Recombinant expression, purification, and kinetic characterization of chondroitinase AC and chondroitinase B from Flavobacterium heparinum

Glycosaminoglycans (GAGs) are a family of complex polysaccharides involved in a diversity of biological processes, ranging from cell signaling to blood coagulation. Chondroitin sulfate (CS) and dermatan sulfate (DS) comprise a biologically important subset of GAGs. Two of the important lyases that degrade CS/DS, chondroitinase AC (EC 4.2.2.5) and chondroitinase B (no EC number), have been isolated and cloned from Flavobacterium heparinum. In this study, we outline an improved methodology for the recombinant expression and purification of these chondroitinases, thus enabling the functional characterization of the recombinant form of the enzymes for the first time. Utilizing an N-terminal 6x histidine tag, the recombinant chondroitinases were produced by two unique expression systems, each of which can be purified to homogeneity in a single chromatographic step. The products of exhaustive digestion of chondroitin-4SO(4) and chondroitin-6SO(4) with chondroitinase AC and dermatan sulfate with chondroitinase B were analyzed by strong-anion exchange chromatography and a novel reverse-polarity capillary electrophoretic technique. In addition, the Michaelis-Menten parameters were determined for these enzymes. With chondroitin-4SO(4) as the substrate, the recombinantly expressed chondroitinase AC has a K(m) of 0.8 microM and a k(cat) of 234 s(-1). This is the first report of kinetic parameters for chondroitinase AC with this substrate. With chondroitin-6SO(4) as the substrate, the enzyme has a K(m) of 0.6 microM and a k(cat) of 480 s(-1). Recombinantly expressed chondroitinase B has a K(m) of 4.6 microM and a k(cat) of 190 s(-1) for dermatan sulfate as its substrate. Efficient recombinant expression of the chondroitinases will facilitate the structure-function characterization of these enzymes and allow for the development of the chondroitinases as enzymatic tools for the fine characterization and sequencing of CS/DS.     

Chondroitinase C from Flavobacterium heparinum.

A chondroitinase that acts upon chondroitin sulfate C and hyaluronic acid was isolated from Flavobacterium heparinum. This enzyme was seperated from constitutional chondroitinase AC and an induced chondroitinase B also present in extracts of F. heparinum previously grown in the presence of chondroitin sulfates A, B or C. The enzyme acts upon chondroitin sulfate C producing tetrasaccharide plus an unsaturated 6-sulfated disaccharide (delta Di-6S), and upon hyaluronic acid producing unsaturated nonsulfated disaccharide (delta Di-OS). Chondroitin sulfate A is also degraded producing oligosaccharides and delta Di-6S but not delta Di-4S. The chondroitinase C is also distinguished from the chondroitinases B and AC by several properties, such as effect of ions, temperature for optimal activity, and susceptibility to increasing salt concentrations. The substrate specificity of the chondroitinase C is different from that of any other chondroitinase or hyaluronidase described so far.     

Crystal structure of chondroitinase B from Flavobacterium heparinum and its complex with a disaccharide product at 1.7 A resolution

Glycosaminoglycans (GAGs) are a family of acidic heteropolysaccharides, including such molecules as chondroitin sulfate, dermatan sulfate, heparin and keratan sulfate. Cleavage of the O-glycosidic bond within GAGs can be accomplished by hydrolases as well as lyases, yielding disaccharide and oligosaccharide products. We have determined the crystal structure of chondroitinase B, a glycosaminoglycan lyase from Flavobacterium heparinum, as well as its complex with a dermatan sulfate disaccharide product, both at 1.7 A resolution. Chondroitinase B adopts the right-handed parallel beta-helix fold, found originally in pectate lyase and subsequently in several polysaccharide lyases and hydrolases. Sequence homology between chondroitinase B and a mannuronate lyase from Pseudomonas sp. suggests this protein also adopts the beta-helix fold. Binding of the disaccharide product occurs within a positively charged cleft formed by loops extending from the surface of the beta-helix. Amino acid residues responsible for recognition of the disaccharide, as well as potential catalytic residues, have been identified. Two arginine residues, Arg318 and Arg364, are found to interact with the sulfate group attached to O-4 of N-acetylgalactosamine. Cleavage of dermatan sulfate likely occurs at the reducing end of the disaccharide, with Glu333 possibly acting as the general base.     

