Abstract
Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even though marine animals are rich in GAGs with unique structures and functions. Herein we isolated a novel GAG lyase for the first time from the marine bacterium Vibrio sp. FC509 and then recombinantly expressed and characterized it. It showed strong lyase activity toward hyaluronan (HA) and chondroitin sulfate (CS) and was designated as HA and CS lyase (HCLase). It exhibited the highest activities to both substrates at pH 8.0 and 0.5 m NaCl at 30 °C. Its activity toward HA was less sensitive to pH than its CS lyase activity. As with most other marine enzymes, HCLase is a halophilic enzyme and very stable at temperatures from 0 to 40 °C for up to 24 h, but its activity is independent of divalent metal ions. The specific activity of HCLase against HA and CS reached a markedly high level of hundreds of thousands units/mg of protein under optimum conditions. The HCLase-resistant tetrasaccharide Δ(4,5)HexUAα1-3GalNAc(6-O-sulfate)β1-4GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate) was isolated from CS-D, the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated d-glucuronic acid (GlcUA) in CS chains. Site-directed mutagenesis indicated that HCLase may work via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid. Thus, the identification of HCLase provides a useful tool for HA- and CS-related research and applications.
Highlights
Glycosaminoglycan (GAG) lyases have been widely isolated from terrestrial but not marine bacteria
Identification of Chondroitin Sulfate-degrading Bacteria— Using the Basal medium (BM) screening plates, 276 bacterial strains were isolated from nine coastal sediment samples
The results obtained from an examination of bacterial growth and consumption of polysaccharides indicated that 15 isolates could degrade more than three types of polysaccharides by utilizing them as the sole carbon sources (Table 2)
Summary
Glycosaminoglycan (GAG) lyases have been widely isolated from terrestrial but not marine bacteria. Results: A novel GAG lyase (HCLase) was identified for the first time from a marine bacterium. After or while CS chains are polymerized by various enzyme complexes, each of which is formed by heterologous combinations of two of the six chondroitin synthase family members [15–17], CS chains are further modified by differential sulfation by specific sulfotransferases at C-2 of GlcUA/L-iduronic acid and/or C-4 and/or C-6 of GalNAc to yield prominent structural diversity [18]. CS and DS chains are often detected as co-polymeric structures (CS-DS) and are more likely to be periodically distributed in cell/tissue-specific manners [14, 21] The introduction of these unique structural domains into CS-DS chains in respective tissues results in different responses to various CS- or DS-binding proteins such as heparin-binding growth factors and cytokines [22].
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