Purification, characterization and specificity of chondroitin lyases and glycuronidase from Flavobacterium heparinum

Abstract

The chondroitin lyases from Flavobacterium heparinum (Cytophaga heparinia) have been widely used in depolymerization of glycosaminoglycan and proteoglycan chondroitin sulphates. Oligosaccharide products derived from chondroitin sulphate can be further degraded by glycuronidases and sulphatases obtained from the same organism. There has been no reported purification of these enzymes to homogeneity nor is there any information on their physical and kinetic characteristics. The absence of pure enzymes has resulted in a lack of understanding of the optimal conditions for their catalytic activity and their substrate specificity. This has limited the use of these enzymes as reagents for preparation of oligosaccharides for structure and activity studies. Reproducible schemes to purify a chondroitin AC lyase, a glycuronidase and chondroitin B lyase from Flavobacterium heparinum to apparent homogeneity are described. Chondroitin AC lyase (chondroitinase AC, EC 4.2.2.5), glycuronidase [chondro-(1-->3)-glycuronidase, no EC number] and chondroitin B lyase (chondroitinase B, no EC number) have M(r) values (assessed by SDS/PAGE) of 74,000, 41,800 and 55,200 respectively, and isoelectric points (determined by isoelectric focusing) of 8.85, 9.28 and 9.05 respectively. Chondroitin lyase AC and B contain pyroglutamic acid at their N-termini precluding their analysis by Edman degradation. Deblocking with pyroglutamate aminopeptidase facilitated the determination of their N-terminal sequences. The kinetic properties of these enzymes have been determined as well as the optimum conditions for their catalytic activity. The specificity of the glycouronidase, determined using 17 different disaccharide substrates, shows that it only acts on unsulphated or 6-O-sulphated 1-->3 linkages. The chondroitin lyases are both endolytic enzymes, and oligosaccharide mapping shows their expected specificity towards the chondroitin and dermatan sulphate polymers.

Crystal Structure of a Bacterial Unsaturated Glucuronyl Hydrolase with Specificity for Heparin

“…Although a UGL that degrades unsaturated chondroitin disaccharide (C⌬6S) was previously isolated as a 1,3-glycuronidase from P. heparinus (37), this enzyme differs from Phep_2238 and Phep_2649 in amino acid composition, isoelectric point, and molecular weight. In contrast, Phep_2830 exhibited enzyme activity toward only unsaturated heparin disaccharides, as described previously (18).…”

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

“…6 While there is no absolute requirement of a metal ion for chondroitin AC lyase activity, various mono-and di-valent metals have been shown to influence enzyme activity. 3 The current study demonstrates that chondroitin AC lyase from Flavobacterium heparinum can also act on chondroitin O -methyl ester. The discovery of this new substrate is significant in that the negatively charged carboxyl group of GlcAp in the natural substrate is replaced with a neutral carboxyl methyl ester, clarifying the role of negative charge at this site in the substrate on enzyme activity.…”

Crystallization and preliminary X-ray analysis of chondroitinase B fromFlavobacterium heparinum

“…However, for the recombinant enzyme puri®cation, only three columns were employed: SP-Sepharose Big Beads (Pharmacia Biotech, Uppsala, Sweden), carboxy-sulfon (40 mm) cation exchange (J. T. Baker Inc., Phillispsburg, NJ, USA) and ceramic hydroxyapatite (20 mm; American International Chemical Inc., Natick, MA, USA). Enzyme activity was measured spectrophotometrically by monitoring the formation of unsaturated oligosaccharide products released from dermatan sulfate at 232 nm (Gu et al, 1995). Chondroitinase B was apparently homogeneous following hydroxyapatite chromatography as determined by SDS±PAGE on a 12%(w/v) separating gel and staining with Coomassie Blue.…”

“…Cells were grown at 296 K in shake¯asks and were harvested when the culture density reached A 600 = 3.0. For puri®cation of chondroitinase B, a procedure similar to that used to purify the wild-type enzyme was used (Gu et al, 1995). However, for the recombinant enzyme puri®cation, only three columns were employed: SP-Sepharose Big Beads (Pharmacia Biotech, Uppsala, Sweden), carboxy-sulfon (40 mm) cation exchange (J. T. Baker Inc., Phillispsburg, NJ, USA) and ceramic hydroxyapatite (20 mm; American International Chemical Inc., Natick, MA, USA).…”

“…Chondroitinase B is a monomeric enzyme of 506 residues with an isoelectric point of 9.05 (Gu et al, 1995). An N-terminal signal sequence of 25 residues is cleaved to yield the mature protein (M r = 53 600 Da) upon export to the F. heparinum periplasmic space.…”