Purification of chondroitinase B and chondroitinase C using glycosaminoglycan-bound AH-Sepharose 4B

Chondroitinase B and chondroitinase C were separated from an extract of Flavobacterium heparinum induced with chondroitin 6-sulfate by using column chromatography on hydroxylapatite. Chondroitinase C was eluted together with the activities of hyaluronidase, delta4,5glycosiduronase, and sulfatase. The latter two activities were eliminated exclusively by passing the crude chondroitinase C fraction through a phosphono-cellulose column pre-equilibrated with 0.07M sodium phosphate buffer (pH 6.8). Chondroitinase C was then purified by affinity chromatography using dermatan sulfate-bound AH-Sepharose 4B coated with the same glycosaminoglycan. Purification of the enzyme was achieved 18-fold and in 73% yield. On the other hand, the activities of delta4,5glycosiduronase and sulfatase were decreased to 50 and 60%, respectively, as compared with those in the crude chondroitinase B fraction, after passing the fraction through a column of phosphono-cellulose pre-equilibrated with 0.1M sodium phosphate buffer (pH 6.8). The remaining activities of these two enzymes were then eliminated from chondroitinase B by affinity chromatography with heparin-bound AH-Sepharose 4B coated with dermatan sulfate. In the affinity chromatography used in the present study, non-covalent coating of the glycosaminoglycan-bound (covalently) AH-Sepharose 4B with the same or another glycosaminoglycan was found to be important.     

Isolation and characterization of an induced chondroitinase ABC from Flavobacterium heparinum

During the investigation of alternative methods for the large scale preparation of chondroitinases AC, B and C from Flavobacterium heparinum, a new chondroitinase activity was observed. This new enzyme, like the other chondroitinases, acts as an eliminase, forming unsaturated sulfated disaccharides from dermatan and chondroitin sulfates. In contrast to the chondroitinases previously described, which are endoglycosidases, this chondroitinase ABC cleaves the glycosidic linkages in an exolytic fashion, beginning at the reducing end of the substrate molecules. The oligosaccharides formed as transient products by the action of either chondroitinases or testicular hyaluronidase upon dermatan and chondroitin sulfates are also rapidly degraded by the chondroitinase ABC, regardless of their size or the presence of delta-4,5 unsaturation in the terminal uronic acid residue. The maximum activity of the chondroitinase ABC occurs at 30 degrees C and at pH 6.0-7.5. Only 15% of the activity was observed at 37 degrees C, indicating that the enzyme is very sensitive to thermal denaturation. It is strongly inhibited by phosphate ions and is also inhibited by the unsaturated disaccharides formed.     

Purification and characterization of novel chondroitin ABC and AC lyases from Bacteroides stercoris HJ-15, a human intestinal anaerobic bacterium.

Two novel chondroitinases, chondroitin ABC lyase (EC 4.2.2.4) and chondroitin AC lyase (EC 4.2.2.5), have been purified from Bacteroides stercoris HJ-15, which was isolated from human intestinal bacteria with glycosaminoglycan degrading enzymes. Chondroitin ABC lyase was purified to apparent homogeneity by a combination of QAE-cellulose, CM-Sephadex C-50, hydroxyapatite and Sephacryl S-300 column chromatography with a final specific activity of 45.7 micromol.min-1.mg-1. Chondroitin AC lyase was purified to apparent homogeneity by a combination of QAE-cellulose, CM-Sephadex C-50, hydroxyapatite and phosphocellulose column chromatography with a final specific activity of 57.03 micromol.min-1.mg-1. Chondroitin ABC lyase is a single subunit of 116 kDa by SDS/PAGE and gel filtration. Chondroitin AC lyase is composed of two identical subunits of 84 kDa by SDS/PAGE and gel filtration. Chondroitin ABC and AC lyases showed optimal activity at pH 7.0 and 40 degrees C, and 5.7-6.0 and 45-50 degrees C, respectively. Both chondroitin lyases were potently inhibited by Cu2+, Zn2+, and p-chloromercuriphenyl sulfonic acid. The purified Bacteroidal chondroitin ABC lyase acted to the greatest extent on chondroitin sulfate A (chondroitin 4-sulfate), to a lesser extent on chondroitin sulfate B (dermatan sulfate) and C (chondroitin 6-sulfate). The purified chondroitin AC lyase acted to the greatest extent on chondroitin sulfate A, and to a lesser extent on chondroitin C and hyaluronic acid. They did not act on heparin and heparan sulfate. These findings suggest that the biochemical properties of these purified chondroitin lyases are different from those of the previously purified chondroitin lyases.     

