Catalytic activity of Mtan catalyzing S-adenosylhomocysteine in Riemerella anatipestifer

Abstract:

[Objective] The sequences of pfs gene (encoding the Mtan protein, also known as Pfs) from different serotypes of Riemerella anatipestifer (RA) were analyzed, and catalytic activity of Mtan was studied. [Methods] The different serotypes of RA pfs gene were amplified by PCR and then the homology of nucleotide sequences was analyzed. The recombinant plasmid, pCold-RA-pfs was constructed, and then expressed in BL21 and the recombinant protein RA-Mtan was purified. Furthermore, the activity of RA-Mtan catalyze S-adenosylhomocysteine (SAH) to produce Homocysteine (HCY) was evaluated by Ellman’s assay, and the activity of AI-2 was detected by Vibrio harveyi reporter strain BB170. [Results] The sequence analysis of pfs indicated that the homology of different serotypes varied from 93.9% to 100%. The SDS-PAGE showed that RA-Mtan was soluble expression in BL21. Moreover, the result suggested that RA-Mtan and recombinant protein LuxS (from Avain pathogenic Escherichia coli) could catalyze SAH to produce 176.7 μmol/L HCY. The reaction products were able to induce luminescence of Vibrio harveyi BB170, demonstrating that recombinant RA-Mtan and LuxS synthesize AI-2 in vitro from SAH. [Conclusion] The RA pfs genes from different serotypes were highly conserved. The RA-Mtan can catalyze SAH to produce HCY, and produce AI-2 with biological activity as well. This study will contribute to further study of the roles of pfs in RA.

Key words:Riemerella anatipestifer, Pfs, S-adenosylhomocysteine, quorum sensing systm, AI-2