Uncovering the detailed mode of cleavage of heparinase I toward structurally defined heparin oligosaccharides.

For a more insightful investigation into the specificity of bacterial heparinase I, a series of structurally well-defined heparin oligosaccharides was synthesized using a highly efficient chemoenzymatic strategy. Apart from the primary cleavage site, five glycosidic linkages of oligosaccharides with varying modifications to obtain secondary cleavage sites were degraded by a high concentration of heparinase I. The reactivity of linkages toward heparinase I was not entirely dependent on the 2- O -sulfated iduronic acid being cleaved or the neighboring 6- O -sulfated glucosamine residues, but it was dependent on higher degrees of sulfation of oligosaccharides and indispensable N -substituted glucosamine adjacent to the cleavable linkage. Moreover, the enzyme demonstrated less preferential cleavage toward glycosidic linkages containing glucuronic acid than those containing iduronic acid of the counterpart oligosaccharides. Biolayer interferometry revealed differences in reactivity that are not completely consistent with different affinities of substrates to enzyme. Our study presented accurate information on the cleavage promiscuity of heparinase I that is crucial for heparin depolymerization. • Heparin oligosaccharides were chemoenzymatically synthesized as substrates of heparinase I. • Five secondary cleavage sites of oligosaccharides were degraded to less extents by heparinase I. • The enzymatic reactivity was dependent on different modification of uronic acid or GlcN. • Heparinase I had less preferential cleavage towards GlcA-formed linkages than IdoA. • BLI proved differences in reactivity are partially consistent with substrate affinity to enzyme. [ABSTRACT FROM AUTHOR]