| Abstract: | Heparan sulfate (HS) is an abundant polysaccharide in the animal kingdom with essentialphysiological functions. HS is composed of sulfated saccharides that are biosynthesized through acomplex pathway involving multiple enzymes. In vivo regulation of this processremains unclear. HS 2-O-sulfotransferase (2OST) is a key enzyme in this pathway.Here, we report the crystal structure of the ternary complex of 2OST, 3′-phosphoadenosine5′-phosphate, and a heptasaccharide substrate. Utilizing site-directed mutagenesis andspecific oligosaccharide substrate sequences, we probed the molecular basis of specificity and 2OSTposition in the ordered HS biosynthesis pathway. These studies revealed that Arg-80, Lys-350, andArg-190 of 2OST interact with the N-sulfo groups near the modification site,consistent with the dependence of 2OST on N-sulfation. In contrast,6-O-sulfo groups on HS are likely excluded by steric and electrostatic repulsionwithin the active site supporting the hypothesis that 2-O-sulfation occurs prior to6-O-sulfation. Our results provide the structural evidence for understanding thesequence of enzymatic events in this pathway. |
