed widely in actinomycetes other than the genus Streptomyces.     

NMR spectroscopy and computational analysis of interaction between Serratia marcescens chitinase B and a dipeptide derived from natural-product cyclopentapeptide chitinase inhibitor argifin

The dipeptide N-acetyl-Arg{N^ω-(N-methylcarbamoyl)}-N-methyl-Phe(2), which is a part of the natural-product cyclopentapeptide chitinase inhibitor argifin (1), inhibits chitinase B from Serratia marcescens (SmChiB) with a half-maximal inhibitory concentration (IC₅₀) of 3.7 μM. Despite the relatively small size of 2, its inhibitory activity is comparable with that of 1 (IC₅₀ = 6.4 μM). To elucidate the basis for this interesting phenomenon, we investigated the interaction between 2 and SmChiB using a combination of nuclear magnetic resonance spectroscopy and computational methods. The transferred nuclear Overhauser effect (TRNOE) experiment obtained structural information on the SmChiB-bound conformation of 2. The binding mode of 2 and SmChiB was modeled by the novel molecular-docking approach proposed in our laboratory, which can explicitly consider water-mediated hydrogen-bonding interactions in protein-ligand interfaces. The SmChiB-bound conformation of 2 in the resulting model satisfied all proton-proton distance constraints derived from the TRNOE experiment, indicating that our model structure of the 2-SmChiB complex is reasonable. A molecular dynamics (MD) simulation examined the stability of the resultant complex structure and suggested that 2 binds to SmChiB in a similar fashion to the binding mode observed for N^ω-(N-methylcarbamoyl)-Arg(1) and N-methyl-Phe(2) of 1 in the crystal structure of the argifin–SmChiB complex. Finally, the binding free energies of 1 and 2 with SmChiB were estimated by the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) method using the MD trajectory. The MM-PBSA calculation suggested that both 1 and 2 bind to SmChiB with similar affinities, which is consistent with their experimental IC₅₀ values. Energetic analysis revealed that the van der Waals interaction of 2 with SmChiB is much less than that of 1, but is completely compensated by the more favorable contribution of solute entropy and the total electrostatic component. The improved total electrostatic component was derived from more favorable electrostatic interactions. Therefore, we conclude that dipeptide 2 was also better optimized against SmChiB than 1 in an electrostatic point of view.     

Fully Deacetylated Chitooligosaccharides Act as Efficient Glycoside Hydrolase Family 18 Chitinase Inhibitors

Small molecule inhibitors against chitinases have potential applications as pesticides, fungicides, and antiasthmatics. Here, we report that a series of fully deacetylated chitooligosaccharides (GlcN)_2–7 can act as inhibitors against the insect chitinase OfChtI, the human chitinase HsCht, and the bacterial chitinases SmChiA and SmChiB with IC₅₀ values at micromolar to millimolar levels. The injection of mixed (GlcN)_2–7 into the fifth instar larvae of the insect Ostrinia furnacalis resulted in 85% of the larvae being arrested at the larval stage and death after 10 days, also suggesting that (GlcN)_2–7 might inhibit OfChtI in vivo. Crystal structures of the catalytic domain of OfChtI (OfChtI-CAD) complexed with (GlcN)_5,6 were obtained at resolutions of 2.0 Å. These structures, together with mutagenesis and thermodynamic analysis, suggested that the inhibition was strongly related to the interaction between the −1 GlcN residue of the inhibitor and the catalytic Glu¹⁴⁸ of the enzyme. Structure-based comparison showed that the fully deacetylated chitooligosaccharides mimic the substrate chitooligosaccharides by binding to the active cleft. This work first reports the inhibitory activity and proposed inhibitory mechanism of fully deacetylated chitooligosaccharides. Because the fully deacetylated chitooligosaccharides can be easily derived from chitin, one of the most abundant materials in nature, this work also provides a platform for developing eco-friendly inhibitors against chitinases.     

Growth of Hyperthermophilic Archaeon Pyrococcus furiosus on Chitin Involves Two Family 18 Chitinases

Pyrococcus furiosus was found to grow on chitin, adding this polysacharide to the inventory of carbohydrates utilized by this hyperthermophilic archaeon. Accordingly, two open reading frames (chiA [Pf1234] and chiB [Pf1233]) were identified in the genome of P. furiosus, which encodes chitinases with sequence similarity to proteins from the glycosyl hydrolase family 18 in less-thermophilic organisms. Both enzymes contain multiple domains that consist of at least one binding domain and one catalytic domain. ChiA (ca. 39 kDa) contains a putative signal peptide, as well as a binding domain (ChiA_BD), that is related to binding domains associated with several previously studied bacterial chitinases. chiB, separated by 37 nucleotides from chiA and in the same orientation, encodes a polypeptide with two different proline-threonine-rich linker regions (6 and 3 kDa) flanking a chitin-binding domain (ChiB_BD [11 kDa]), followed by a catalytic domain (ChiB_cat [35 kDa]). No apparent signal peptide is encoded within chiB. The two chitinases share little sequence homology to each other, except in the catalytic region, where both have the catalytic glutamic acid residue that is conserved in all family 18 bacterial chitinases. The genes encoding ChiA, without its signal peptide, and ChiB were cloned and expressed in Escherichia coli. ChiA exhibited no detectable activity toward chitooligomers smaller than chitotetraose, indicating that the enzyme is an endochitinase. Kinetic studies showed that ChiB followed Michaelis-Menten kinetics toward chitotriose, although substrate inhibition was observed for larger chitooligomers. Hydrolysis patterns on chitooligosaccharides indicated that ChiB is a chitobiosidase, processively cleaving off chitobiose from the nonreducing end of chitin or other chitooligomers. Synergistic activity was noted for the two chitinases on colloidal chitin, indicating that these two enzymes work together to recruit chitin-based substrates for P. furiosus growth. This was supported by the observed growth on chitin as the sole carbohydrate source in sulfur-free media.     

