Characterization and Structural Analysis of a Novel Carbohydrate-Binding Module from Family 96 with Chondroitin Sulfate-Specific Binding Capacity.

Chondroitin sulfate (CS) is the predominant glycosaminoglycan within the human body and is widely applied in various industries. Carbohydrate-binding modules (CBMs) possessing the capacity for carbohydrate recognition are verified to be important tools for polysaccharide investigation. Only one CS-specific CBM, PhCBM100, has hitherto been characterized. In the present study, two CBM96 domains present in the same putative PL8_3 chondroitin AC lyase were discovered and recombinantly expressed. The results of microtiter plate assays and affinity gel electrophoresis assays showed that the two corresponding proteins, DmCBM96-1 and DmCBM96-2, bind specifically to CSs. The crystal structure of DmCBM96-1 was determined at a 2.20 Å resolution. It adopts a ß-sandwich fold comprising two antiparallel ß-sheets, showing structural similarities to TM6-N4, which is the founding member of the CBM96 family. Site mutagenesis analysis revealed that the residues of Arg27, Lys45, Tyr51, Arg53, and Arg157 are critical for CS binding. The characterization of the two CBM96 proteins demonstrates the diverse ligand specificity of the CBM96 family and provides promising tools for CS investigation.

A novel chondroitin AC lyase from Pedobacter xixiisoli: Cloning, expression, characterization and the application in the preparation of oligosaccharides.

Chondroitin AC lyase can efficiently hydrolyze chondroitin sulfate (CS) to low molecule weight chondroitin sulfate, which has been widely used in clinical therapy, including anti-tumor, anti-oxidation, hypolipidemic, and anti-inflammatory. In this work, a novel chondroitin AC lyase from Pedobacter xixiisoli (PxchonAC) was cloned and overexpressed in Escherichia coli BL21 (DE3). The characterization of PxchonAC showed that it has specific activities on chondroitin sulfate A, Chondroitin sulfate C and hyaluronic acid with 428.77, 270.57, and 136.06 U mg-1, respectively. The Km and Vmax of PxchonAC were 0.61 mg mL-1 and 670.18 U mg-1 using chondroitin sulfate A as the substrate. The enzyme had a half-life of roughly 660 min at 37 °C in the presence of Ca2+ and remained a residual activity of 54 % after incubated at 4 °C for 25 days. Molecular docking revealed that Asn123, His223, Tyr232, Arg286, Arg290, Asn372, and Glu374 were mainly involved in the substrate binding. The enzymatic hydrolysis product was analyzed by gel permeation chromatography, demonstrating PxchonAC could hydrolyze CS efficiently.

Identification and biochemical characterization of a novel chondroitin sulfate/dermantan sulfate lyase from Photobacterium sp.

Chondroitin sulfate (CS)/dermatan sulfate (DS) lyases play important roles in structural and functional studies of CS/DS. In this study, a novel CS/DS lyase (enCSase) was identified from the genome of the marine bacterium Photobacterium sp. QA16. This enzyme is easily heterologously expressed and purified as highly active form against various CS, DS and hyaluronic acid (HA). Under the optimal conditions, the specific activities of this enzyme towards CSA, CSC, CSD, CSE, DS and HA were 373, 474, 171, 172, 141 and 97 U/mg of proteins, respectively. As an endolytic enzyme, enCSase degrades HA to unsaturated hexa- and tetrasaccharides but CS/DS to unsaturated tetra- and disaccharides as the final products. Sequencing analysis showed that the structures of tetrasaccharides in the final products of CS variants were not unique but were highly variable, indicating the randomness of substrate degradation by this enzyme. Further studies showed that the smallest substrate of enCSase was octasaccharide for HA but hexasaccharide for CS/DS, which could explain why this enzyme cannot degrade HA hexa- and tetrasaccharides and CS/DS tetrasaccharides further. It is believed that enCSase may be a very useful tool for structural and functional studies and related applications of CS/DS and HA.

Cloning and characterization of a novel chondroitinase ABC categorized into a new subfamily of polysaccharide lyase family 8.

