Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Kim, Woojoo E., Ishikawa, Fumihiro, Re, Rebecca N., Suzuki, Takehiro, Dohmae, Naoshi, Kakeya, Hideaki, Tanabe, Genzoh, Burkart, Michael D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: RSC 2022
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
_version_ 1784665208223956992
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
work_keys_str_mv AT kimwoojooe developingcrosslinkersspecificforepimerizationdomaininnrpsinitiationmodulestoevaluatemechanism
AT ishikawafumihiro developingcrosslinkersspecificforepimerizationdomaininnrpsinitiationmodulestoevaluatemechanism
AT rerebeccan developingcrosslinkersspecificforepimerizationdomaininnrpsinitiationmodulestoevaluatemechanism
AT suzukitakehiro developingcrosslinkersspecificforepimerizationdomaininnrpsinitiationmodulestoevaluatemechanism
AT dohmaenaoshi developingcrosslinkersspecificforepimerizationdomaininnrpsinitiationmodulestoevaluatemechanism
AT kakeyahideaki developingcrosslinkersspecificforepimerizationdomaininnrpsinitiationmodulestoevaluatemechanism
AT tanabegenzoh developingcrosslinkersspecificforepimerizationdomaininnrpsinitiationmodulestoevaluatemechanism
AT burkartmichaeld developingcrosslinkersspecificforepimerizationdomaininnrpsinitiationmodulestoevaluatemechanism