An active sulfotransferase (ST, residues 558-882) domain of the human heparan sulfate N-deacetylase/N-sulfotransferase (hHSNST) has been identified by aligning the amino acid sequence of hHSNST to that of mouse estrogen sulfotransferase (EST). The bacterially expressed ST domain transfers the 5'-sulfuryl group of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to only deacetylated heparin with an efficiency similar to that previously reported for the purified rat HSNST. Moreover, the K(m,PAPS) (2.1 microM) of the ST domain is also similar to that of the rat enzyme. Lys48 is a key residue in mEST catalysis. The residue corresponding to Lys48 is conserved in all known heparan sulfate sulfotransferases (Lys614 in the ST domain of hHSNST). Mutation of Lys614 to Ala abolishes N-sulfotransferase activity, indicating an important catalytic role of Lys614 in the ST domain. Crystals of the ST domain have been grown (orthorhombic space group P2(1)2(1)2) with diffraction to 2.5 A resolution.