Oligopeptides production by a method involving an enzymatic reaction and a subsequent chemical reaction
September 15-19, 2019
We previously reported that an amide bond is unexpectedly formed1) by an acyl-CoA synthetase, AcsA, which plays an essential role in acid utilization in the nitrile-degrative pathway2). Although AcsA essentially catalyzes the formation of a carbon-sulfur bond (the ligation of an acid with CoA), it surprisingly synthesized N-acyl-l-cysteine when a suitable acid and l-cysteine are used as substrates. Furthermore, this unexpected enzyme activity was also observed for acetyl-CoA synthetase and firefly luciferase, both of which belong to the same superfamily of adenylate-forming enzymes. However, the mechanism underlying the carbon-nitrogen bond synthesis remained unknown.
Next, we succeeded in producing N-(D-alanyl)-l-cysteine (a dipeptide) from D-alanine and l-cysteine by using DltA, which is homologous to the adenylation domain of nonribosomal peptide synthetase (NRPS) and belongs to the superfamily of adenylate-forming enzymes. To elucidate the mechanism of these surprising reaction, DltA was used. When cysteine derivatives with a protected amino group N-Boc-l-Cys was used instead of l-cysteine, we confirmed the formation of an thioester intermediate. Thereby, we proposed the following unprecedented reaction mechanism underlying these carbon-nitrogen bond synthetic reactions by the thioester-bond-synthesizing enzymes: (i) the formation of S-acyl-l-cysteine as an intermediate via its “enzymatic activity” and (ii) subsequent “chemical“ S→N acyl transfer in the intermediate, resulting in peptide formation3). Step (ii) of this reaction mechanism is identical to the corresponding reaction in native chemical ligation, a method of chemical peptide synthesis, whereas step (i) is not.
We predicted that enzymes belonging to the superfamily of adenylate-forming enzymes can synthesize peptide/amide compounds by the same mechanism. Accordingly, we tried to express and purify DhbE, a stand-alone adebylation domain of NRPS, for production of valuable peptide/amide compounds. The purified DhbE synthesized N-aromatic acyl-l-cysteine4).
Here, we reported the first demonstration of the N-acylation by “internal” adenylation domains in the multidomain enzyme DhbF. The adenylation domain of NRPS originally is responsible for its selective substrate recognition and activation of the substrate. DhbF is an NRPS involved in bacillibactin synthesis and consists of multiple domains (adenylation domain, condensation domain, peptidyl carrier protein domain, and thioesterase domain). DhbFA1 and DhbFA2 (here named) are “internal” adenylation domains in DhbF. Here, we firstly succeeded in expressing and purifying “internal” adenylation domain DhbFA1 or DhbFA2 separately. When glycine and l-cysteine were used as substrates of DhbFA1, the formation of N-glycyl-l-cysteine (Gly-Cys) was observed. When l-threonine and l-cysteine were used as substrates of DhbFA2, N-l-threonyl-l-cysteine (Thr-Cys) was formed. Furthermore, DhbFA1 or DhbFA2 synthesizes not only dipeptides but also various oligopeptides. Because many adenylation domains that could activate the respective substrates are present in the natural world, we can synthesize various peptides or amides by using adenylation domains or enzymes belonging to the superfamily of adenylate-forming enzymes.
1. Abe, T. et al., J. Biol. Chem. 283, 11312-11321 (2008).
2. Hashimoto, Y. et al., J. Biol. Chem. 280, 8660-8667 (2005).
3. Abe, T. et al., J. Biol. Chem. 291, 1735-1750 (2016).
4. Abe, T. et al., J. Antibiot. 70, 435-442 (2017).
Tomoko Abe, Kenta Kobayashi, Kiwamu Shiiba, and Michihiko Kobayashi, "Oligopeptides production by a method involving an enzymatic reaction and a subsequent chemical reaction" in "Enzyme Engineering XXV", Huimin Zhao, University of Illinois at Urbana-Champaign, USA John Wong, Pfizer, USA Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/enzyme_xxv/20