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The structure of EntE with 2-nitrobenzoyl adenylate analog
Structural highlights
FunctionENTE_ECOLI Activates the carboxylate group of 2,3-dihydroxy-benzoate (2,3-DHB), via ATP-dependent PPi exchange reactions, to the acyladenylate. Then, catalyzes the acylation of holo-EntB with 2,3-DHB adenylate, preparing that molecule for amide bond formation with L-serine. Publication Abstract from PubMedAdenylation (A) domains act as the gatekeepers of non-ribosomal peptide synthetases (NRPSs) ensuring the activation and thioesterification of the correct amino acid/aryl acid building blocks. Aryl acid building blocks are most commonly observed in iron-chelating siderophores, but are not limited to them. The non-ribosomal codes toward aryl acid substrates are poorly understood. Very little is known about the reprogramming of aryl acid A-domains. Here we show that a single asparagine-to-glycine mutation in an aryl acid A-domain creates novel enzyme specificities toward a wide range of non-native aryl acids. The engineered catalyst is capable of activating the non-native aryl acids functionalized with nitro, cyano, bromo, and iodo, even though no enzymatic activity of wild-type enzyme was observed toward these substrates. Co-crystal structures with non-hydrolysable aryl-AMP analogues revealed the origins of substrate promiscuity expansion, highlighting an enlarged substrate binding pocket of the enzyme. Our finding may be exploited to produce diversified aryl acid-containing natural products and serve as a template for further directed evolution in combinatorial biosynthesis. An engineered aryl acid adenylation domain with a capacious active site microenvironment.,Ishikawa F, Miyanaga A, Kitayama H, Nakamura S, Nakanishi I, Kudo F, Eguchi T, Tanabe G Angew Chem Int Ed Engl. 2019 Apr 3. doi: 10.1002/anie.201900318. PMID:30945421[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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