title: Deciphering the Mode of Action of the Processive Polysaccharide Modifying Enzyme Dermatan Sulfate Epimerase 1 by Hydrogen-Deuterium Exchange Mass Spectrometry.
abstract: Distinct from template-directed biosynthesis of nucleic acids and proteins, the enzymatic synthesis of heterogeneous polysaccharides is a complex process that is difficult to study using common analytical tools. Therefore, the mode of action and processivity of those enzymes are largely unknown. Dermatan sulfate epimerase 1 (DS-epi1) is the predominant enzyme during the formation of iduronic acid residues in the glycosaminoglycan dermatan sulfate. Using recombinant DS-epi1 as a model enzyme, we describe a tandem mass spectrometry-based method to study the mode of action of polysaccharide processing enzymes. The enzyme action on the substrate was monitored by hydrogen-deuterium exchange mass spectrometry and the sequence information was then fed into mathematical models with two different assumptions of the mode of action for the enzyme: processive reducing end to non-reducing end, and processive non-reducing end to reducing end. Model data was scored by correlation to experimental data and it was found that DS-epi1 attacks its substrate on a random position, followed by a processive mode of modification towards the non-reducing end and that the substrate affinity of the enzyme is negatively affected by each additional epimerization event. It could also be shown that the smallest active substrate was the reducing end uronic acid in a tetrasaccharide and that octasaccharides and longer oligosaccharides were optimal substrates. The method of using tandem mass spectrometry to generate sequence information of the complex enzymatic products in combination within silicomodeling can be potentially applied to study the mode of action of other enzymes involved in polysaccharide biosynthesis.
authors.affiliation: Department of Experimental Medical Science, Lund University, Sweden.
authors.forename: Emil
authors.initials: E
authors.lastname: Tykesson
authors.affiliation: Department of Biochemistry, Boston University, Massachusetts, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Mao
authors.affiliation: Department of Experimental Medical Science, Lund University, Sweden.
authors.forename: Marco
authors.initials: M
authors.lastname: Maccarana
authors.affiliation: Department of Biochemistry, Boston University, Massachusetts, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Pu
authors.affiliation: Department of Chemistry and Biochemistry, and Center for Quantitative Obesity Research, Montclair State University, New Jersey, USA.
authors.forename: Jinshan
authors.initials: J
authors.lastname: Gao
authors.affiliation: Department of Biochemistry, Boston University, Massachusetts, USA.
authors.forename: Cheng
authors.initials: C
authors.lastname: Lin
authors.affiliation: Department of Biochemistry, Boston University, Massachusetts, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Zaia
authors.affiliation: Department of Experimental Medical Science, Lund University, Sweden.
authors.forename: Gunilla
authors.initials: G
authors.lastname: Westergren-Thorsson
authors.affiliation: Department of Chemistry, Lund University, Sweden.
authors.forename: Ulf
authors.initials: U
authors.lastname: Ellervik
authors.affiliation: S3IT, University of Zurich, Switzerland.
authors.forename: Lars
authors.initials: L
authors.lastname: Malmström
authors.affiliation: Department of Experimental Medical Science, Lund University, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Malmström