|17 (Research article)
|Pacheco, Benny; Maccarana, Marco; Goodlett, David; Malmström, Anders; Malmström, Lars
|Identification of the active site of DS-epimerase 1 and requirement of N-glycosylation for enzyme function.
|J Biol Chem (2009) 284 1741-7
|41 citations (journal impact: 5.33)
|Dermatan sulfate is a highly sulfated polysaccharide and has a variety of biological functions in development and disease. Iduronic acid domains in dermatan sulfate which are formed by the action of two DS-epimerases have a key role in mediating these functions. We have identified the catalytic site and three putative catalytic residues in DS-epimerase 1 His205 Tyr261 and His450 by tertiary structure modeling and amino acid conservation to heparinase II. These residues were systematically mutated to alanine or more conserved residues which resulted in complete loss of epimerase activity. Based on these data and the close relationship between lyase and epimerase reactions we propose a model where His450 functions as a general base abstracting the C5-proton from glucuronic acid. Subsequent cleavage of the glycosidic linkage by Tyr261 generates a 45-unsaturated hexuronic intermediate which is protonated at the C5-carbon by His205 from the side of the sugar plane opposite to the side of previous proton abstraction. Concomitant recreation of the glycosidic linkage ends the reaction generating iduronic acid. In addition we show that proper N-glycosylation of DS-epimerase 1 is required for enzyme activity. This study represents the first description of the structural basis for epimerization by a glycosaminoglycan epimerase.