Identification and characterization of a chondroitin synthase from Avibacterium paragallinarum
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Avibacterium paragallinarum is a Gram-negative bacterium that causes infectious coryza in chicken. It was reported that the capsule polysaccharides extracted from Av. paragallinarum genotype A contained chondroitin. Chondroitin synthase of Av. paragallinarum (ApCS) encoded by one gene within the presumed capsule biosynthesis gene cluster exhibited considerable homology to identified bacterial chondroitin synthases. Herein, we report the identification and characterization of ApCS. This enzyme indeed displays chondroitin synthase activity involved in the biosynthesis of the capsule. ApCS is a bifunctional protein catalyzing the elongation of the chondroitin chain by alternatively transferring the glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) residues from their nucleotide forms to the non-reducing ends of the saccharide chains. GlcA with a para-nitrophenyl group (pNP) could serve as the acceptor for ApCS; this enzyme shows a stringent donor tolerance when the acceptor is as small as this monosaccharide. Then, UDP-GalNAc and GlcA-pNP were injected sequentially through the chip-immobilized chondroitin synthases, and the surface plasmon resonance data demonstrated that the up-regulated extent caused by the binding of the donor is one possibly essential factor in successful polymerization reaction. This conclusion will, therefore, enhance the understanding of the mode of action of glycosyltransferase. Surprisingly, high activity at near-zero temperature as well as weak temperature dependence of this novel bacterial chondroitin synthase indicate that ApCS was a cold-active enzyme. From all accounts, ApCS becomes the fourth known bacterial chondroitin synthase, and the potential applications in artificial chondroitin sulfate and glycosaminoglycan synthetic approaches make it an attractive glycosyltransferase for further investigation.
KeywordsChondroitin sulfate Avibacterium paragallinarum Chondroitin synthase Substrate specificity Cold-active enzyme
JS designed and coordinated the work. TW carried out the experiments. TW and CZ carried out the HPLC analysis. DM, YL, and HZ carried out the donor and acceptor experiments. SZ conducted the MS analysis. TW and CM conducted the NMR analysis. Tian-Tian Wang purified KfoC. JS and TW wrote the manuscript. All authors have read and approved the final manuscript.
This work was supported in part by the National Natural Science Foundation of China (Project no. 31770845), as well as the Projects of Science and Technology Department of Shandong Province (Project nos. 2015ZDJS04002, ZR2015QL007, and ZR2017PH072).
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Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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