Biochemical characterization of the chondroitinase B active site

Chondroitinase B from Flavobacterium heparinum is the only known lyase that cleaves the glycosaminoglycan, dermatan sulfate (DS), as its sole substrate. A recent co-crystal structure of chondroitinase B with a disaccharide product of DS depolymerization has provided some insight into the location of the active site and suggested potential roles of some active site residues in substrate binding and catalysis. However, this co-crystal structure was not representative of the actual enzyme-substrate complex, because the disaccharide product did not have the right length or the chemical structure of the minimal substrate (tetrasaccharide) involved in catalysis. Therefore, only a limited picture of the functional role of active site residues in DS depolymerization was presented in previous structural studies. In this study, by docking a DS tetrasaccharide into the proposed active site of the enzyme, we have identified novel roles of specific active site amino acids in the catalytic function of chondroitinase B. Our conformational analysis also revealed a unique, symmetrical arrangement of active site amino acids that may impinge on the catalytic mechanism of action of chondroitinase B. The catalytic residues Lys-250, Arg-271, His-272, and Glu-333 along with the substrate binding residues Arg-363 and Arg-364 were mutated using site-directed mutagenesis, and the kinetics and product profile of each mutant were compared with recombinant chondroitinase B. Mutating Lys-250 to alanine resulted in inactivation of the enzyme, potentially attributable to the role of the residue in stabilizing the carbanion intermediate formed during enzymatic catalysis. The His-272 and Glu-333 mutants showed diminished enzymatic activity that could be indicative of a possible role for one or both residues in the abstraction of the C-5 proton from the galactosamine. In addition, the Arg-364 mutant had an altered product profile after exhaustive digestion of DS, suggesting a role for this residue in defining the substrate specificity of chondroitinase B.     

Crystal structure of Proteus vulgaris chondroitin sulfate ABC lyase I at 1.9A resolution

Chondroitin Sulfate ABC lyase I from Proteus vulgaris is an endolytic, broad-specificity glycosaminoglycan lyase, which degrades chondroitin, chondroitin-4-sulfate, dermatan sulfate, chondroitin-6-sulfate, and hyaluronan by beta-elimination of 1,4-hexosaminidic bond to unsaturated disaccharides and tetrasaccharides. Its structure revealed three domains. The N-terminal domain has a fold similar to that of carbohydrate-binding domains of xylanases and some lectins, the middle and C-terminal domains are similar to the structures of the two-domain chondroitin lyase AC and bacterial hyaluronidases. Although the middle domain shows a very low level of sequence identity with the catalytic domains of chondroitinase AC and hyaluronidase, the residues implicated in catalysis of the latter enzymes are present in chondroitinase ABC I. The substrate-binding site in chondroitinase ABC I is in a wide-open cleft, consistent with the endolytic action pattern of this enzyme. The tryptophan residues crucial for substrate binding in chondroitinase AC and hyaluronidases are lacking in chondroitinase ABC I. The structure of chondroitinase ABC I provides a framework for probing specific functions of active-site residues for understanding the remarkably broad specificity of this enzyme and perhaps engineering a desired specificity. The electron density map showed clearly that the deposited DNA sequence for residues 495-530 of chondroitin ABC lyase I, the segment containing two putative active-site residues, contains a frame-shift error resulting in an incorrectly translated amino acid sequence.     

Role of arginine 292 in the catalytic activity of chondroitin AC lyase from Flavobacterium heparinum

Chondroitin AC lyase (chondroitinase EC 4.2.2.5), an eliminase from Flavobacterium heparinum, cleaves chondroitin sulfate glycosaminoglycans (GAGs) at 1,4 glycosidic linkages between N-acetylgalactosamine and glucuronic acid residues. Cleavage occurs through beta-elimination in a random endolytic action pattern. Crystal structures of chondroitin AC lyase (wild type) complexed with oligosaccharides reveal a binding site within a narrow and shallow protein channel, suggesting several amino acids as candidates for the active site residues. Site-specific mutagenesis studies on residues within the active-site tunnel revealed that only the Arg to Ala 292 mutation (R292A) retained activity. Furthermore, structural data suggested that R292 was primarily involved in recognition of N-acetyl or O-sulfo moieties of galactosamine residues and did not directly participate in catalysis. The current study demonstrates that the R292A mutation affords approximately 10-fold higher K(m) values but no significant change in V(max), consistent with hypothesis that R292 is involved in binding the O-sulfo moiety of the saccharide residues. Change in chondroitin sulfate viscosity, as a function of its enzymatic cleavage, affords a shallower concave curve for the R292A mutant, suggesting its action pattern is neither purely random endolytic nor purely random exolytic. Product studies using gel electrophoresis confirm the altered action pattern of this mutant. Thus, these data suggest that the R292A mutation effectively reduces binding affinity, making it possible for the oligosaccharide chain, still bound after initial endolytic cleavage, to slide through the tunnel to the catalytic site for subsequent, processive, step-wise, exolytic cleavage.     