Chondroitinases degrade chondroitin sulfate (CS) into oligosaccharides, of which the biological activities have vital roles in various fields. Some chondroitinases in polysaccharide lyase family 8 (PL8) have been classified into four subfamilies (PL8_1, PL8_2, PL8_3, and PL8_4) based on their sequence similarity and substrate specificities. In this study, a gene, vpa_0049, was cloned from marine bacterium Vibrio sp. QY108. The encoded protein, Vpa_0049, did not belong to the four existing subfamilies in PL8 based on phylogenetic analysis. Vpa_0049 could degrade various glycosaminoglycans (CS-A, CS-B, CS-C, CS-D, and HA) into unsaturated disaccharides in an endolytic manner, which was different from PL8 lyases of four existing subfamilies. The maximum activity of Vpa_0049 on different glycosaminoglycan substrates appeared at 30-37 °C and pH 7.0-8.0 in the presence of NaCl. Vpa_0049 showed approximately 50% of maximum activity towards CS-B and HA at 0 °C. It was stable in alkaline conditions (pH 8.0-10.6) and 0-30 °C. Our study provides a new broad-substrate chondroitinase and presents an in-depth understanding of PL8.

Cloning and Characterization of a Chondroitin AC Exolyase from Arthrobacter sp. SD-04.

Glycosaminoglycans (GAGs) and their low-molecular weight derivates have received considerable interest in terms of their potential clinical applications, and display a wide variety of pharmacological and pharmacokinetic properties. Structurally distinct GAG chains can be prepared by enzymatic depolymerization. A variety of bacterial chondroitin sulfate (CS) lyases have been identified, and have been widely used as catalysts in this process. Here, we identified a putative chondroitin AC exolyase gene, AschnAC, from an Arthrobacter sp. strain found in a CS manufacturing workshop. We expressed the enzyme, AsChnAC, recombinantly in Escherichia coli, then purified and characterized it in vitro. The enzyme indeed displayed exolytic cleavage activity toward HA and various CSs. Removing the putative N-terminal secretion signal peptide of AsChnAC improved its expression level in E. coli while maintaining chondroitin AC exolyase activity. This novel catalyst exhibited its optimal activity in the absence of added metal ions. AsChnAC has potential applications in preparation of low-molecular weight GAGs, making it an attractive catalyst for further investigation.

Expression, purification and characterisation of chondroitinase AC II with glyceraldehyde-3-phosphate dehydrogenase tag and chaperone (GroEs-GroEL) from Arthrobacter sp. CS01.

In this study, chondroitinase (ChSase) AC II from Arthrobacter sp. CS01 was cloned, expressed in Escherichia coli BL21 (DE3), purified and characterised. To assist in protein folding and improve on high protein aggregation rates, two strategies involving chaperones and fusion tags were chosen to increase enzyme activity and improve enzymatic properties. ChSase AC II enzyme activity increased from 3.12 to 9.15 U/ml with chaperone GroEs-GroEL, and the specific activity increased from 19.8 to 25.74 U/mg with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) tag. ChSase AC II and GAPDH-ChSase AC II displayed maximum activities at 37 °C and 40 °C, at pH 6.5 and 7.0, respectively. GAPDH-ChSase AC II activity remained above 69.8% after incubation at 40 °C for 120 min, and ChSase AC II activity remained approximately 32.1% under the same conditions, indicating that ChSase AC II thermostability was enhanced by the GAPDH tag. These properties suggested that the enzymes are promising prospects in medical and industrial applications.

Insights into the structural characteristics and substrate binding analysis of chondroitin AC lyase (PsPL8A) from Pedobacter saltans.

The structure of chondroitin AC lyase (PsPL8A) of family 8 polysaccharide lyase was characterized. Modeled PsPL8A structure showed, it contains N-terminal (α/α)6 incomplete toroidal fold and a layered ß sandwich structure at C-terminal. Ramchandran plot displayed 98.5% residues in favoured and 1.2% in generously allowed region. Secondary structure of PsPL8A by CD revealed 27.31% α helices 22.7% ß sheets and 49.9% random coils. Protein melting study showed, PsPL8A completely unfolds at 60°C. SAXS analysis showed, PsPL8A is fully folded in solution form. The ab initio derived dummy model of PsPL8A superposed well with its modeled structure excluding some α-helices and loop region. Structural superposition and docking analysis showed, N153, W105, H203, Y208, Y212, R266 and E349 were involved in catalysis. Mutants N153A, H203A, Y212F, R266A and E349A created by SDM revealed no residual activity. Isothermal titration calorimetry analysis of Y212F and H203A with C4S polysaccharide, showed moderate binding by Y212F (Ka=9.56±3.81×105) and no binding with H203A, showing active contribution of Y212 in substrate binding. Residues Y212 and H203 or R266 might act as general base and general acid respectively. Residues N153 and E349 are likely contributing in charge neutralization and stabilizing enolate anion intermediate during ß-elimination.

