title: Uncovering the Catalytic Direction of Chondroitin AC Exolyase: FROM THE REDUCING END TOWARDS THE NON-REDUCING END.
abstract: Glycosaminoglycans (GAGs) are polysaccharides that play vital functional roles in numerous biological processes, and compounds belonging to this class have been implicated in a wide variety of diseases. Chondroitin AC lyase (ChnAC) (EC 4.2.2.5) catalyzes the degradation of various GAGs, including chondroitin sulfate and hyaluronic acid, to give the corresponding disaccharides containing an Δ(4)-unsaturated uronic acid at their non-reducing terminus. ChnAC has been isolated from various bacteria and utilized as an enzymatic tool for study and evaluating the sequencing of GAGs. Despite its substrate specificity and the fact that its crystal structure has been determined to a high resolution, the direction in which ChnAC catalyzes the cleavage of oligosaccharides remain unclear. Herein, we have determined the structural cues of substrate depolymerization and the cleavage direction of ChnAC using model substrates and recombinant ChnAC protein. Several structurally defined oligosaccharides were synthesized using a chemoenzymatic approach and subsequently cleaved using ChnAC. The degradation products resulting from this process were determined by mass spectrometry. The results revealed that ChnAC cleaved the β1,4-glycosidic linkages between glucuronic acid and glucosamine units when these bonds were located on the reducing end of the oligosaccharide. In contrast, the presence of a GlcNAc-α-1,4-GlcA unit at the reducing end of the oligosaccharide prevented ChnAC from cleaving the GalNAc-β1,4-GlcA moiety located in the middle or at the non-reducing end of the chain. These interesting results therefore provide direct proof that ChnAC cleaves oligosaccharide substrates from their reducing end toward their non-reducing end. This conclusion will therefore enhance our collective understanding of the mode of action of ChnAC.
authors.affiliation: From the Key Laboratory of Chemical Biology of Natural Products (Ministry of Education), Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China and.
authors.forename: Feng-Xin
authors.initials: FX
authors.lastname: Yin
authors.affiliation: From the Key Laboratory of Chemical Biology of Natural Products (Ministry of Education), Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China and National Glycoengineering Research Center, Shandong University, Jinan 250012, China fswang@sdu.edu.cn.
authors.forename: Feng-Shan
authors.initials: FS
authors.lastname: Wang
authors.affiliation: From the Key Laboratory of Chemical Biology of Natural Products (Ministry of Education), Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China and National Glycoengineering Research Center, Shandong University, Jinan 250012, China shengjuzheng@sdu.edu.cn.
authors.forename: Ju-Zheng
authors.identifier: http://orcid.org/0000-0003-2856-9162
authors.initials: JZ
authors.lastname: Sheng
mesh_terms: D000837:Anion Exchange Resins; D001173:Arthrobacter / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002240:Carbohydrate Sequence; D002808:Chondroitin Lyases / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
chemical_list: D000837:Anion Exchange Resins; D001426:Bacterial Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D002808:Chondroitin Lyases
keywords: Arthrobacter species; Catalytic Direction; Chondroitin exolyase; chondroitin sulfate; elimination reaction; glycosaminoglycan; mass spectrometry (MS); oligosaccharide; structurally defined substrate; substrate specificity