SCREENING THE SPECIFIC SUBSTRATES OF ADENYLATION DOMAIN FROM MARINE ACTINOMYCETES BY FLUORESCENCE QUENCHING AND ISOTHERMAL TITRATION CALORIMETRY

• Authors: MING LEI , ZHIJUAN ZHAO , KE LIU , YANLI LIU , HUI ZHANG , XUE WU , SIYING GONG , YANLING MA , HAOBIN ZHAO , JINLIN LIU , JINRONG MIN , CHAO QI
• Languages of publication: EN

Abstracts

EN

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.

Keywords

EN

A domain; Aspartic acid; Fluorescence quenching (FQ); Isothermal titration calorimetry (ITC); Marine actinomycetes; static quenching

• Journal: Acta Poloniae Pharmaceutica - Drug Research
• Year: 2018
• Volume: 75
• Issue: 6
• Pages: 1287-1292

Dates

published

2018-12-31

Contributors

author

MING LEI

author

ZHIJUAN ZHAO

author

KE LIU

author

YANLI LIU

author

HUI ZHANG

author

XUE WU

author

SIYING GONG

author

YANLING MA

author

HAOBIN ZHAO

author

JINLIN LIU

author

JINRONG MIN

author

CHAO QI

Identifiers

DOI

10.32383/appdr/89805