Cloning and Expression of Recombinant Chondroitinase ACII and Its Comparison to the Arthrobacter aurescens Enzyme.

Chondroitin sulfates are the glycosaminoglycan chains of proteoglycans critical in the normal development and pathophysiology of all animals. Chondroitinase ACII, a polysaccharide lyase originally isolated from Arthrobacter aurescens IAM 110 65, which is widely used in the analysis and study of chondroitin structure, is no longer commercially available. The aim of the current study is to prepare recombinant versions of this critical enzyme for the glycobiology research community. Two versions of recombinant chondroitinase ACII are prepared in Escherichia coli, and their activity, stability, specificity, and action pattern are examined, along with a non-recombinant version secreted by an Arthrobacter strain. The recombinant enzymes are similar to the enzyme obtained from Arthrobacter for all examined properties, except for some subtle specificity differences toward uncommon chondroitin sulfate substrates. These differences are believed to be due to either post-translational modification of the Arthrobacter-secreted enzyme or other subtle structural differences between the recombinant and natural enzymes. The secreted chondroitinase can serve as a suitable replacement for the original enzyme that is currently unavailable, while the recombinant ones can be applied generally in the structural determination of most standard chondroitin sulfates.

High Nutrient Concentration Can Induce Virulence Factor Expression and Cause Higher Virulence in an Environmentally Transmitted Pathogen.

Environmentally transmitted opportunistic pathogens shuttle between two substantially different environments: outside-host and within-host habitats. These environments differ from each other especially with respect to nutrient availability. Consequently, the pathogens are required to regulate their behavior in response to environmental cues in order to survive, but how nutrients control the virulence in opportunistic pathogens is still poorly understood. In this study, we examined how nutrient level in the outside-host environment affects the gene expression of putative virulence factors of the opportunistic fish pathogen Flavobacterium columnare. The impact of environmental nutrient concentration on bacterial virulence was explored by cultivating the bacteria in various nutrient conditions, measuring the gene expression of putative virulence factors with RT-qPCR and, finally, experimentally challenging rainbow trout (Oncorhynchus mykiss) fry with these bacteria. Our results show that increased environmental nutrient concentration can increase the expression of putative virulence genes, chondroitinase (cslA) and collagenase, in the outside-host environment and may lead to more rapid fish mortality. These findings address that the environmental nutrients may act as significant triggers of virulence gene expression and therefore contribute to the interaction between an environmentally transmitted opportunistic pathogen and its host.

Uncovering the Catalytic Direction of Chondroitin AC Exolyase: FROM THE REDUCING END TOWARDS THE NON-REDUCING END.

Glycosaminoglycans (GAGs) are polysaccharides that play vital functional roles in numerous biological processes, and compounds belonging to this class have been implicated in a wide variety of diseases. Chondroitin AC lyase (ChnAC) (EC 4.2.2.5) catalyzes the degradation of various GAGs, including chondroitin sulfate and hyaluronic acid, to give the corresponding disaccharides containing an Δ(4)-unsaturated uronic acid at their non-reducing terminus. ChnAC has been isolated from various bacteria and utilized as an enzymatic tool for study and evaluating the sequencing of GAGs. Despite its substrate specificity and the fact that its crystal structure has been determined to a high resolution, the direction in which ChnAC catalyzes the cleavage of oligosaccharides remain unclear. Herein, we have determined the structural cues of substrate depolymerization and the cleavage direction of ChnAC using model substrates and recombinant ChnAC protein. Several structurally defined oligosaccharides were synthesized using a chemoenzymatic approach and subsequently cleaved using ChnAC. The degradation products resulting from this process were determined by mass spectrometry. The results revealed that ChnAC cleaved the ß1,4-glycosidic linkages between glucuronic acid and glucosamine units when these bonds were located on the reducing end of the oligosaccharide. In contrast, the presence of a GlcNAc-α-1,4-GlcA unit at the reducing end of the oligosaccharide prevented ChnAC from cleaving the GalNAc-ß1,4-GlcA moiety located in the middle or at the non-reducing end of the chain. These interesting results therefore provide direct proof that ChnAC cleaves oligosaccharide substrates from their reducing end toward their non-reducing end. This conclusion will therefore enhance our collective understanding of the mode of action of ChnAC.

