Expression, Purification And Determination About Specific Substrates Of Sare0718, A Protein Of Adenylation Domain From Marine Actinomycete Salinispora Arenicola CNS-205

Non-ribosomal peptide synthetases (NRPSs) synthetize non-ribosomal peptides (NRPs), a general class of natural products with biological value. And adenylation domains (A domains), which are essential modules for NRPSs, play a critical role in the diversity of NRPs. A domain recognizes the cognate substrate and activates it as its aminoacyl adenylate. Salinispora arenicola CNS-205 (S.arenicola CNS-205) is a representative strain of the firstly-obligate marine actinomyces, which have been reported before. There are a large number of cryptic secondary metabolite gene clusters in the genome of S.arenicola CNS-205. In this study, a pGEX-2T-sare0718 recombinant plasmid (the gene sare0718 was cloned from Salinispora arenicola CNS-205, S.arenicola CNS-205) was transformed and expressed as a fusion protein GST-Sare0718. Fluorescence quenching (FQ) and isothermal titration calorimetry (ITC) were conducted to investigate the binding of 20 common amino acids to GST-Sare0718.The results of FQ revealed that aspartic acid is the specific substrate of A domain from S.arenicola CNS-205. The intrinsic fluorescence of GST-Sare0718 is quenched steadily by addition of aspartic acid (Asp) and glutamine (Glu) through static quenching mechanism. The binding sites n, which are n(Asp)= 1.16 and n(Gln)=0.94, are closed to 1. It indicates that there is one biding site on GST-Sare0718. And the binding constants Ka are Ka(Asp)=1.504×106 L/mol and Ka(Gin)=1.468×105 L/mol. Ka(Asp)≥Ka(Gln).Similarly, we confirm Asp preference of GST-Sare0718 by ITC. Asp is also the specific substrate of A domain from S.arenicola CNS-205. Analyzed by the software "NanoAnalyze", the results of ITC titration show that Asp and Glu can combine with GST-Sare0718. And the binding constants Ka are Ka(Asp)=1.227×105 L/mol and Ka(Gln)=1.629×104 L/mol. Ka(Asp)≥Ka(Gln).These results are same as that of FQ.The research can enrich the substrates of A domain from marine actinomycetes. In addition, the experimental data indicates that the prediction system, "the specificity-conferring code" from terrestrial actinomycetes, is not suitable for marine actinomycetes. So it is urgent to set up a special predictive system for marine actinomycetes. Moreover, the same reaction systemis studied by two methods-FQ and ITC, researching the interactions between the small molecules and proteins. It makes the results much more accurate and persuasive.