Chitinolytic activity of phylogenetically diverse Bacillus cereus sensu lato from natural environments

Bacillus cereus s.l., Gram-positive endospore-forming bacilli, persist ubiquitously in different natural habitats and play various ecological roles. Nevertheless, although chitin is one of the most abundant polymer on Earth, the study of the ability of B. cereus s.l. to hydrolyze this polymer were limited to individual B. cereus and B. thuringiensis strains only. Thus, to fill this gap in this research we focused on (i) the linkage between the capability to chitin degradation and the phylogenetic relatedness of B. cereus s.l. strains, and (ii) the genetic background of chitinolytic properties of these bacilli. Our results showed that chitin degradation is common among the B. cereus group members, yet strains clustered into particular phylogenetic groups differ in their chitinolytic capacity. Separate clustering of chitinolytic and non-chitinolytic strains in the phylogenetic tree indicates the ecotypic structure of these isolates. Two proteins belonging to subfamily A (ChiA) and subfamily B (ChiB) of the glycoside hydrolase GH18 family exhibited simultaneous chitobiosidase and endochitinase activities, and are responsible for chitin utilization by environmental B. cereus s.l. isolates.     

Molecular cloning of the gene encoding a novel beta-N-acetylhexosaminidase from a marine bacterium, Alteromonas sp. strain O-7, and characterization of the cloned enzyme

We have reported that the chitinolytic system of Alteromonas sp. strain O-7 consists of chitinases (ChiA, ChiB, and ChiC), a chitinase-like enzyme (ChiD), beta-N-acetylglucosaminidases (GlcNAcasesA, GlcNAcaseB, and GlcNAcaseC), and a novel transglycosylative enzyme (Hex99). The gene encoding a beta-hexosaminidase with an unusual substrate specificity (hex86), located upstream of the hex99 gene, was cloned and sequenced. The gene encoded a protein of 761 amino acids with a calculated molecular mass of 86,758 Da. The deduced amino acid sequence of Hex86 showed sequence similarity with beta-hexosaminidases belonging to family 20. The hex86 gene was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. The enzyme rapidly cleaved p-nitrophenyl-beta-N-acetyl-D-glucosaminide and slowly cleaved p-nitrophenyl-beta-N-acetyl-D-galactosaminide. Unexpectedly, the enzyme did not hydrolyzed chitin oligosaccharides under the assay conditions for synthetic glycosides. However, after prolonged incubation with excessive quantities of the enzyme, Hex86 hydrolyzed chitin oligosaccharides. These results indicate that Hex86 is a novel enzyme that prefers p-nitrophenyl-beta-N-acetyl-D-glucosaminide to chitin oligosaccharides as a substrate.     

Kinetic studies on the hydrolysis of N-acetylated and N-deacetylated derivatives of 4-methylumbelliferyl chitobioside by the family 18 chitinases ChiA and ChiB from Serratia marcescens

Kinetic analyses of the hydrolysis reactions of N-acetylated and N-deacetylated derivatives of 4-methylumbelliferyl chitobioside [(GlcNAc)(2)-UMB (1), GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)(2)-UMB (4)] by ChiA and ChiB from Serratia marcescens were performed. Both enzymes released UMB from all compounds apart from 4. The S-v curves of the hydrolyses of 1 by ChiA and ChiB both exhibited atypical kinetic patterns, and the shapes of the two S-v curves were different from one another. However, both curve shapes were explained by assuming some of the enzyme present formed complexes with multiple molecules of the substrate. Conversely, the S-v curves generated in the cleavage of 2 and 3 by ChiA exhibited typical Michaelis-Menten profiles. Both enzymes hydrolysed 2 with an approximately 14-fold higher K(m) value relative to 1, indicating that the N-acetyl group was recognised at the -2 subsite. The k(cat) value obtained with ChiA was identical to the k(cat) value observed for 1. However, the k(cat) value for ChiB was one-fourth that of 1, suggesting that the removal of the N-acetyl group caused an increase in the formation of a non-productive ES-complex. ChiA and ChiB hydrolysed 3 with 5- and 20-fold greater K(m) values relative to 1, respectively, and 60- and 30-fold smaller k(cat) values relative to 1, respectively. The reaction mechanism of family 18 chitinases is discussed based upon the results obtained from the hydrolysis of these compounds.