Gene deletion strategy to examine the involvement of the two chondroitin lyases in Flavobacterium columnare virulence.

Flavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (PompA) was fused to sacB to construct a counterselectable marker for F. columnare. F. columnare carrying PompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB, were deleted. The ΔcslA and ΔcslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (ΔcslA ΔcslB) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G4 and of the chondroitin lyase-deficient ΔcslA ΔcslB mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of F. columnare but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum Bacteroidetes.

Chondroitin Lyase from a Marine Arthrobacter sp. MAT3885 for the Production of Chondroitin Sulfate Disaccharides.

Chondroitin sulfate (CS) saccharides from cartilage tissues have potential application in medicine or as dietary supplements due to their therapeutic bioactivities. Studies have shown that depolymerized CS saccharides may display enhanced bioactivity. The objective of this study was to isolate a CS-degrading enzyme for an efficient production of CS oligo- or disaccharides. CS-degrading bacteria from marine environments were enriched using in situ artificial support colonization containing CS from shark cartilage as substrate. Subsequently, an Arthrobacter species (strain MAT3885) efficiently degrading CS was isolated from a CS enrichment culture. The genomic DNA from strain MAT3885 was pyro-sequenced by using the 454 FLX sequencing technology. Following assembly and annotation, an orf, annotated as family 8 polysaccharide lyase genes, was identified, encoding an amino acid sequence with a similarity to CS lyases according to NCBI blastX. The gene, designated choA1, was cloned in Escherichia coli and expressed downstream of and in frame with the E. coli malE gene for obtaining a high yield of soluble recombinant protein. Applying a dual-tag system (MalE-Smt3-ChoA1), the MalE domain was separated from ChoA1 with proteolytic cleavage using Ulp1 protease. ChoA1 was defined as an AC-type enzyme as it degraded chondroitin sulfate A, C, and hyaluronic acid. The optimum activity of the enzyme was at pH 5.5-7.5 and 40 °C, running a 10-min reaction. The native enzyme was estimated to be a monomer. As the recombinant chondroitin sulfate lyase (designated as ChoA1R) degraded chondroitin sulfate efficiently compared to a benchmark enzyme, it may be used for the production of chondroitin sulfate disaccharides for the food industry or health-promoting products.

A novel eliminase from a marine bacterium that degrades hyaluronan and chondroitin sulfate.

Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even though marine animals are rich in GAGs with unique structures and functions. Herein we isolated a novel GAG lyase for the first time from the marine bacterium Vibrio sp. FC509 and then recombinantly expressed and characterized it. It showed strong lyase activity toward hyaluronan (HA) and chondroitin sulfate (CS) and was designated as HA and CS lyase (HCLase). It exhibited the highest activities to both substrates at pH 8.0 and 0.5 m NaCl at 30 °C. Its activity toward HA was less sensitive to pH than its CS lyase activity. As with most other marine enzymes, HCLase is a halophilic enzyme and very stable at temperatures from 0 to 40 °C for up to 24 h, but its activity is independent of divalent metal ions. The specific activity of HCLase against HA and CS reached a markedly high level of hundreds of thousands units/mg of protein under optimum conditions. The HCLase-resistant tetrasaccharide Δ(4,5)HexUAα1-3GalNAc(6-O-sulfate)ß1-4GlcUA(2-O-sulfate)ß1-3GalNAc(6-O-sulfate) was isolated from CS-D, the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated d-glucuronic acid (GlcUA) in CS chains. Site-directed mutagenesis indicated that HCLase may work via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid. Thus, the identification of HCLase provides a useful tool for HA- and CS-related research and applications.

The crystal structure of novel chondroitin lyase ODV-E66, a baculovirus envelope protein.