Significance of ribosomal ribonucleic acid synthesis for control of the G1 period in the cell cycle of the heterobasidiomycetous yeast Rhodosporidium toruloides.

Bacteroides thetaiotaomicron, a gram-negative anaerobe found in human colons, could utilize chondroitin sulfate, a tissue mucopolysaccharide, as its sole source of carbohydrate. The enzymes responsible for the breakdown of chondroitin sulfate by B. thetaiotaomicron were similar to those produced by Proteus vulgaris and Flavobacterium heparinum and included a lyase (EC 4.2.2.4), which degraded chondroitin sulfate into sulfated disaccharides, sulfatases (EC 3.1.6.4), which removed the sulfate residues, and a glucuronidase, which broke the unsulfated disaccharides into monosaccharide components. Chondroitin sulfate lyase, the first enzyme in the breakdown sequence, was not extracellular. It appeared to be located in the periplasmic space since lyase activity was released by treatment with ethylenediaminetetraacetate and lysozyme. Moreover, sodium polyanethole sulfonate, a high-molecular-weight inhibitor of chondroitin lyase, did not inhibit breakdown of chondroitin sulfate by intact bacteria. The sulfatase and glucuronidase appeared to be intracellular. None of these enzymes was strongly bound to membranes, and none of the steps in the breakdown of chondroitin sulfate was sensitive to oxygen.     

Substrate specificity studies of Flavobacterium chondroitinase C and heparitinases towards the glycosaminoglycan--protein linkage region. Use of a sensitive analytical method developed by chromophore-labeling of linkage glycoserines using dimethylaminoazobenzenesulfonyl chloride.

Bacterial chondroitinases and heparitinases are potentially useful tools for structural studies of chondroitin sulfate and heparin/heparan sulfate. Substrate specificities of Flavobacterium chondroitinase C, as well as heparitinases I and II, towards the glycosaminoglycan-protein linkage region -HexA-HexNAc-GlcA-Gal-Gal-Xyl-Ser (where HexA represents glucuronic acid or iduronic acid and HexNAc represents N-acetylgalactosamine or N-acetylglucosamine) were investigated using various structurally defined oligosaccharides or oligosaccharide-serines derived from the linkage region. In the case of oligosaccharide-serines, they were labeled with a chromophore dimethylaminoazobenzenesulfonyl chloride (DABS-Cl), which stably reacted with the amino group of the serine residue and rendered high absorbance for microanalysis. Chondroitinase C cleaved the GalNAc bond of the pentasaccharides or hexasaccharides derived from the linkage region of chondroitin sulfate chains and tolerated sulfation of the C-4 or C-6 of the GalNAc residue and C-6 of the Gal residues, as well as 2-O-phosphorylation of the Xyl residue. In contrast, it did not act on the GalNAc-GlcA linkage when attached to a 4-O-sulfated Gal residue. Heparitinase I cleaved the innermost glucosaminidic bond of the linkage region oligosaccharide-serines of heparin/heparan sulfate irrespective of substitution by uronic acid, whereas heparitinase II acted only on the glucosaminidic linkages of the repeating disaccharide region, but not on the innermost glucosaminidic linkage. These defined specificities of chondroitinase C, as well as heparitinases I and II, will be useful for preparation and structural analysis of the linkage oligosaccharides.     

