ITC-based kinetics assay for NIS synthetases.
ITC
Iterative enzymology
Kinetics assay
NIS synthetases
Journal
Methods in enzymology
ISSN: 1557-7988
Titre abrégé: Methods Enzymol
Pays: United States
ID NLM: 0212271
Informations de publication
Date de publication:
2024
2024
Historique:
medline:
19
8
2024
pubmed:
19
8
2024
entrez:
18
8
2024
Statut:
ppublish
Résumé
NIS Synthetases are a widely distributed, novel superfamily of enzymes critical to stealth siderophore production-small molecules increasingly associated with virulence. Study of these enzymes for inhibition or utilization in biosynthesis of new antibiotics has been hindered by multiple kinetics assays utilizing different limiting reporters or relying on product dissociation as a precursor to signal. We present a label free, continuous readout assay optimized for NIS Synthetase systems utilizing an isothermal titration calorimetry instrument. This assay has been tested in an iterative system comparing multiple turnovers on a single substrate to a single bond formation event and is able to delineate these complex kinetics well. The ITC-based kinetic assay is the first label-free assay for the NIS field, which may allow for more detailed kinetic comparisons in the future, and may also have broader use for iterative enzymes in general.
Identifiants
pubmed: 39155121
pii: S0076-6879(24)00326-4
doi: 10.1016/bs.mie.2024.06.017
pii:
doi:
Substances chimiques
Peptide Synthases
EC 6.3.2.-
Bacterial Proteins
0
Siderophores
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
75-87Informations de copyright
Copyright © 2024. Published by Elsevier Inc.