Chondroitin lyases have been known as pathogenic bacterial enzymes that degrade chondroitin. Recently, baculovirus envelope protein ODV-E66 was identified as the first reported viral chondroitin lyase. ODV-E66 has low sequence identity with bacterial lyases at <12%, and unique characteristics reflecting the life cycle of baculovirus. To understand ODV-E66's structural basis, the crystal structure was determined and it was found that the structural fold resembled that of polysaccharide lyase 8 proteins and that the catalytic residues were also conserved. This structure enabled discussion of the unique substrate specificity and the stability of ODV-E66 as well as the host specificity of baculovirus.

Chondroitinase activity can be transduced by a lentiviral vector in vitro and in vivo.

The bacterial enzyme chondroitinase ABC (ChABC), which cleaves chondroitin sulfate glycosaminoglycan chains, can degrade inhibitory scar tissue formed following spinal cord injury, thereby promoting axonal growth and regeneration. However, delivering the active enzyme for prolonged periods presents practical limitations. To overcome these problems, we prepared a lentiviral vector (LV) encoding chondroitinase AC (Chase) together with the green fluorescent protein (GFP) reporter (Chase/LV) and demonstrated its expression and enzymatic activity in vitro and in vivo. Neural precursor cells infected with Chase/LV expressed the GFP reporter at levels that increased dramatically with time in culture. Enzymatic activity from the supernatant of the infected cells was demonstrated by dot blot assay using an antibody that recognizes the digested form of CSPG and was compared with the bacterial ChABC enzyme. Chick DRG cultures plated adjacent to the CSPG border and incubated with supernatant from Chase/LV-infected cells showed neurites growing into the CSPG area, a response similar to that after treatment with ChABC. In contrast, in control cultures, the neurites turned to avoid the inhibitory CSPG interface. Degradation of CSPG in these cultures was confirmed by specific CSPG antibodies. A single injection of Chase/LV into the spinal cord resulted in sustained secretion of the enzyme, whose activity was detected for 8 weeks by expression of GFP and evidence of the digested form of CSPG. This study demonstrates the efficacy of the Chase/LV vector and its potential as a therapeutic tool to reduce scar inhibition and promote axonal growth and repair following central nervous system injury.

Cloning and expression of chondroitinase AC from Bacteroides stercoris HJ-15.

Enzymes that degrade glycosaminoglycans (GAGs) can help reveal the biological roles, structure, and mechanisms of GAGs. We cloned chondroitinase AC, which can degrade chondroitin sulfates A and C, from the genomic library of Bacteroides stercoris HJ-15 isolated from human intestine. The probe (1.4 kb) for the chondroitinase AC gene was prepared from the PCR product of the primers produced using two internal amino acid sequences of chondroitinase AC purified from B. stercoris HJ-15. Using this probe, a chondroitinase AC-positive, 4 kb DNA fragment was selected from pKF3 vector gene libraries containing 2.5-4.5 kb DNA fragments digested with HindIII. The amino acid sequence of the cloned chondroitinase AC showed 41% homology to that of Flavobacterium heparinum. The cloned chondroitinase AC gene was expressed under the T7 promoter of the expression vector, pET-26b+, in Escherichia coli BL21(DE3) and purified using His bind column chromatography. The expressed chondroitinase AC potently degraded chondroitin sulfates A and C.

Mammalian-produced chondroitinase AC mitigates axon inhibition by chondroitin sulfate proteoglycans.

Chondroitin sulfate proteoglycans (CSPGs) are up-regulated following spinal cord injury and are partly responsible for failed regeneration. Experimental paradigms in vivo that degrade chondroitin sulfate glycosaminoglycan chains with the bacterial enzyme, chondroitinase, greatly enhance the ability of axons to regenerate through the glial scar. Unfortunately, enthusiasm for this treatment paradigm is diminished by the lack of a minimally invasive and sustained delivery method. To address these deficits, we have engineered a Tet-On adenoviral vector encoding chondroitinase AC and have characterized its enzymatic function in vitro. U373 human astrocytoma cells were transduced with adenovirus and subsequently induced with doxycycline to secrete enzymatically active chondroitinase as detected by western blot and kinetic analyses. Enzymatic activity demonstrated biological relevance in studies where neurite outgrowth into and across CSPG-adsorbed regions pre-treated with conditioned media from chondroitinase secreting astrocytes was significantly increased compared with untreated controls (p < 0.0001). We also measured important parameters of enzyme activity including: pH, temperature, and enzyme stability that are fundamental to harnessing the true therapeutic potential of this approach. The use of resident cells for continuous secretion of CSPG-degrading enzymes at the site of the glial scar promises to be of greater clinical relevance than contemporary methods.

