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.
Highlights
Specific for each position [3]
The presence of its epimer ␣-Liduronic acid (IdoA) is critical in mediating several biological functions of dermatan sulfate, such as the binding to hepatocyte growth factor and heparin cofactor II [1]
Based on the modeled enzyme and site-directed mutagenesis, we propose a mechanism of epimerization involving two histidines (His-205 and His-450) and one tyrosine (Tyr-261) as putative catalytic amino acids with the formation of a 4,5-unsaturated hexuronic acid intermediate
Summary
Specific for each position [3]. The degree of epimerization and sulfation and the position of the sulfate groups vary during development and among tissues and are regulated by growth factors [4]. Based on sequence similarity with the DS-epimerase, a second epimerase has been identified, and its activity has been established.. The polymer level stereochemical inversion of the C5 carboxyl group of GlcA from an equatorial to an axial position, thereby forming the epimer IdoA, is a chemically challenging reaction. It requires the abstraction of the C5 proton, which has a very high pKa, and the subsequent reprotonation has to take place from the opposite side of the sugar plane. Based on the modeled enzyme and site-directed mutagenesis, we propose a mechanism of epimerization involving two histidines (His-205 and His-450) and one tyrosine (Tyr-261) as putative catalytic amino acids with the formation of a 4,5-unsaturated hexuronic acid intermediate
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
