Heparan Sulfate/HeparinN-Deacetylase/N-Sulfotransferase

Abstract

GlycosaminoglycanN-acetylglucosaminylN-deacetylases/N-sulfotransferases are structurally related enzymes that play an important role in the biosynthesis of heparan sulfate and heparin. They are dual catalytic, single membrane-spanning polypeptides of approximately 850–880 amino acids that catalyze the N-deacetylation ofN-acetylglucosamine of glycosaminoglycans followed byN-sulfation of the same sugar. On the basis of homologies of these proteins with other N-acetylglucosaminylN-deacetylases involved in the biosynthesis of chitin and putative deacetylases from bacteria, we have constructed two soluble chimeras between protein A and the amino- and carboxyl-terminal halves of the above mastocytoma holoenzyme. The carboxyl-terminal chimera half (amino acids 479–880) was able to catalyze the N-sulfation of glucosamine of heparan sulfate with a similar affinity for its two substrates, adenosine 3′-phosphate 5′-phosphosulfate and heparan sulfate, as the holoenzyme. However, the reaction only occurred at 30 °C and not at 37 °C, both temperatures at which the holoenzyme was active. The V _max of the chimera was 10–20-fold slower than that of the holoenzyme. Soluble chimeras between protein A and amino acids 43–521 and 43–680 of the holoenzyme were unable to catalyze the N-deacetylation of the bacterial N-acetylglucosaminyl-glucuronic acid polymer K5 under conditions where the holoenzyme was active. The recent appearance in genome data banks of homologs to the N-sulfotransferase domain and now the direct demonstration that this domain catalyzes this reaction raises the possibility that both N-deacetylation and N-sulfation activities of the holoenzyme might have emerged as gene fusions during evolution.