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.     

Degradation of even-numbered reduced and non-reduced hyaluronate oligosaccharides with D-glucuronic acid or N-acetyl-D-glucosamine as non-reducing terminal by chondroitin ABC and AC lyases.

Chondroitin ABC and AC lyases split hexosaminidic linkages in galactosaminoglycans and hyaluronic acid. Even-numbered oligosaccharides from hyaluronic acid with either D-glucuronic acid or N-acetylglucosamine in non-reducing position were used, prior to and after reduction with sodium borohydride, as substrates for chondroitin ABC and AC lyases. These substrates allowed elucidation of the effects of the nearest neighborhood of the bond to be split on the action of the enzymes. The results indicate that chondroitin ABC lyase acts strictly as an endolyase towards hyaluronate and requires the presence of a disaccharide in both reducing and non-reducing positions of the endohexosaminidic bond to be split. None of the hexosaminidic bonds of the tetrasaccharide GlcNAc-GlcUA-GlcNAc-GlcUA is split by chondroitin ABC lyase. In contrast chondroitin AC lyase acts also as an exoglycosidase towards hyaluronate and recognizes only the amino sugar and the uronic acid residue that are linked via the hexosaminidic bond which is split. Thus, the N-acetylglucosamine and glucuronic acid residues at both ends of a tetrasaccharide with the structure GlcNAc-GlcUA-GlcNAc-GlcUA are liberated.     

Preparation of the methyl ester of hyaluronan and its enzymatic degradation

A methyl ester of hyaluronan in which the carboxyl groups were fully esterified was prepared using trimethylsilyl diazomethane. This derivative, while not depolymerized by hyaluronan lyases or hyaluronan hydrolases, was a substrate for both chondroitin ACI lyase (EC 4.2.2.5) from Flavobacterium heparinum and chondroitin ACII lyase (EC 4.2.2.5) from Arthrobacter aurescens. The major product isolated in these depolymerization reactions was methyl alpha-L-threo-hex-4-enepyranosyluronate-(1-->3)-2-acetamido-2-deoxy-alpha,beta-D-glucopyranoside as determined by 1H NMR spectroscopy and MALDITOF mass spectrometry.     

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.     

A 3D-structural model of unsulfated chondroitin from high-field NMR: 4-sulfation has little effect on backbone conformation

The glycosaminoglycan chondroitin sulfate is essential in human health and disease but exactly how sulfation dictates its 3D-strucutre at the atomic level is unclear. To address this, we have purified homogenous oligosaccharides of unsulfated chondroitin (with and without ¹⁵N-enrichment) and analysed them by high-field NMR to make a comparison published chondroitin sulfate and hyaluronan 3D-structures. The result is the first full assignment of the tetrasaccharide and an experimental 3D-model of the hexasaccharide (PDB code 2KQO). In common with hyaluronan, we confirm that the amide proton is not involved in strong, persistent inter-residue hydrogen bonds. However, in contrast to hyaluronan, a hydrogen bond is not inferred between the hexosamine OH-4 and the glucuronic acid O5 atoms across the β(1→3) glycosidic linkage. The unsulfated chondroitin bond geometry differs slightly from hyaluronan by rotation about the β(1→3) ψ dihedral (as previously predicted by simulation), while the β(1→4) linkage is unaffected. Furthermore, comparison shows that this glycosidic linkage geometry is similar in chondroitin-4-sulfate. We therefore hypothesise that both hexosamine OH-4 and OH-6 atoms are solvent exposed in chondroitin, explaining why it is amenable to sulfation and hyaluronan is not, and also that 4-sulfation has little effect on backbone conformation. Our conclusions exemplify the value of the 3D-model presented here and progress our understanding of glycosaminoglycan molecular properties.     

Formation of dermatan sulfate by cultured human skin fibroblasts. Effects of sulfate concentration on proportions of dermatan/chondroitin

