Journal
BIOCHEMISTRY
Volume 41, Issue 23, Pages 7424-7434Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi012147o
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- NCI NIH HHS [CA 090940] Funding Source: Medline
- NIGMS NIH HHS [5T32 GM 08334, GM 057073] Funding Source: Medline
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The soil bacterium Flavobacterium heparinum produces several enzymes that degrade heparan sulfate glycosaminoglycans (HSGAGs) in a sequence-specific manner. Among others, these enzymes include the heparinases and an unusual glycuronidase that hydrolyzes the unsaturated Delta4,5 uronic acid at the nonreducing end of oligosaccharides resulting from prior heparinase eliminative cleavage. We report here the molecular cloning of the Delta4,5 glycuronidase gene from the flavobacterial genome and its recombinant expression in Escherichia coli as a highly active enzyme. We also report the biochemical and kinetic characterization of this enzyme, including an analysis of its substrate specificity. We find that the Delta4,5 glycuronidase discriminates on the basis of both the glycosidic linkage and the sulfation pattern within its saccharide substrate. In particular, we find that the glycuronidase displays a strong preference for 1-->4 linkages, making this enzyme specific to heparin/heparan sulfate rather than 1-->3 linked glycosaminoglycans such as chondroitin/dermatan sulfate or hyaluronan. Finally, we demonstrate the utility of this enzyme in the sequencing of heparinase-derived HSGAG oligosaccharides.
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