Ishikawa, Fumihiro Nohara, Maya Nakamura, Shinya Nakanishi, Isao Tanabe, Genzoh Precise Probing of Residue Roles by NRPS Code Swapping: Mutation, Enzymatic Characterization, Modeling, and Substrate Promiscuity of Aryl Acid Adenylation Domains Aryl acids are most commonly found in iron-scavenging siderophores but are not limited to them. The nonribosomal peptide synthetase (NRPS) codes of aryl acids remain poorly elucidated relative to those of amino acids. Here, we defined more precisely the role of active-site residues in aryl acid adenylation domains (A-domains) by gradually grafting the NRPS codes used for salicylic acid (Sal) into an archetypal aryl acid A-domain, EntE [specific for the substrate 2,3-dihydroxybenzoic acid (DHB)]. Enzyme kinetics and modeling studies of these EntE variants demonstrated that the NRPS code residues at positions 236, 240, and 339 collectively regulate the substrate specificity toward DHB and Sal. Furthermore, the EntE variants exhibited the ability to activate the non-native aryl acids 3-hydroxybenzoic acid, 3-aminobenzoic acid, 3-fluorobenzoic acid, and 3-chlorobenzoic acid. These studies enhance our knowledge of the NRPS codes of aryl acids and could be exploited to reprogram aryl acid A-domains for non-native aryl acids. archetypal aryl acid A-domain;nonribosomal peptide synthetase;aryl acids;3- fluorobenzoic acid;3- aminobenzoic acid;NRPS code residues;Adenylation Domains Aryl acids;non-native aryl acids;DHB;reprogram aryl acid A-domains;non-native aryl acids 3- hydroxybenzoic acid;NRPS Code Swapping;3- chlorobenzoic acid;NRPS codes;aryl acid adenylation domains;EntE variants 2020-01-15
    https://acs.figshare.com/articles/journal_contribution/Precise_Probing_of_Residue_Roles_by_NRPS_Code_Swapping_Mutation_Enzymatic_Characterization_Modeling_and_Substrate_Promiscuity_of_Aryl_Acid_Adenylation_Domains/11608542
10.1021/acs.biochem.9b00748.s001