N-Acetylgalactosamine 4,6-O-sulfate residues mediate binding and activation of heparin cofactor II by porcine mucosal dermatan sulfate.

Dermatan sulfate (DS) accelerates the inhibition of thrombin by heparin cofactor II (HCII). A hexasaccharide consisting of three l-iduronic acid 2-O-sulfate (IdoA2SO3)-->N-acetyl-D-galactosamine 4-O-sulfate (GalNAc4SO3) subunits was previously isolated from porcine skin DS and shown to bind HCII with high affinity. DS from porcine intestinal mucosa has a much lower content of this disaccharide but activates HCII with potency similar to that of porcine skin DS. Therefore, we sought to characterize oligosaccharides from porcine mucosal DS that interact with HCII. DS was partially depolymerized with chondroitinase ABC, and oligosaccharides containing 2-12 monosaccharide units were isolated. The oligosaccharides were then fractionated by anion-exchange and affinity chromatography on HCII-Sepharose, and the disaccharide compositions of selected fractions were determined. We found that the smallest oligosaccharides able to bind HCII were hexasaccharides. Oligosaccharides 6-12 units long that lacked uronic acid (UA)2SO3 but contained one or two GalNAc4,6SO3 residues bound, and binding was proportional to both oligosaccharide size and number of GalNAc4,6SO3 residues. Intact DS and bound dodecasaccharides contained predominantly IdoA but little D-glucuronic acid. Decasaccharides and dodecasaccharides containing one or two GalNAc4,6SO3 residues stimulated thrombin inhibition by HCII and prolonged the clotting time of normal but not HCII-depleted human plasma. These data support the hypothesis that modification of IdoA-->GalNAc4SO3 subunits in the DS polymer by either 2-O-sulfation of IdoA or 6-O-sulfation of GalNAc can generate molecules with HCII-binding sites and anticoagulant activity.

An atypical approach identifies TYR234 as the key base catalyst in chondroitin AC lyase.

Chondroitin AC lyase from Flavobacterium heparinum catalyses the degradation of chondroitin by an anionic E1cb elimination mechanism that involves proton abstraction from C5 of glucuronic acid. The lyase also carries out efficient proton transfer to a sugar nitronate anion, which was designed originally as an inhibitor of the enzyme, with a second-order rate constant of kcat/Km=2.7x10(6) M(-1) s(-); this is very similar to that of the natural chondroitin substrate (kcat/Km=1.3x10(6) M(-1) s(-1)). Studies with this nitronate should therefore provide insight into the proton-transfer step (general base catalysis) within this mechanism. Indeed, the Tyr234Phe mutant of the enzyme was essentially inactive with the natural substrate and correspondingly did not catalyse proton transfer to the nitronate, thereby implicating this residue as the general base catalyst. Parallel studies designed to identify the acid catalyst were carried out by using a substrate with a 2,4-dinitrophenol leaving group that needs no acid assistance for departure. These results are consistent with Tyr234 also playing the role of acid catalyst. Not only do these studies confirm the suspected role of Tyr234, but also they validate a new methodology for identification of acid/base catalysts in lyases and epimerases of this type. In addition a structural and mechanistic rationale is provided for different active-site acid/base configurations in syn and anti lyases.

Gene cloning and functional analysis of glycosaminoglycan-degrading enzyme chondroitin AC lyase from Flavobacterium columnare G4.

The chondroitin AC lyase gene, cslA, was cloned for the first time from the fish bacterial pathogen F. columnare G4. From the first transcription initiation site, the cslA extends 2620 nucleotides to the end of the 3' region. The open reading frame of cslA transcript has 2286 nucleotides encoding 762 amino acids with a 16 residues long signal peptide at the N-terminus. The gene, cslA was then successfully expressed in Escherichia coli and recombinant chondroitin AC lyase, rChonAC was purified, with its lytic activity analyzed. Zymography analysis copolymerized with chondroitin sulphate revealed the lytic activity of rChonAC and also the crude native ChonAC isolated from periplamic space of cultured F. columnare G4. The low level of lytic activity observed in crude native ChonAC may be due possibly to the low level of expression of this gene in the cultured condition. The expression and the role of this virulence factor is of interest for further research on the pathogenesis of F. columnare.