[3H,35S]Dermatan/chondroitin sulfate glycosaminoglycans produced during culture of fibroblasts in medium containing varying concentrations of sulfate were tested for their susceptibility to chondroitin ABC lyase and chondroitin AC lyase. Chondroitin ABC lyase completely degraded [3H]hexosamine-labeled and [35S] sulfate-labeled dermatan/chondroitin sulfate to disaccharides. Chondroitin AC lyase treatment of the labeled glycosaminoglycans produced different results. With this enzyme, dermatan/chondroitin sulfate formed at high concentrations of sulfate yielded small glycosaminoglycans and larger oligosaccharides but almost no disaccharide. This indicated that the dermatan/chondroitin sulfate co-polymer contained mostly iduronic acid with only an occasional glucuronic acid. As the medium sulfate concentration was progressively lowered, there was a concomitant increase in the susceptibility to degradation by chondroitin AC lyase. Thus, the labeled glycosaminoglycans formed at the lowest concentration of sulfate yielded small oligosaccharides including substantial amounts of disaccharide. The smaller chondroitin AC lyase-resistant [3H,35S]dermatan/chondroitin sulfate oligosaccharides were analyzed by gel filtration. Results indicated that, in general, the iduronic acid-containing disaccharide residues present in the undersulfated [3H,35S]glycosaminoglycan were sulfated, whereas the glucuronic acid-containing disaccharide residues were non-sulfated. This work confirms earlier reports that there is a relationship between epimerization and sulfation. Moreover, it demonstrates that medium sulfate concentration is critical in determining the proportions of dermatan to chondroitin (iduronic/glucuronic acid) produced by cultured cells.     

Biosynthesis of chondroitin/dermatan sulfate

Chondroitin sulfate and dermatan sulfate are synthesized as galactosaminoglycan polymers containing N-acetylgalactosmine alternating with glucuronic acid. The sugar residues are sulfated to varying degrees and positions depending upon the tissue sources and varying conditions of formation. Epimerization of any of the glucuronic acid residues to iduronic acid at the polymer level constitutes the formation of dermatan sulfate. Chondroitin/dermatan glycosaminoglycans are covalently attached by a common tetrasaccharide sequence to the serine residues of core proteins while they are adherent to the inner surface of endoplasmic reticulum/Golgi vesicles. Addition of the first sugar residue, xylose, to core proteins begins in the endoplasmic reticulum, followed by the addition of two galactose residues by two distinct glycosyl transferases in the early cis/medial regions of the Golgi. The linkage tetrasaccharide is completed in the medial/trans Golgi by the addition of the first glucuronic acid residue, followed by transfer of N-acetylgalactosamine to initiate the formation of a galactosaminoglycan rather than a glucosaminoglycan. This specific N-acetylgalactosaminyl transferase is different from the chondroitin synthase involved in generation of the repeating disaccharide units to form the chondroitin polymer. Sulfation of the chondroitin polymer by specific sulfotransferases occurs as the polymer is being formed. All the enzymes in the pathway for synthesis have been cloned, with the exception of the glucuronyl to iduronyl epimerase involved in the formation of dermatan residues.     

Liquid chromatography-mass spectrometry to study chondroitin lyase action pattern

Liquid chromatography-mass spectrometry was applied to determine the action pattern of different chondroitin lyases. Two commercial enzymes, chondroitinase ABC (Proteus vulgaris) and chondroitinase ACII (Arthrobacter aurescens), having action patterns previously determined by viscosimetry and gel electrophoresis were first examined. Next, the action patterns of recombinant lyases, chondroitinase ABC from Bacteroides thetaiotaomicron (expressed in Escherichia coli) and chondroitinase AC from Flavobacterium heparinum (expressed in its original host), were examined. Chondroitin sulfate A (CS-A, also known as chondroitin-4-sulfate) was used as the substrate for these four lyases. Aliquots taken at various time points were analyzed. The products of chondroitinase ABC (P. vulgaris) and chondroitinase AC (F. heparinum) contained unsaturated oligosaccharides of sizes ranging from disaccharide to decasaccharide, demonstrating that both are endolytic enzymes. The products afforded by chondroitinase ABC (B. thetaiotaomicron) and chondroitinase ACII (A. aurescens) contained primarily unsaturated disaccharide. These two exolytic enzymes showed different minor products, suggesting some subtle specificity differences between the actions of these two exolytic lyases on chondroitin sulfate A.     

