Abstract
Sulfation patterns along glycosaminoglycan (GAG) chains dictate their functional role. The N-deacetylase N-sulfotransferase family (NDST) catalyzes the initial downstream modification of heparan sulfate and heparin chains by removing acetyl groups from subsets of N-acetylglucosamine units and, subsequently, sulfating the residual free amino groups. These enzymes transfer the sulfuryl group from 3′-phosphoadenosine-5′-phosphosulfate (PAPS), yielding sulfated sugar chains and 3′-phosphoadenosine-5′-phosphate (PAP). For the N-sulfotransferase domain of NDST1, Lys833 has been implicated to play a role in holding the substrate glycan moiety close to the PAPS cofactor. Additionally, Lys833 together with His716 interact with the sulfonate group, stabilizing the transition state. Such a role seems to be shared by Lys614 through donation of a proton to the bridging oxygen of the cofactor, thereby acting as a catalytic acid. However, the relevance of these boundary residues at the hydrophobic cleft is still unclear. Moreover, whether Lys833, His716 and Lys614 play a role in both glycan recognition and glycan sulfation remains elusive. In this study we evaluate the contribution of NDST mutants (Lys833, His716 and Lys614) to dynamical effects during sulfate transfer using comprehensive combined docking and essential dynamics. In addition, the binding location of the glycan moiety, PAPS and PAP within the active site of NDST1 throughout the sulfate transfer were determined by intermediate state analysis. Furthermore, NDST1 mutants unveiled Lys833 as vital for both the glycan binding and subsequent N-sulfotransferase activity of NDST1.
Highlights
Sulfotransferases (STs) are a large family of enzymes that catalyze sulfate conjugation to carbohydrates, proteins, and a variety of metabolic compounds
For the docking experiments, the sulfuryl group was added to the phosphoadenosine 59-phosphate (PAP) molecule before the disaccharide docking, resulting in a specular approach of catalytic residues to the substrate
The interaction modes of the a-GlcN(1R4)-glucuronic acid (GlcA) and NST are shown in Fig. 2, Fig. S1 and the distances listed in Table 1, where only the mutated amino acids are displayed
Summary
Sulfotransferases (STs) are a large family of enzymes that catalyze sulfate conjugation to carbohydrates, proteins, and a variety of metabolic compounds. A conserved histidine deprotonates this acceptor, prompting the sulfur atom for the PAPS attack [9,10], building a negative charge on the bridging oxygen atom from PAPS and so assisting its dissociation by interaction with the conserved serine [7,9] While it is still unknown whether this mechanism occurs in a sequential or random manner, recent reports have demonstrated the influence of many residues in this process, notably, two lysine residues stabilize the transition state by interacting with the bridging oxygen between the sulfate and phosphate groups of PAPS [12,13]
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