title: Amide compound synthesis by adenylation domain of bacillibactin synthetase.
abstract: The adenylation domain of nonribosomal peptide synthetase (NRPS) is responsible for the selective substrate recognition and its activation (as an acyl-O-AMP intermediate) during ATP consumption. DhbE, a stand-alone adenylation domain, acts on an aromatic acid, 2,3-dihydroxybenzoic acid (DHB). This activation is the initial step of the synthesis of bacillibactin that is a high-affinity small-molecule iron chelator also termed siderophore. Subsequently, the activated DHB is transferred and attached covalently to a peptidyl carrier protein domain via a thioester bond. Adenylation domains belong to the superfamily of adenylate-forming enzymes including acetyl-CoA synthetase, acyl-CoA synthetase and firefly luciferase. We previously reported a novel N-acylation reaction for an acyl-CoA synthetase (AcsA) that originally catalyzes the formation of a thioester bond between an acid and CoA, yielding acyl-CoA. This novel reaction was also confirmed for acetyl-CoA synthetase and firefly luciferase, but not yet for an adenylation domain. Here, we for the first time demonstrated the synthesis of N-acyl-L-cysteine by a stand-alone adenylation domain, DhbE. When DHB and L-cysteine were used as substrates of DhbE, N-DHB-L-cysteine was formed. A Vmaxvalue of 0.0156±0.0008 units mg-1and Kmvalues of 150±18.3 mM for L-cysteine and 0.0579±0.0260 mM for DHB were obtained in this novel reaction. Furthermore, DhbE synthesized N-benzoyl-L-cysteine when benzoic acid and L-cysteine were used as substrates. Through the N-acylation reaction of DhbE, we also succeeded in the synthesis of N-aromatic acyl compounds that have never previously been reported to be produced by this enzymatic method.
authors.affiliation: Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The University of Tsukuba, Ibaraki, Japan.|Division of Life Science and Engineering, School of Science and Engineering, Tokyo Denki University, Saitama, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Abe
authors.affiliation: Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The University of Tsukuba, Ibaraki, Japan.
authors.forename: Yoshiteru
authors.initials: Y
authors.lastname: Hashimoto
authors.affiliation: Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The University of Tsukuba, Ibaraki, Japan.
authors.forename: Sayaka
authors.initials: S
authors.lastname: Sugimoto
authors.affiliation: Division of Life Science and Engineering, School of Science and Engineering, Tokyo Denki University, Saitama, Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Kobayashi
authors.affiliation: Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The University of Tsukuba, Ibaraki, Japan.
authors.forename: Takuto
authors.initials: T
authors.lastname: Kumano
authors.affiliation: Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The University of Tsukuba, Ibaraki, Japan.
authors.forename: Michihiko
authors.initials: M
authors.lastname: Kobayashi
mesh_terms: D000106:Acetate-CoA Ligase / Q000378:metabolism; D000215:Acylation; D000225:Adenine / Q000737:chemistry*; D000577:Amides / Q000138:chemical synthesis*; D019817:Benzoic Acid / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D009842:Oligopeptides / Q000096:biosynthesis*; D013379:Substrate Specificity
chemical_list: D000577:Amides; D009842:Oligopeptides; C430721:bacillibactin; D019817:Benzoic Acid; D008156:Luciferases; D008025:Ligases; D003066:Coenzyme A Ligases; D000106:Acetate-CoA Ligase; D000225:Adenine; D003545:Cysteine