Chondroitin C lyase [4.2.2.] is unable to cleave fructosylated sequences inside the partially fructosylated <Emphasis Type="Italic">Escherichia coli</Emphasis> K4 polymer

Chondroitin C lyase was demonstrated to be unable to act on fructosylated sequences inside a partially fructosylated polysaccharide having the chondroitin backbone structure, the Escherichia coli K4 polymer, using different analytical approaches. Chondroitin C lyase produced various unsaturated oligosaccharides by acting on an approximately 27%-fructosylated K4 polymer. The online HPLC-ESI-MS approach showed the disaccharide nature of the main species produced by chondroitinase C as DeltaHexA-GalNAc. Furthermore, the non-digested sequences inside the K4 polymer were demonstrated to be oligosaccharides bearing a fructose for each glucuronic acid unit. In fact, unsaturated fully fructosylated oligomers, from tetrasaccharide to decasaccharide (DeltaHexA(Fru)-GalNAc-[GlcA(Fru)-GalNAc](n) with n between 1 and 4), at decreasing percentages, were produced by the enzyme. These results clearly indicate that chondroitinase C cleaved the innermost glucuronic acid-N-acetylgalactosamine linkage without affecting the 1,4 glycosidic linkage between fructosylated glucuronic acid and N-acetylgalactosamine residues, confirming that the 3-O-fructosylation of the GlcA residue renders the polysaccharide resistant to the enzyme action. This novel specific activity of chondroitinase C was also useful for the production of discrete microgram amounts of fully fructosylated oligomers, from 4- to 10-mers, from E. coli K4 for possible further studies and applications.     

Crystal structure of chondroitin AC lyase, a representative of a family of glycosaminoglycan degrading enzymes.

Glycosaminoglycans (GAGs), highly sulfated polymers built of hexosamine-uronic acid disaccharide units, are major components of the extracellular matrix, mostly in the form of proteoglycans. They interact with a large array of proteins, in particular of the blood coagulation cascade. Degradation of GAGs in mammalian systems occurs by the action of GAG hydrolases. Bacteria express a large number of GAG-degrading lyases that break the hexosamine-uronic acid bond to create an unsaturated sugar ring. Flavobacterium heparinum produces at least five GAG lyases of different specificity. Chondroitin AC lyase (chondroitinase AC, 75 kDa) is highly active toward chondroitin 4-sulfate and chondroitin-6 sulfate. Its crystal structure has been determined to 1.9 A resolution. The enzyme is composed of two domains. The N-terminal domain of approximately 300 residues contains mostly alpha-helices which form a doubly-layered horseshoe (a subset of the (alpha/alpha)6 toroidal topology). The approximately 370 residues long C-terminal domain is made of beta-strands arranged in a four layered beta-sheet sandwich, with the first two sheets having nine strands each. This fold is novel and has no counterpart in full among known structures. The sequence of chondroitinase AC shows low level of homology to several hyaluronate lyases, which likely share its fold. The shape of the molecule, distribution of electrostatic potential, the pattern of conservation of the amino acids and the results of mutagenesis of hyaluronate lyases, indicate that the enzymatic activity resides primarily within the N-terminal domain. The most likely candidate for the catalytic base is His225. Other residues involved in catalysis and/or substrate binding are Arg288, Arg292, Lys298 and Lys299.     

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.     

Studies on the chemistry of arterial wall, XVII. Metabolic characteristics of different types of chondroitin sulfate-dermatan sulfate hybrids in arterial tissue.

1) Chondroitin sulfate and dermatan sulfate of bovine arterial tissue exist as copolymers with a varying degree of hybridization between chondroitin and dermatan sulfates. A fraction rich in dermatan sulfate hybridized with 20% chondroitin sulfate (termed DS-rich hybrid) and a fraction rich in chondroitin sulfate containing 17% DS as copolymer constituent (CS-rich hybrid) can be isolated by the subfractionation of the arterial tissue CS-DS preparation. 2) When arterial tissue segments were preincubated with [14C]glucosamine, 95% of the radioactivity incorporated into the glycosaminoglycans was found to be present in the galactosamine moiety of all of the CS-DS subfractions, whereas the relative proportion of 14C radioactivity incorporated into the galactosamine and uronic acid components was 51:49 following preincubation with [14C]glucose. In both experiments the specific radioactivity of the DS-rich hybrids was twice as high as that of the CS-rich hybrids. 3) Enzymatic degradation of the hybrid CS-DS subfractions by chondroitin AC and ABC lyases revealed that the specific radioactivity of the CS and DS disaccharide units released from the DS-rich hybrids was twice as high as those isolated from the CS-rich hybrids, but within each hybrid fraction the galactosamine moieties of the CS and DS units and their glucuronic and iduronic acid components exhibited equal specific radioactivities. 4) The results strongly support the assumption that distinct compartments exist for the formation of hybrid CS-DS proteoglycans with different proportions of CS and DS.     

