Uncovering the Catalytic Direction of Chondroitin AC Exolyase

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 ⌬ 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 ␤1,4-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-␣-1,4-GlcA unit at the reducing end of the oligosaccharide prevented ChnAC from cleaving the GalNAc-␤1,4-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.

Cloning, Expression, and Characterization of a New Glycosaminoglycan Lyase from Microbacterium sp. H14

“…The characterized enzymes with the highest similarity to HCLaseM are chondroitin lyase (GenBank ANC28180.1) from Arthrobacter sp. GAG (45%) [15], chondroitin lyase (AFM38168.1) from Arthrobacter sp. MAT3885 (44%), and chondroitin lyase (PDB 1RWA_A) from Arthrobacter aurescens (43%) [16,17].…”

Chemoenzymatic Synthesis of Homogeneous Heparan Sulfate and Chondroitin Sulfate Chimeras

“…Enzymatic synthesis of hyaluronan-chondroitin chimeric oligosaccharides has been reported, 22 and the oligosaccharides were used to study the mechanism of action of chondroitin lyases. 23 However, the structures of hyaluronan-chondroitin oligosaccharides lack sulfo groups and thereby do not represent HS-CS chimeric oligosaccharides. In the present studies, the synthesis of seven 12-mers carrying low to high sulfation levels was achieved.…”

Cascade synthesis of uridine-5′-diphosphate glucuronic acid by coupling multiple whole cells expressing hyperthermophilic enzymes

“…The product concentration in our system was determined by polyamine-based anion exchange–high-performance liquid chromatography (PAMN-HPLC) (Fig. 4a) [32], and the identity of UDP-Glc was confirmed by electrospray ionization-mass spectrometry (ESI–MS) as described previously [33] (Fig. 4b).…”