Using engineered 6-O-sulfotransferase to improve the synthesis of anticoagulant heparin

Abstract

Heparan sulfate (HS) and heparin are sulfated polysaccharides exhibiting diverse physiological functions. HS 6-O-sulfotransferase (6-OST) is a HS biosynthetic enzyme that transfers a sulfo group to the 6-OH position of...

Deciphering the substrate recognition mechanisms of the heparan sulfate 3-O-sulfotransferase-3

“…Structurally guided mutagenesis within the substrate binding pocket of 6-OST and 2-OST have led to greater control of chemoenzymatic synthesis. 27,28 This study provides clues to regulating 3-OST-3 specificity, generating complex sulfation patterns beyond what is available using wild-type enzyme. The availability of structurally homogeneous HS oligosaccharides with unique sulfation patterns should offer a useful tool to examine the individualized effects of specific sulfated carbohydrate sequences in a biological system.…”

“…Structurally guided mutagenesis within the substrate binding pocket of 6-OST and 2-OST have led to greater control of chemoenzymatic synthesis 27,28 . This study provides clues to regulating 3-OST-3 specificity, generating complex sulfation patterns beyond what is available using wild-type enzyme.…”

From Steroid and Drug Metabolism to Glycobiology, Using Sulfotransferase Structures to Understand and Tailor Function

“…Based on crystal structures of sulfotransferases engaged with oligosaccharide substrates, some of these enzymes have been engineered via site-directed mutagenesis to alter their specificities to improve control of the chemoenzymatic pathway (Liu et al, 2014;Liu et al, 2012;Xu et al, 2008;Xu et al, 2017b;Yi et al, 2020). Particularly, a triple mutation of zf6-OST-3 designed based on the crystal structures, allows for sulfation only on the non-reducing terminal glucosamine, providing fine-tuned control of 6-O-sulfation in the chemoenzymatic synthesis pathway (Yi et al, 2020). Cytosolic sulfotransferases are also being investigated for their ability to generate sulfated products for biological reagents and drug discovery (Lamb et al, 2006;Matsumura et al, 2018;Shi et al, 2012;Shimohira et al, 2017;Xie et al, 2020).…”

Investigation of the biological functions of heparan sulfate using a chemoenzymatic synthetic approach

“…As an example, we demonstrated that a 6-OST-3 mutant, 6-OST-3 R112E/R206E/R329E, adds additional selectivity to introduce the 6- O -sulfation. 29 Three positively charged amino acid residues, including R112, R206 and R329, are used by the enzyme to balance the negative charge from substrates or products with multiple 6- O -sulfo groups. Substituting these positively charge amino acid residues with negatively charged residues removes the enzyme's ability to transfer multiple 6- O -sulfo groups to an oligosaccharide substrate.…”

“…The 6-OST-3 mutant expands the ability to synthesize partially 6- O -sulfated oligosaccharides. 29 Numerous crystal structures of other HS sulfotransferases have been reported, revealing how the enzymes interact with substrates. 30–33 The structural information will guide engineering efforts to create the sulfotransferases with desired substrate specificities.…”