High-resolution crystal structure of Arthrobacter aurescens chondroitin AC lyase: an enzyme-substrate complex defines the catalytic mechanism

Chondroitin lyases (EC 4.2.2.4 and EC 4.2.2.5) are glycosaminoglycan-degrading enzymes that act as eliminases. Chondroitin lyase AC from Arthrobacter aurescens (ArthroAC) is known to act on chondroitin 4-sulfate and chondroitin 6-sulfate but not on dermatan sulfate. Like other chondroitin AC lyases, it is capable of cleaving hyaluronan. We have determined the three-dimensional crystal structure of ArthroAC in its native form as well as in complex with its substrates (chondroitin 4-sulfate tetrasaccharide, CS(tetra) and hyaluronan tetrasaccharide) at resolution varying from 1.25 A to 1.9A. The primary sequence of ArthroAC has not been previously determined but it was possible to determine the amino acid sequence of this enzyme from the high-resolution electron density maps and to confirm it by mass spectrometry. The enzyme-substrate complexes were obtained by soaking the substrate into the crystals for varying lengths of time (30 seconds to ten hours) and flash-cooling the crystals. The electron density map for crystals soaked in the substrate for as short as 30 seconds showed the substrate clearly and indicated that the ring of central glucuronic acid assumes a distorted boat conformation. This structure strongly supports the lytic mechanism where Tyr242 acts as a general base that abstracts the proton from the C5 position of glucuronic acid while Asn183 and His233 neutralize the charge on the glucuronate acidic group. Comparison of this structure with that of chondroitinase AC from Flavobacterium heparinum (FlavoAC) provides an explanation for the exolytic and endolytic mode of action of ArthroAC and FlavoAC, respectively.     

Action pattern of polysaccharide lyases on glycosaminoglycans

The action pattern of polysaccharide lyases on glycosaminoglycansubstrates was examined using viscosimetric measurements andgradient polyacrylamide gel electrophoresis (PAGE). Heparinlyase I (heparinase, EC 4.2.2.7 [EC] ) and heparin lyase II (no ECnumber) both acted on heparin in a random endolytic fashion.Heparin lyase II showed an ideal endolytic action pattern onheparan sulphate, while heparin lyase I decreased the molecularweight of heparan sulphate more slowly. Heparin lyase III (heparitinase,EC 4.2.2.8 [EC] ) acted endolytically only on heparan sulphate anddid not cleave heparin. Chondroitin ABC lyase (chondroitinaseABC, EC 4.2.2.4 [EC] ) from Proteus vulgaris acted endolytically onchondroitin-6-sulphate (chondroitin sulphate C) and dermatansulphate at nearly identical initial rates, but acted on chondroitin-4-sulphate(chondroitin sulphate A) at a reduced rate, decreasing its molecularweight much more slowly. Two chondroitin AC lyases (chondroitinaseAC, both EC 4.2.2.5 [EC] ) were examined towards chondroitin-4- and-6-sulphates. The exolytic action of chondroitin AC lyase Afrom Arthrobacter aurescens on both chondroitin-4- and -6-sulphateswas demonstrated viscosimetrically and confirmed using bothgradient PAGE and gel permeation chromatography. ChondroitinAC lyase F from Flavobacterium heparinum (Cytophagia heparinia)acted endolytically on the same substrates. Chondroitin B lyase(chondroitinase B, no EC number) from F.heparinum acted endolyticallyon dermatan sulphate giving a nearly identical action patternas observed for chondroitin ABC lyase acting on dermatan sulphate. action pattern chondroitin lyase glycosaminoglycan heparin lyase.     

Isolation and characterization of an induced chondroitinase ABC from Flavobacterium heparium

During the investigation of alternative methods for the large sclae preparation of chondroitinases AC, B and C from Flavobacterium heparinum, a new chondroitinase activity was observed. This new enzyme, like the other chondroitinases, acts as an eliminase, forming unsaturated sulfated disaccharides from dermatan and chondroitin sulfates. In contrast tot he chondroitinases previously described, which are endoglycosidases, this chondroitinase ABC cleaves the glycosidic linkages in an exolytic fashiom, beginning at the reducing end of the substrate molecules. The oligosaccharides formed as transient products by the action of either chondroitinases or testicular hyaluronidase upon dermatan and chontroitin sulfates are also rapidly degraded by the chondroitinase ABC, regardless of their size or the presence of Δ-4,5 unsaturation in the terminal uronic acid residue. The maximum activity of the chondroitinase ABC occurs at 30°C and at pH 6.0–7.5. Only 15% of the activity was observed at 37°C, indicating that the enzyme is very sensitive to thermal denaturation. It is stronly inhibited by phosphate ions and is also inhibited by the unsaturated disaccharides formed.     

Crystallization and some properties of chondroitinase from Arthrobacter aurescens.

