title: Identification of the active site of DS-epimerase 1 and requirement of N-glycosylation for enzyme function.
abstract: 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, His-205, Tyr-261, and His-450, 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 His-450 functions as a general base abstracting the C5 proton from glucuronic acid. Subsequent cleavage of the glycosidic linkage by Tyr-261 generates a 4,5-unsaturated hexuronic intermediate, which is protonated at the C5 carbon by His-205 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.
authors.affiliation: Department of Experimental Medical Science, Lund University, Biomedical Center D12, SE-221 84 Lund, Sweden. benny.pacheco@med.lu.se
authors.forename: Benny
authors.initials: B
authors.lastname: Pacheco
authors.forename: Marco
authors.initials: M
authors.lastname: Maccarana
authors.forename: David R
authors.initials: DR
authors.lastname: Goodlett
authors.forename: Anders
authors.initials: A
authors.lastname: Malmström
authors.forename: Lars
authors.initials: L
authors.lastname: Malmström
mesh_terms: D019943:Amino Acid Substitution; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000502:physiology*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003871:Dermatan Sulfate / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology
chemical_list: D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D003871:Dermatan Sulfate; D011133:Polysaccharide-Lyases; C114663:heparinase II; C405383:DSE protein, human