Lessons learned in engineering interrupted adenylation domains when attempting to create trifunctional enzymes from three independent monofunctional ones

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

doi:10.1039/d0ra05490a

Lessons learned in engineering interrupted adenylation domains when attempting to create trifunctional enzymes from three independent monofunctional ones

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

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

1 Scopus citations

Abstract

Interrupted adenylation (A) domains are fascinating examples of multifunctional enzymes. They are found in nonribosomal peptide synthetases (NRPSs), which biosynthesize nonribosomal peptides (NRPs), a major class of medically relevant natural products (NPs). Interrupted A domains contain the catalytic portion of another domain within them, typically a methylation (M) domain, thus combining both adenylation and methylation capabilities. In recent years, interrupted A domains have demonstrated tremendous enzyme engineering potential as they are able to be constructed artificially in a laboratory setting by combining the A and M domains of two separate NRPS proteins. A recent discovery and characterization of a naturally occurring interrupted A domain that harbored two M domains back-to-back, a trifunctional protein, showed the ingenuity of Nature to bothN- andO-methylate amino acids, the building blocks of NRPs. Since we have shown that a single M domain could be added to an uninterrupted A domain to create an artificial interrupted A domain, we set out to investigate if: (i) an A domain could be engineered to contain two back-to-back M domains and (ii) the added M domains would have to reflect the pattern in Nature, a side chain (O-) methylating M domain (M_s) followed by a backbone (N-) methylating M domain (M_b), or if the order of the M domains could be reversed. To address these questions, we set out to create our own AM_sM_bA and AM_bM_sA engineered interrupted A domains. We evaluated these engineered proteins connected (incis) and/or disconnected (intrans) from the native thiolation (T) domain, through a series of radiometric assays, high performance liquid chromatography (HPLC), and mass spectrometry (MS) for adenylation, loading, and methylation ability. We found that although adenylation activity was preserved in both versions (AM_sM_bA and AM_bM_sA), addition of the M domains, in natural and unnatural order, did not result in the desired added methylation capability. This study offers valuable insights into the limits of constructing engineered interrupted A domains as potential tools for modifications of NRPs.

Original language	English
Pages (from-to)	34299-34307
Number of pages	9
Journal	RSC Advances
Volume	10
Issue number	56
DOIs	https://doi.org/10.1039/d0ra05490a
State	Published - Sep 15 2020

Bibliographical note

Funding Information:
This work was supported by a National Science Foundation (NSF) CAREER Award MCB-1149427 (to S. G.-T.) and by startup funds from the University of Kentucky College of Pharmacy (to S. G.-T.). T. A. L. was in part supported by a 2018–2019 and a 2019–2020 Pharmaceutical Sciences Excellence in Graduate Achievement Fellowship from the College of Pharmacy at the University of Kentucky as well as a 2019–2020 Pre-doctoral Fellowship in Pharmaceutical Sciences from the American Foundation of Pharmaceutical Education (AFPE). We thank Dr Nishad Tham-ban Chandrika for synthesizing the N,O-dimethyl-L-Ser15 used as a standard in the MS and MS2 studies presented herein. We thank Dr Atefeh Garzan for synthesizing the N-methyl-L-Cys9 used in the HPLC studies presented in this manuscript.

Funding Information:
This work was supported by a National Science Foundation (NSF) CAREER Award MCB-1149427 (to S. G.-T.) and by startup funds from the University of Kentucky College of Pharmacy (to S. G.-T.). T. A. L. was in part supported by a 2018-2019 and a 2019-2020 Pharmaceutical Sciences Excellence in Graduate Achievement Fellowship from the College of Pharmacy at the University of Kentucky as well as a 2019-2020 Pre-doctoral Fellowship in Pharmaceutical Sciences from the American Foundation of Pharmaceutical Education (AFPE). We thank Dr Nishad Thamban Chandrika for synthesizing theN,O-dimethyl-l-Ser used as a standard in the MS and MS studies presented herein. We thank Dr Atefeh Garzan for synthesizing theN-methyl-l-Cys used in the HPLC studies presented in this manuscript.

Publisher Copyright:
© The Royal Society of Chemistry 2020.

