Identification and characterization of a chondroitin synthase from Avibacterium paragallinarum
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.
- Cimini D, Iacono ID, Carlino E, Finamore R, Restaino OF, Diana P, Bedini E, Schiraldi C (2017) Engineering S. equi subsp. zooepidemicus towards concurrent production of hyaluronic acid and chondroitin biopolymers of biomedical interest. AMB Express 7:61. https://doi.org/10.1186/s13568-017-0364-7 CrossRefPubMedPubMedCentralGoogle Scholar
- Esko JD, Kimata K, Lindahl U (2009) Proteoglycans and sulfated glycosaminoglycans. In: Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME (eds) Essentials of Glycobiology, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (NY)Google Scholar
- Feller G, Gerday C (2003) Psychrophilic enzymes: hot topics in cold adaptation. Nat Rev Microbiol 1:nrmicro773. doi: https://doi.org/10.1038/nrmicro773
- Handel TM, Johnson Z, Crown SE, Lau EK, Proudfoot AE (2005) Regulation of protein function by glycosaminoglycans—as exemplified by chemokines. Annu Rev Biochem 74:385–410. https://doi.org/10.1146/annurev.biochem.72.121801.161747 CrossRefPubMedGoogle Scholar
- Lairson LL, Henrissat B, Davies GJ, Withers SG (2008) Glycosyltransferases: structures, functions, and mechanisms. Annu Rev Biochem 77:521–555. https://doi.org/10.1146/annurev.biochem.76.061005.092322 CrossRefPubMedGoogle Scholar
- Masuko S, Bera S, Green DE, Weïwer M, Liu J, DeAngelis PL, Linhardt RJ (2012) Chemoenzymatic synthesis of uridine diphosphate-GlcNAc and uridine diphosphate-GalNAc analogs for the preparation of unnatural glycosaminoglycans. J Org Chem 77:1449–1456. https://doi.org/10.1021/jo202322k CrossRefPubMedPubMedCentralGoogle Scholar
- Otto NJ, Green DE, Masuko S, Mayer A, Tanner ME, Linhardt RJ, DeAngelis PL (2012) Structure/function analysis of Pasteurella multocida heparosan synthases toward defining enzyme specificity and engineering novel catalysts. J Biol Chem 287:7203–7212. https://doi.org/10.1074/jbc.M111.311704 CrossRefPubMedPubMedCentralGoogle Scholar
- Requena D, Chumbe A, Torres M, Alzamora O, Ramirez M, Valdivia-Olarte H, Gutierrez AH, Izquierdo-Lara R, Saravia LE, Zavaleta M, Tataje-Lavanda L, Best I, Fernández-Sánchez M, Icochea E, Zimic M, Fernández-Díaz M (2013) Genome sequence and comparative analysis of Avibacterium paragallinarum. Bioinformation 9:528–536. https://doi.org/10.6026/97320630009528 CrossRefPubMedPubMedCentralGoogle Scholar
- Santiago M, Ramírez-Sarmiento CA, Zamora RA, Parra LP (2016) Discovery, molecular mechanisms, and industrial applications of cold-active enzymes. Front Microbiol 7. https://doi.org/10.3389/fmicb.2016.01408
- Sasisekharan R, Raman R, Prabhakar V (2006) Glycomics approach to structure-function relationships of glycosaminoglycans. Annu Rev Biomed Eng 8:181–231. https://doi.org/10.1146/annurev.bioeng.8.061505.095745 CrossRefPubMedGoogle Scholar
- Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. https://doi.org/10.1093/nar/22.22.4673 CrossRefPubMedPubMedCentralGoogle Scholar
- Varki A, Cummings RD, Esko JD, Stanley P, Hart GW, Aebi M, Darvill AG, Kinoshita T, Packer NH, Prestegard JH, Schnaar RL, Seeberger PH (eds) (2015) Essentials of Glycobiology, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (NY)Google Scholar
- Xu Y, Cai C, Chandarajoti K, Hsieh P-H, Li L, Pham TQ, Sparkenbaugh EM, Sheng J, Key NS, Pawlinski R, Harris EN, Linhardt RJ, Liu J (2014) Homogeneous low-molecular-weight heparins with reversible anticoagulant activity. Nat Chem Biol 10:248–250. https://doi.org/10.1038/nchembio.1459 CrossRefPubMedPubMedCentralGoogle Scholar
- Xu Y, Chandarajoti K, Zhang X, Pagadala V, Dou W, Hoppensteadt DM, Sparkenbaugh EM, Cooley B, Daily S, Key NS, Severynse-Stevens D, Fareed J, Linhardt RJ, Pawlinski R, Liu J (2017) Synthetic oligosaccharides can replace animal-sourced low–molecular weight heparins. Sci Transl Med 9:eaan5954. https://doi.org/10.1126/scitranslmed.aan5954 CrossRefPubMedGoogle Scholar