Chemical and Pharmaceutical Bulletin
Online ISSN : 1347-5223
Print ISSN : 0009-2363
ISSN-L : 0009-2363
Activity, Binding, and Modeling Studies of a Reprogrammed Aryl Acid Adenylation Domain with an Enlarged Substrate Binding Pocket
Fumihiro IshikawaHinano KitayamaShinya NakamuraKatsuki TakashimaIsao NakanishiGenzoh Tanabe
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2021 Volume 69 Issue 2 Pages 222-225


The gatekeeping adenylation (A) domain of the non-ribosomal peptide synthetase (NRPS) selectively incorporates specific proteinogenic/non-proteinogenic amino acid into a growing peptide chain. The EntE of the enterobactin NRPS is a discrete aryl acid A-domain with 2,3-dihydroxybenzoic acid (DHB) substrate specificity. Reprogrammed EntE N235G variant possesses an enlarged substrate recognition site, and is capable of accepting non-native aryl acids. Biochemical characterization of this unique substrate recognition site should provide a better understanding of activi-site microenvironments. Here, we synthesized a non-hydrolysable adenylate analogue with 2-aminobenzoic acid (2-ABA), 3-aminobenzoic acid (3-ABA), and 4-aminobenzoic acid (4-ABA) and used them to calculate the apparent inhibition constants (Kiapp.). Dose–response experiments using 3-ABA-sulfamoyladenosine (AMS) provided Kiapp. values of 596 nM for wild-type EntE and 2.4 nM for the N235G variants. These results suggest that 3-amino group of benzoic acid plays an important role in substrate recognition by the N235G variant. These findings would help designing aryl acid substrates with substituents at the 2- and 3-positions.

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