ASJC Scopus subject areas

Chemistry (all)
Chemical Engineering (all)

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10.1039/d0ra05490a

Cite this

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title = "Lessons learned in engineering interrupted adenylation domains when attempting to create trifunctional enzymes from three independent monofunctional ones",

abstract = "Interrupted adenylation (A) domains are fascinating examples of multifunctional enzymes. They are found in nonribosomal peptide synthetases (NRPSs), which biosynthesize nonribosomal peptides (NRPs), a major class of medically relevant natural products (NPs). Interrupted A domains contain the catalytic portion of another domain within them, typically a methylation (M) domain, thus combining both adenylation and methylation capabilities. In recent years, interrupted A domains have demonstrated tremendous enzyme engineering potential as they are able to be constructed artificially in a laboratory setting by combining the A and M domains of two separate NRPS proteins. A recent discovery and characterization of a naturally occurring interrupted A domain that harbored two M domains back-to-back, a trifunctional protein, showed the ingenuity of Nature to bothN- andO-methylate amino acids, the building blocks of NRPs. Since we have shown that a single M domain could be added to an uninterrupted A domain to create an artificial interrupted A domain, we set out to investigate if: (i) an A domain could be engineered to contain two back-to-back M domains and (ii) the added M domains would have to reflect the pattern in Nature, a side chain (O-) methylating M domain (Ms) followed by a backbone (N-) methylating M domain (Mb), or if the order of the M domains could be reversed. To address these questions, we set out to create our own AMsMbA and AMbMsA engineered interrupted A domains. We evaluated these engineered proteins connected (incis) and/or disconnected (intrans) from the native thiolation (T) domain, through a series of radiometric assays, high performance liquid chromatography (HPLC), and mass spectrometry (MS) for adenylation, loading, and methylation ability. We found that although adenylation activity was preserved in both versions (AMsMbA and AMbMsA), addition of the M domains, in natural and unnatural order, did not result in the desired added methylation capability. This study offers valuable insights into the limits of constructing engineered interrupted A domains as potential tools for modifications of NRPs.",

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

note = "Funding Information: This work was supported by a National Science Foundation (NSF) CAREER Award MCB-1149427 (to S. G.-T.) and by startup funds from the University of Kentucky College of Pharmacy (to S. G.-T.). T. A. L. was in part supported by a 2018–2019 and a 2019–2020 Pharmaceutical Sciences Excellence in Graduate Achievement Fellowship from the College of Pharmacy at the University of Kentucky as well as a 2019–2020 Pre-doctoral Fellowship in Pharmaceutical Sciences from the American Foundation of Pharmaceutical Education (AFPE). We thank Dr Nishad Tham-ban Chandrika for synthesizing the N,O-dimethyl-L-Ser15 used as a standard in the MS and MS2 studies presented herein. We thank Dr Atefeh Garzan for synthesizing the N-methyl-L-Cys9 used in the HPLC studies presented in this manuscript. Funding Information: This work was supported by a National Science Foundation (NSF) CAREER Award MCB-1149427 (to S. G.-T.) and by startup funds from the University of Kentucky College of Pharmacy (to S. G.-T.). T. A. L. was in part supported by a 2018-2019 and a 2019-2020 Pharmaceutical Sciences Excellence in Graduate Achievement Fellowship from the College of Pharmacy at the University of Kentucky as well as a 2019-2020 Pre-doctoral Fellowship in Pharmaceutical Sciences from the American Foundation of Pharmaceutical Education (AFPE). We thank Dr Nishad Thamban Chandrika for synthesizing theN,O-dimethyl-l-Ser used as a standard in the MS and MS studies presented herein. We thank Dr Atefeh Garzan for synthesizing theN-methyl-l-Cys used in the HPLC studies presented in this manuscript. Publisher Copyright: {\textcopyright} The Royal Society of Chemistry 2020.",

year = "2020",

month = sep,

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doi = "10.1039/d0ra05490a",

language = "English",

volume = "10",

pages = "34299--34307",

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T1 - Lessons learned in engineering interrupted adenylation domains when attempting to create trifunctional enzymes from three independent monofunctional ones

AU - Lundy, Taylor A.

