Issue 5, 2019

Probing the limits of interrupted adenylation domains by engineering a trifunctional enzyme capable of adenylation, N-, and S-methylation

Abstract

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 M3S domain from TioS(A3aM3SA3b) between the a8–a9 region of a mono-interrupted A domain, TioN(AaMNAb), that already contained a side chain methylating MN domain between its a2–a3 region. We evaluated the di-interrupted A domain TioN(AMNAM3SA) 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.

Graphical abstract: Probing the limits of interrupted adenylation domains by engineering a trifunctional enzyme capable of adenylation, N-, and S-methylation

Supplementary files

Article information

Article type
Paper
Submitted
01 Dec. 2018
Accepted
07 Janv. 2019
First published
15 Janv. 2019

Org. Biomol. Chem., 2019,17, 1169-1175

Author version available

Probing the limits of interrupted adenylation domains by engineering a trifunctional enzyme capable of adenylation, N-, and S-methylation

T. A. Lundy, S. Mori and S. Garneau-Tsodikova, Org. Biomol. Chem., 2019, 17, 1169 DOI: 10.1039/C8OB02996B

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements