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 V value of 0.0156±0.0008 units mg and K values 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.

Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

“…This variant lacks the Ser689 attachment site to the PPant group that is involved in thioester formation and therefore would only allow the formation of the acyl adenylate and not the thioester. [26][27][28][29][30] Our current results are not able to establish if this reaction is enzyme catalyzed. [26][27][28][29][30] Our current results are not able to establish if this reaction is enzyme catalyzed.…”

“…Conversion to ilepcimide 20 (79 %) was still observed with this variant, suggesting that adenylation activity alone is sufficient for amidation, presumably by nucleophilic attack onto the acyl adenylate ( Figure 2B), ar eaction which has been observed with other adenylating and phosphorylating enzymes,including NRPS and glutamine synthetase. [26][27][28][29][30] Our current results are not able to establish if this reaction is enzyme catalyzed.…”

Synthesis of d -Amino Acid-Containing Dipeptides Using the Adenylation Domains of Nonribosomal Peptide Synthetase

“…In this paper, the combined reaction of the enzymatic adenylation of amino acids and the subsequent chemical reaction with nucleophiles was applied to provide a broad range of D-amino acid-containing dipeptides. The previous studies using this system undertook the synthesis of amide compounds (10,11) or LL-dipeptides (19)(20)(21). Here, we extended and verified this application to D-amino acid-containing-dipeptide synthesis.…”

Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

“…This variant lacks the Ser689 attachment site to the PPant group that is involved in thioester formation and therefore would only allow the formation of the acyl adenylate and not the thioester. Conversion to ilepcimide 20 (79 %) was still observed with this variant, suggesting that adenylation activity alone is sufficient for amidation, presumably by nucleophilic attack onto the acyl adenylate (Figure B), a reaction which has been observed with other adenylating and phosphorylating enzymes, including NRPS and glutamine synthetase . Our current results are not able to establish if this reaction is enzyme catalyzed.…”