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Precise Probing of Residue Roles by NRPS Code Swapping: Mutation, Enzymatic Characterization, Modeling, and Substrate Promiscuity of Aryl Acid Adenylation Domains
journal contribution
posted on 2020-01-15, 03:03 authored by Fumihiro Ishikawa, Maya Nohara, Shinya Nakamura, Isao Nakanishi, Genzoh TanabeAryl
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.
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archetypal aryl acid A-domainnonribosomal peptide synthetasearyl acids3- fluorobenzoic acid3- aminobenzoic acidNRPS code residuesAdenylation Domains Aryl acidsnon-native aryl acidsDHBreprogram aryl acid A-domainsnon-native aryl acids 3- hydroxybenzoic acidNRPS Code Swapping3- chlorobenzoic acidNRPS codesaryl acid adenylation domainsEntE variants
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