The adenylation (A) domains found in nonribosomal peptide synthetases (NRPSs) exhibit tremendous plasticity. Some A domains have been shown to display the ability to contain within them the catalytic portion of an auxiliary domain, most commonly that of a methyltransferase (M) enzyme. This unique feature of A domains interrupted by M domains allows them to possess bifunctionality, where they can both adenylate and methylate an amino acid substrate. Additionally, these types of inserted M domains are able to selectively carry out either backbone or side chain methylation of amino acids. Interruptions with M domains are naturally found to occur either between the a2-a3 or the a8-a9 of the ten conserved motifs of A domains. Herein, we set out to answer the following question: Can one A domain support two different M domain interruptions occurring in two different locations (a2-a3 and a8-a9) of the A domain and possess the ability to adenylate an amino acid and methylate it on both its side chain and backbone? To answer this question we added a backbone methylating M 3S domain from TioS(A 3a M 3S A 3b ) between the a8-a9 region of a mono-interrupted A domain, TioN(A a M N A b ), that already contained a side chain methylating M N domain between its a2-a3 region. We evaluated the di-interrupted A domain TioN(AM N AM 3S A) with a series of radiometric and mass spectrometry assays and found that this engineered enzyme was indeed capable of all three activities. These findings show that production of an active trifunctional di-interrupted A domain is possible and represents an exciting new avenue for future nonribosomal peptide (NRP) derivatization.
|Number of pages||7|
|Journal||Organic and Biomolecular Chemistry|
|State||Published - 2019|
Bibliographical noteFunding Information:
This project was supported by startup funds from the College of Pharmacy at the University of Kentucky (to S. G.-T.) and by a National Science Foundation CAREER Award MCB-1149427 (to S. G.-T.). T. A. L. was in part supported by a 2018–2019 Pharmaceutical Sciences Excellence in Graduate Achievement Fellowship from the College of Pharmacy at the University of Kentucky. S. M. is a recipient of a 2018 long-term visit fellowship from the Yamada Science Foundation, Japan. We thank Dr. Atefeh Garzan for providing the N-Me-L-Cys and N,S-diMe-L-Cys for which the synthesis was previously published in ref. 17. We thank Jarrod W. Creameans who, under the supervision of T. A. L., constructed the ptioN(AMNAM3SA)-pET28a and ptioN(AMNAM3SA)D818A-pET28a plasmids and also expressed and purified the TioN(AMNAM3SA)D818A enzyme. We also thank Scott Kinison for training of T. A. L. and his help in developing the MS and MS2 methods.
©2019 The Royal Society of Chemistry.
ASJC Scopus subject areas
- Physical and Theoretical Chemistry
- Organic Chemistry