Specificity and action pattern of heparanase Bp, a β-glucuronidase from Burkholderia pseudomallei

The specificity and action pattern of a β-glucuronidase derived from the pathogenic bacteria Burkholderia pseudomallei and expressed in Escherichia coli as a recombinant protein has been evaluated. While this enzyme shows activity on a number of glycosaminoglycans, our study has focused on its action on heparin, heparan sulfate and their biosynthetic intermediates as well as chemoenzymatically synthesized, structurally defined heparan sulfate oligosaccharides. These heparin/heparan sulfate (HP/HS) substrates examined varied in size and structure, but all contained an uronic acid (UA) residue β-(1→4) linked to a glucosamine residue. On the substrates tested, this enzyme (heparanase Bp) acted only on a glucuronic acid residue β-(1→4) linked to an N-acetylglucosamine, N-sulfoglucosamine or N-acetyl-6-O-sulfoglucosamine residue. A substrate was required to have a length of pentasaccharide or longer and heparanase Bp acted with a random endolytic action pattern on HP/HS. The specificity and glycohydrolase mechanism of action of heparanase Bp resembles mammalian heparanase and is complementary to the bacterial heparin lyases, which act through an eliminase mechanism on a glucosamine residue (1→4) linked to a UA residue, suggesting its utility as a tool for the structural determination of HP/HS as well as representing a possible model for the medically relevant mammalian heparanase. The utility heparanase Bp was demonstrated by the oligosaccharide mapping of heparin, which afforded resistant intact highly sulfated domains ranging from tetrasaccharide to >28-mer with a molecular weight >9000.

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

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.