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

Taylor A. Lundy; Shogo Mori; Sylvie Garneau-Tsodikova

doi:10.1039/c8ob02996b

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

Taylor A. Lundy, Shogo Mori, Sylvie Garneau-Tsodikova

Research output: Contribution to journal › Article › peer-review

8 Scopus citations

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 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.

Original language	English
Pages (from-to)	1169-1175
Number of pages	7
Journal	Organic and Biomolecular Chemistry
Volume	17
Issue number	5
DOIs	https://doi.org/10.1039/c8ob02996b
State	Published - 2019

Bibliographical note

Funding 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.

Publisher Copyright:
©2019 The Royal Society of Chemistry.

ASJC Scopus subject areas

Biochemistry
Physical and Theoretical Chemistry
Organic Chemistry

Access to Document

10.1039/c8ob02996b

Cite this

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title = "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 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.",

author = "Lundy, {Taylor A.} and Shogo Mori and Sylvie Garneau-Tsodikova",

note = "Funding 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. Publisher Copyright: {\textcopyright}2019 The Royal Society of Chemistry.",

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doi = "10.1039/c8ob02996b",

language = "English",

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pages = "1169--1175",

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T1 - Probing the limits of interrupted adenylation domains by engineering a trifunctional enzyme capable of adenylation,

T2 - N -, and S -methylation

AU - Lundy, Taylor A.

AU - Mori, Shogo

AU - Garneau-Tsodikova, Sylvie

N1 - Funding 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. Publisher Copyright: ©2019 The Royal Society of Chemistry.

PY - 2019

Y1 - 2019

N2 - 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.

AB - 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.

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