Chondroitin sulfate (CS) and dermatan sulfate (DS) are glycosaminoglycans whose diverse biological functions are largely determined by specific sulfation patterns. The GlcA/IdoA-GalNAc4S (A/iA unit), where GlcA, IdoA, GalNAc and 4S denote D-glucuronic acid, L-iduronic acid, N-acetylgalactosamine and 4-O-sulfation, respectively, is the most prevalent sulfation motif in animal tissues and participates in a broad spectrum of physiological and pathological processes. However, chondroitinase A (CSase A), a lyase capable of selectively degrading CS/DS rich in A/iA units, has not yet been identified, which has limited our understanding of the functional roles of such structures in CS/DS. Here, we report the first discovery of a CSase A from the metagenome of sediments near the Haima cold seep. This enzyme belongs to polysaccharide lyase family 8 and exhibits strict selectivity for A/iA units, yielding disaccharides composed almost exclusively of the 4-O-sulfated disaccharide, with no other sulfated disaccharide variants detectable even under exhaustive digestion. Structural analysis of resistant tetrasaccharides and hexasaccharides, and degradation assays with structure-defined oligosaccharides demonstrate that CSase A exclusively cleaves the β-1,4 glycosidic bond preceding a disaccharide unit containing GalNAc or GalNAc4S, thereby defining its rigorous substrate specificity. Site-directed mutagenesis and activity profiling identified Glu-579, Arg-480, Tyr-427, and His-420 as residues essential for catalysis, supporting a proposed tyrosine-histidine-mediated acid-base mechanism. The discovery of CSase A provides a unique molecular tool for dissecting the functional roles of A/iA units in CS/DS-related biology and disease.
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