Structures of Streptococcus pneumoniae hyaluronate lyase in complex with chondroitin and chondroitin sulfate disaccharides. Insights into specificity and mechanism of action

Streptococcus pneumoniae hyaluronate lyase is a surface enzyme of this Gram-positive bacterium. The enzyme degrades hyaluronan and chondroitin/chondroitin sulfates by cleaving the beta1,4-glycosidic linkage between the glycan units of these polymeric substrates. This degradation helps spreading of this bacterial organism throughout the host tissues and facilitates the disease process caused by pneumococci. The mechanism of this degradative process is based on beta-elimination, is termed proton acceptance and donation, and involves selected residues of a well defined catalytic site of the enzyme. The degradation of hyaluronan alone is thought to proceed through a processive mode of action. The structures of complexes between the enzyme and chondroitin as well as chondroitin sulfate disaccharides allowed for the first detailed insights into these interactions and the mechanism of action on chondroitins. This degradation of chondroitin/chondroitin sulfates is nonprocessive and is selective for the chondroitin sulfates only with certain sulfation patterns. Chondroitin sulfation at the 4-position on the nonreducing site of the linkage to be cleaved or 2-sulfation prevent degradation due to steric clashes with the enzyme. Evolutionary studies suggest that hyaluronate lyases evolved from chondroitin lyases and still retained chondroitin/chondroitin sulfate degradation abilities while being specialized in the degradation of hyaluronan. The more efficient processive degradation mechanism has come to be preferred for the unsulfated substrate hyaluronan.     

The synthesis of a novel thio-linked disaccharide of chondroitin as a potential inhibitor of polysaccharide lyases

A thio-linked disaccharide based on the structure of the glycosaminoglycan chondroitin was synthesized as a potential inhibitor of chondroitin AC lyase from Flavobacterium heparinum for structural analysis of the active site. Instead it was found to be a slow substrate, thereby demonstrating that lyases, in contrast to glycosidases, can cleave thioglycoside links between sugars.     

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.     

Enzymatic determination of free glucuronic acid with glucuronolactone reductase. I. Isolation and purification of glucuronolactone reductase from rat kidney

Glucuronolactone reductase [EC 1.1.1.20] from rat kidney was purified over 300-fold by ammonium sulfate fractionation, chromatography on DEAE-cellulose and hydroxylapatite columns, and preparative isoelectric focusing. The substrate specificity of the enzyme in the reduction reaction was broad, and hexuronic acid was one of the best substrates among monosaccharides. Km values for D-glucuronic acid, D-glucuronolactone, D-galacturonic acid, and L-iduronic acid were 6, 9, 4, and 6 mM, respectively. An investigation of the activity for aldose led to the finding that triose and tetrose served as good substrates for this enzyme. However, the activity for aldopentose or aldohexose was less than 1% of that for D-glucuronic acid at the same concentration. The enzyme was inactive towards most hexosamines (galactosamine, mannosamine, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine, but not glucosamine), meso-inositol, D-fructose, and tetrasaccharides from hyaluronic acid and chondroitin 4-sulfate. Trisaccharides from hyaluronic acid and chondroitin 6-sulfate which possess glucuronic acid at the reducing end were poor substrates for the enzyme and the activity towards these 4-substituted glucuronic acids was less than 3% of that towards non-substituted glucuronic acid.     

Baculovirus envelope protein ODV-E66 is a novel chondroitinase with distinct substrate specificity

Chondroitin sulfate is a linear polysaccharide of alternating D-glucuronic acid and N-acetyl-D-galactosamine residues with sulfate groups at various positions of the sugars. It interacts with and regulates cytokine and growth factor signal transduction, thus influencing development, organ morphogenesis, inflammation, and infection. We found chondroitinase activity in medium conditioned by baculovirus-infected insect cells and identified a novel chondroitinase. Sequence analysis revealed that the enzyme was a truncated form of occlusion-derived virus envelope protein 66 (ODV-E66) of Autographa californica nucleopolyhedrovirus. The enzyme was a novel chondroitin lyase with distinct substrate specificity. The enzyme was active over a wide range of pH (pH 4-9) and temperature (30-60 °C) and was unaffected by divalent metal ions. The ODV-E66 truncated protein digested chondroitin most efficiently followed by chondroitin 6-sulfate. It degraded hyaluronan to a minimal extent but did not degrade dermatan sulfate, heparin, and N-acetylheparosan. Further analysis using chemo-enzymatically synthesized substrates revealed that the enzyme specifically acted on glucuronate residues in non-sulfated and chondroitin 6-sulfate structures but not in chondroitin 4-sulfate structures. These results suggest that this chondroitinase is useful for detailed structural and compositional analysis of chondroitin sulfate, preparation of specific chondroitin oligosaccharides, and study of baculovirus infection mechanism.     

