SCREENING THE SPECIFIC SUBSTRATES OF ADENYLATION DOMAIN FROM MARINE ACTINOMYCETES BY FLUORESCENCE QUENCHING AND ISOTHERMAL TITRATION CALORIMETRY
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Adenylation domain (A domain) is a model of non-ribosomal peptide synthetases (NRPs), responsible for binding and activating the substrates-amino acids. 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 protein GST-Sare0718. Fluorescence quenching (FQ) was conducted to investigate the binding of 20 common amino acids to GST-Sare0718, then isothermal titration calorimetry (ITC) also be used[changed]. The results of FQ revealed that intrinsic fluorescence of GST-Sare0718 is quenched steadily by addition of aspartic acid (Asp) and glutamine (Gln) through static quenching mechanism. The binding constants Ka are Asp (1.504×106 L/mol) ≥ Gln (1.468×105 L/mol). And there is one binding site on the protein GST-Sare0718. We confirmed Asp preference of GST-Sare0718 by ITC. Therefore, Asp is the specific substrate. In addition, the experimental data indicates that the prediction system, “the specificity-conferring code”, is not suitable for marine actinomycetes. So it is urgent to set up a special predictive system for marine actinomycetes.
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