AU - Mori, Shogo

AU - Garneau-Tsodikova, Sylvie

N1 - Funding Information: This work was supported by a National Science Foundation (NSF) CAREER Award MCB-1149427 (to S. G.-T.) and by startup funds from the University of Kentucky College of Pharmacy (to S. G.-T.). T. A. L. was in part supported by a 2018–2019 and a 2019–2020 Pharmaceutical Sciences Excellence in Graduate Achievement Fellowship from the College of Pharmacy at the University of Kentucky as well as a 2019–2020 Pre-doctoral Fellowship in Pharmaceutical Sciences from the American Foundation of Pharmaceutical Education (AFPE). We thank Dr Nishad Tham-ban Chandrika for synthesizing the N,O-dimethyl-L-Ser15 used as a standard in the MS and MS2 studies presented herein. We thank Dr Atefeh Garzan for synthesizing the N-methyl-L-Cys9 used in the HPLC studies presented in this manuscript. Funding Information: This work was supported by a National Science Foundation (NSF) CAREER Award MCB-1149427 (to S. G.-T.) and by startup funds from the University of Kentucky College of Pharmacy (to S. G.-T.). T. A. L. was in part supported by a 2018-2019 and a 2019-2020 Pharmaceutical Sciences Excellence in Graduate Achievement Fellowship from the College of Pharmacy at the University of Kentucky as well as a 2019-2020 Pre-doctoral Fellowship in Pharmaceutical Sciences from the American Foundation of Pharmaceutical Education (AFPE). We thank Dr Nishad Thamban Chandrika for synthesizing theN,O-dimethyl-l-Ser used as a standard in the MS and MS studies presented herein. We thank Dr Atefeh Garzan for synthesizing theN-methyl-l-Cys used in the HPLC studies presented in this manuscript. Publisher Copyright: © The Royal Society of Chemistry 2020.

PY - 2020/9/15

Y1 - 2020/9/15

N2 - Interrupted adenylation (A) domains are fascinating examples of multifunctional enzymes. They are found in nonribosomal peptide synthetases (NRPSs), which biosynthesize nonribosomal peptides (NRPs), a major class of medically relevant natural products (NPs). Interrupted A domains contain the catalytic portion of another domain within them, typically a methylation (M) domain, thus combining both adenylation and methylation capabilities. In recent years, interrupted A domains have demonstrated tremendous enzyme engineering potential as they are able to be constructed artificially in a laboratory setting by combining the A and M domains of two separate NRPS proteins. A recent discovery and characterization of a naturally occurring interrupted A domain that harbored two M domains back-to-back, a trifunctional protein, showed the ingenuity of Nature to bothN- andO-methylate amino acids, the building blocks of NRPs. Since we have shown that a single M domain could be added to an uninterrupted A domain to create an artificial interrupted A domain, we set out to investigate if: (i) an A domain could be engineered to contain two back-to-back M domains and (ii) the added M domains would have to reflect the pattern in Nature, a side chain (O-) methylating M domain (Ms) followed by a backbone (N-) methylating M domain (Mb), or if the order of the M domains could be reversed. To address these questions, we set out to create our own AMsMbA and AMbMsA engineered interrupted A domains. We evaluated these engineered proteins connected (incis) and/or disconnected (intrans) from the native thiolation (T) domain, through a series of radiometric assays, high performance liquid chromatography (HPLC), and mass spectrometry (MS) for adenylation, loading, and methylation ability. We found that although adenylation activity was preserved in both versions (AMsMbA and AMbMsA), addition of the M domains, in natural and unnatural order, did not result in the desired added methylation capability. This study offers valuable insights into the limits of constructing engineered interrupted A domains as potential tools for modifications of NRPs.

AB - Interrupted adenylation (A) domains are fascinating examples of multifunctional enzymes. They are found in nonribosomal peptide synthetases (NRPSs), which biosynthesize nonribosomal peptides (NRPs), a major class of medically relevant natural products (NPs). Interrupted A domains contain the catalytic portion of another domain within them, typically a methylation (M) domain, thus combining both adenylation and methylation capabilities. In recent years, interrupted A domains have demonstrated tremendous enzyme engineering potential as they are able to be constructed artificially in a laboratory setting by combining the A and M domains of two separate NRPS proteins. A recent discovery and characterization of a naturally occurring interrupted A domain that harbored two M domains back-to-back, a trifunctional protein, showed the ingenuity of Nature to bothN- andO-methylate amino acids, the building blocks of NRPs. Since we have shown that a single M domain could be added to an uninterrupted A domain to create an artificial interrupted A domain, we set out to investigate if: (i) an A domain could be engineered to contain two back-to-back M domains and (ii) the added M domains would have to reflect the pattern in Nature, a side chain (O-) methylating M domain (Ms) followed by a backbone (N-) methylating M domain (Mb), or if the order of the M domains could be reversed. To address these questions, we set out to create our own AMsMbA and AMbMsA engineered interrupted A domains. We evaluated these engineered proteins connected (incis) and/or disconnected (intrans) from the native thiolation (T) domain, through a series of radiometric assays, high performance liquid chromatography (HPLC), and mass spectrometry (MS) for adenylation, loading, and methylation ability. We found that although adenylation activity was preserved in both versions (AMsMbA and AMbMsA), addition of the M domains, in natural and unnatural order, did not result in the desired added methylation capability. This study offers valuable insights into the limits of constructing engineered interrupted A domains as potential tools for modifications of NRPs.

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Lessons learned in engineering interrupted adenylation domains when attempting to create trifunctional enzymes from three independent monofunctional ones

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