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Developing crosslinkers specific for epimerization domain in NRPS initiation modules to evaluate mechanism
Nonribosomal peptide synthetases (NRPSs) are complex multi-modular enzymes containing catalytic domains responsible for the loading and incorporation of amino acids into natural products. These unique molecular factories can produce peptides with nonproteinogenic d-amino acids in which the epimeriza...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
RSC
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8905534/ https://www.ncbi.nlm.nih.gov/pubmed/35359491 http://dx.doi.org/10.1039/d2cb00005a |
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author | Kim, Woojoo E. Ishikawa, Fumihiro Re, Rebecca N. Suzuki, Takehiro Dohmae, Naoshi Kakeya, Hideaki Tanabe, Genzoh Burkart, Michael D. |
author_facet | Kim, Woojoo E. Ishikawa, Fumihiro Re, Rebecca N. Suzuki, Takehiro Dohmae, Naoshi Kakeya, Hideaki Tanabe, Genzoh Burkart, Michael D. |
author_sort | Kim, Woojoo E. |
collection | PubMed |
description | Nonribosomal peptide synthetases (NRPSs) are complex multi-modular enzymes containing catalytic domains responsible for the loading and incorporation of amino acids into natural products. These unique molecular factories can produce peptides with nonproteinogenic d-amino acids in which the epimerization (E) domain catalyzes the conversion of l-amino acids to d-amino acids, but its mechanism remains not fully understood. Here, we describe the development of pantetheine crosslinking probes that mimic the natural substrate l-Phe of the initiation module of tyrocidine synthetase, TycA, to elucidate and study the catalytic residues of the E domain. Mechanism-based crosslinking assays and MALDI-TOF MS were used to identify both H743 and E882 as the crosslinking site residues, demonstrating their roles as catalytic bases. Mutagenesis studies further validated these results and allowed the comparison of reactivity between the catalytic residues, concluding that glutamate acts as the dominant nucleophile in the crosslinking reaction, resembling the deprotonation of the C(α)-H of amino acids in the epimerization reaction. The crosslinking probes employed in these studies provide new tools for studying the molecular details of E domains, as well as the potential to study C domains. In particular, they would elucidate key information for how these domains function and interact with their substrates in nature, further enhancing the knowledge needed to assist combinatorial biosynthetic efforts of NRPS systems to produce novel compounds. |
format | Online Article Text |
id | pubmed-8905534 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | RSC |
record_format | MEDLINE/PubMed |
spelling | pubmed-89055342022-03-30 Developing crosslinkers specific for epimerization domain in NRPS initiation modules to evaluate mechanism Kim, Woojoo E. Ishikawa, Fumihiro Re, Rebecca N. Suzuki, Takehiro Dohmae, Naoshi Kakeya, Hideaki Tanabe, Genzoh Burkart, Michael D. RSC Chem Biol Chemistry Nonribosomal peptide synthetases (NRPSs) are complex multi-modular enzymes containing catalytic domains responsible for the loading and incorporation of amino acids into natural products. These unique molecular factories can produce peptides with nonproteinogenic d-amino acids in which the epimerization (E) domain catalyzes the conversion of l-amino acids to d-amino acids, but its mechanism remains not fully understood. Here, we describe the development of pantetheine crosslinking probes that mimic the natural substrate l-Phe of the initiation module of tyrocidine synthetase, TycA, to elucidate and study the catalytic residues of the E domain. Mechanism-based crosslinking assays and MALDI-TOF MS were used to identify both H743 and E882 as the crosslinking site residues, demonstrating their roles as catalytic bases. Mutagenesis studies further validated these results and allowed the comparison of reactivity between the catalytic residues, concluding that glutamate acts as the dominant nucleophile in the crosslinking reaction, resembling the deprotonation of the C(α)-H of amino acids in the epimerization reaction. The crosslinking probes employed in these studies provide new tools for studying the molecular details of E domains, as well as the potential to study C domains. In particular, they would elucidate key information for how these domains function and interact with their substrates in nature, further enhancing the knowledge needed to assist combinatorial biosynthetic efforts of NRPS systems to produce novel compounds. RSC 2022-01-27 /pmc/articles/PMC8905534/ /pubmed/35359491 http://dx.doi.org/10.1039/d2cb00005a Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by-nc/3.0/ |
spellingShingle | Chemistry Kim, Woojoo E. Ishikawa, Fumihiro Re, Rebecca N. Suzuki, Takehiro Dohmae, Naoshi Kakeya, Hideaki Tanabe, Genzoh Burkart, Michael D. Developing crosslinkers specific for epimerization domain in NRPS initiation modules to evaluate mechanism |
title | Developing crosslinkers specific for epimerization domain in NRPS initiation modules to evaluate mechanism |
title_full | Developing crosslinkers specific for epimerization domain in NRPS initiation modules to evaluate mechanism |
title_fullStr | Developing crosslinkers specific for epimerization domain in NRPS initiation modules to evaluate mechanism |
title_full_unstemmed | Developing crosslinkers specific for epimerization domain in NRPS initiation modules to evaluate mechanism |
title_short | Developing crosslinkers specific for epimerization domain in NRPS initiation modules to evaluate mechanism |
title_sort | developing crosslinkers specific for epimerization domain in nrps initiation modules to evaluate mechanism |
topic | Chemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8905534/ https://www.ncbi.nlm.nih.gov/pubmed/35359491 http://dx.doi.org/10.1039/d2cb00005a |
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