A strain of Arthrobacter aurescens which secretes a large amount of chondroitinase into a culture broth, was isolated from soil. The chondroitinase was purified 380-fold over culture broth in 24% yield and crystallized. Some properties of the purified enzyme were studied and described: thermal stability (below 45 degrees), pH stability (pH 4.9 to 7.4), optimum temperature (50 degrees), and optimum pH (pH 6.0). Chrondroitin sulfate A and C, chondroitin, and hyaluronic acid were split by the enzyme but dermatan sulfate could not be. The initial rates of enzymic degradation of chondroitin sulfate C, chondroitin, and hyaluronic acid were 1.1, 1.95, and 3.2, respectively, compared to that of chondroitin sulfate A. When the enzyme was allowed to act on chondroitin sulfate A and C, the reducing power and the ultraviolet absorption at 232 nm increased proportionally to the decrease in viscosity of the substrate solution. Finally these substrates were degraded to the extent of 100% to disaccharides. By the enzyme action the main products from chondroitin sulfate A and C were deta 4,5-unsaturated disaccharides, which were identified as 2-acetamido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose and 2-acet-amido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose by paper chromatography, ultraviolet absorption spectroscophy, and infrared spectroscopy. Thus it is suggested that the chondroitinase is a chondroitin sulfate A and C lyase, one of the hyaluronate lyases (EC 4.2.99.1).     

The catalytic machinery of chondroitinase ABC I utilizes a calcium coordination strategy to optimally process dermatan sulfate

The chondroitinases are bacterial lyases that specifically cleave chondroitin sulfate and/or dermatan sulfate glycosaminoglycans. One of these enzymes, chondroitinase ABC I from Proteus vulgaris, has the broadest substrate specificity and has been widely used to depolymerize these glycosaminoglycans. Biochemical and structural studies to investigate the active site of chondroitinase ABC I have provided important insights into the catalytic amino acids. In this study, we demonstrate that calcium, a divalent ion, preferentially increases the activity of chondroitinase ABC I toward dermatan versus chondroitin substrates in a concentration-dependent manner. Through biochemical and biophysical investigations, we have established that chondroitinase ABC I binds calcium. Experiments using terbium, a fluorescent calcium analogue, confirm the specificity of this interaction. On the basis of theoretical structural models of the enzyme-substrate complexes, specific amino acids that could potentially play a role in calcium coordination were identified. These amino acids were investigated through site-directed mutagenesis studies and kinetic assays to identify possible mechanisms for calcium-mediated processing of the dermatan substrate in the active site of the enzyme.     

The structure of chondroitin B lyase complexed with glycosaminoglycan oligosaccharides unravels a calcium-dependent catalytic machinery

Chondroitinase B from Pedobacter heparinus is the only known enzyme strictly specific for dermatan sulfate and is a widely used enzymatic tool for the structural characterization of glycosaminoglycans. This beta-helical polysaccharide lyase belongs to family PL-6 and cleaves the beta(1,4) linkage of dermatan sulfate in a random manner, yielding 4,5-unsaturated dermatan sulfate disaccharides as the product. The previously reported structure of its complex with a dermatan sulfate disaccharide product identified the -1 and -2 subsites of the catalytic groove. We present here the structure of chondroitinase B complexed with several dermatan sulfate and chondroitin sulfate oligosaccharides. In particular, the soaking of chondroitinase B crystals with a dermatan sulfate hexasaccharide results in a complex with two dermatan sulfate disaccharide reaction products, enabling the identification of the +2 and +1 subsites. Unexpectedly, this structure revealed the presence of a calcium ion coordinated by sequence-conserved acidic residues and by the carboxyl group of the l-iduronic acid at the +1 subsite. Kinetic and site-directed mutagenesis experiments have subsequently demonstrated that chondroitinase B absolutely requires calcium for its activity, indicating that the protein-Ca(2+)-oligosaccharide complex is functionally relevant. Modeling of an intact tetrasaccharide in the active site of chondroitinase B provided a better understanding of substrate specificity and the role of Ca(2+) in enzymatic activity. Given these results, we propose that the Ca(2+) ion neutralizes the carboxyl moiety of the l-iduronic acid at the cleavage site, whereas the conserved residues Lys-250 and Arg-271 act as Br?nsted base and acid, respectively, in the lytic degradation of dermatan sulfate by chondroitinase B.     

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.