Structural Determinants of Substrate Recognition and Catalysis by Heparan Sulfate Sulfotransferases

Heparan
sulfate (HS) and heparin contain imprinted “sulfation
codes”, which dictate their diverse physiological and pathological
functions. A group of orchestrated biosynthetic enzymes cooperate
in polymerizing and modifying HS chains. The biotechnological development
of enzymes that can recreate this sulfation pattern on synthetic heparin
is challenging, primarily due to the paucity of quantitative data
for sulfotransferase enzymes. Herein, we identified critical structural
characteristics that determine substrate specificity and shed light
on the catalytic mechanism of sugar sulfation of two HS sulfotransferases,
2-O-sulfotransferase (HS2ST) and 6-O-sulfotransferase (HS6ST). Two
sets of molecular clamps in HS2ST recognize appropriate substrates;
these clamps flank the acceptor binding site on opposite sides. The
hexuronic epimers, and not their puckers, have a critical influence
on HS2ST selectivity. In contrast, HS6ST recognizes a broader range
of substrates. This promiscuity is granted by a conserved tryptophan
residue, W210, that positions the acceptor within the active site
for catalysis by means of strong electrostatic interactions. Lysines
K131 and K132 act in concert with a second tryptophan, W153, shedding
water molecules from within the active site, thus providing HS6ST
with a binding preference toward 2-O-sulfated substrates. QM/MM calculations
provided valuable mechanistic insights into the catalytic process,
identifying that the sulfation of both HS2ST and HS6ST follows a SN2-like
mechanism. When they are taken together, our findings reveal the molecular
basis of how these enzymes recognize different substrates and catalyze
sugar sulfation, enabling the generation of enzymes that could create
specific heparin epitopes.