Chondroitin O-methyl ester: an unusual substrate for chondroitin AC lyase

Chondroitin O-methyl ester was depolymerized by chondroitin AC lyase (EC 4.2.2.5) from Flavobacterium heparinum. The major product isolated from the depolymerization reaction was found to be methyl alpha-L-threo-hex-4-enopyranosyluronate-(1-->4)-2-acetamido-2-deoxy-alpha,beta-D-galactopyranoside.     

Composite active site of chondroitin lyase ABC accepting both epimers of uronic acid

Enzymes have evolved as catalysts with high degrees of stereospecificity. When both enantiomers are biologically important, enzymes with two different folds usually catalyze reactions with the individual enantiomers. In rare cases a single enzyme can process both enantiomers efficiently, but no molecular basis for such catalysis has been established. The family of bacterial chondroitin lyases ABC comprises such enzymes. They can degrade both chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans at the nonreducing end of either glucuronic acid (CS) or its epimer iduronic acid (DS) by a beta-elimination mechanism, which commences with the removal of the C-5 proton from the uronic acid. Two other structural folds evolved to perform these reactions in an epimer-specific fashion: (alpha/alpha)(5) for CS (chondroitin lyases AC) and beta-helix for DS (chondroitin lyases B); their catalytic mechanisms have been established at the molecular level. The structure of chondroitinase ABC from Proteus vulgaris showed surprising similarity to chondroitinase AC, including the presence of a Tyr-His-Glu-Arg catalytic tetrad, which provided a possible mechanism for CS degradation but not for DS degradation. We determined the structure of a distantly related Bacteroides thetaiotaomicron chondroitinase ABC to identify additional structurally conserved residues potentially involved in catalysis. We found a conserved cluster located approximately 12 A from the catalytic tetrad. We demonstrate that a histidine in this cluster is essential for catalysis of DS but not CS. The enzyme utilizes a single substrate-binding site while having two partially overlapping active sites catalyzing the respective reactions. The spatial separation of the two sets of residues suggests a substrate-induced conformational change that brings all catalytically essential residues close together.     

Purification and characterization of a 3'-phosphoadenylylsulfate:chondroitin 6-sulfotransferase from arterial tissue

A 3'-phosphoadenylylsulfate:chondroitin sulfotransferase (EC 2.8.2.5) was purified to homogeneity (about 760-fold) from the cytosolic fraction of calf arterial tissue by Con A-Sepharose, ion exchange and affinity chromatography. The enzyme has a molecular mass of 38000 Da, optimal activity at pH 6.0 (100%) and 7.25 (75%), requires divalent cations for maximal activity (Mn2+ greater than Mg2+, Ca2+) and exhibits specificity towards desulfated chondroitin sulfate and oligosaccharides derived therefrom. The enzyme transfers sulfate groups from [35S]phosphoadenylylsulfate exclusively to C-6 OH groups of N-acetylgalactosamine units of the acceptor substrates. Maximal sulfate transfer occurs at 2mM chondroitin disaccharide units (100%), the transfer rates decreasing with decreasing chain length in the order deca (55%), octa (17%) and hexasaccharides (4%). Lineweaver-Burk plots revealed equal maximal velocities for chondroitin, deca-, octa- and hexasaccharide, but decreasing Km values. Chondroitin 4-sulfate has 21% of the acceptor potency exhibited by chondroitin, whereas dermatan sulfate, heparan sulfate and hyaluronate and the chondroitin tetrasaccharide showed no acceptor properties. Analysis of the reaction products formed by prolonged enzymatic sulfation of a reduced chondroitin hexasaccharide [GlcA-GalNAc]2-GlcA-GalNAc-ol revealed that the preterminal N-acetylgalactosamine from the non-reducing end and the internal N-acetylgalactosamine but not the N-acetylgalactosaminitol were sulfated and that no hexasaccharide disulfate was formed by the action of chondroitin 6-sulfotransferase. Chondroitin 6-sulfotransferase is considered to possess a binding region capable of accommodating a nonsulfated oligosaccharide sequence of at least six sugars and is believed to act in the course of chondroitin sulfate synthesis in cooperation with, but shortly after, the enzymes involved in the chain elongation reaction.