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Siderophore biosynthesis: a substrate specificity assay for nonribosomal peptide synthetase-independent siderophore synthetases involving trapping of acyl-adenylate intermediates with hydroxylamine
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Kadi, Nadia and Challis, Gregory L. (2009) Siderophore biosynthesis: a substrate specificity assay for nonribosomal peptide synthetase-independent siderophore synthetases involving trapping of acyl-adenylate intermediates with hydroxylamine. Methods in Enzymology, Vol.458 . pp. 431-457. doi:10.1016/S0076-6879(09)04817-4
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Official URL: http://dx.doi.org/10.1016/S0076-6879(09)04817-4
Abstract
Siderophores are an important group of structurally diverse natural products that play key roles in ferric iron acquisition in most microorganisms. Two major pathways exist for siderophore biosynthesis. One is dependent on nonribosomal peptide synthetase (NRPS) multienzymes. The enzymology of several NRPS-dependent pathways to structurally diverse siderophores has been intensively studied for more than 10 years and is generally well understood. The other major pathway is NRPS-independent. It relies on a novel family of synthetase enzymes that until recently has received very little attention. Over the last 2 years, these enzymes have begun to be intensively investigated and several examples have now been characterized. In this article, we give an overview of the enzymology of NRPS-dependent and NRPS-independent pathways for siderophore biosynthesis, using selected examples to highlight key features.
An important facet of many studies of the enzymology of siderophore biosynthesis has been to investigate the substrate specificity of the synthetase enzymes involved. For NRPS-dependent pathways, the ATP-pyrophophate exchange assay has been widely used to investigate the substrate specificity of adenylation domains within the synthetase multienzymes. This assay is ineffective for NRPS-independent siderophore (NIS) synthetases, probably because pyrophosphate is not released from the enzyme after the carboxylic acid substrate and ATP react to form an acyl adenylate. An alternative assay for enzymes that form acyl adenylates involves trapping of the activated carboxyl group with hydroxylamine to form a hydroxamic acid that can be converted to its ferric complex and detected spectrophotometrically. This assay has not been widely used for NRPS adenylation domains. Here, we show that it is an effective assay for examining the carboxylic acid substrate specificity of NIS synthetases. Application of the assay to the type B NIS synthetase AcsA shows that it is selective for alpha-ketoglutaric acid, confirming a bioinformatics-based prediction of the substrate specificity of this enzyme.
| Item Type: | Journal Item | ||||
|---|---|---|---|---|---|
| Subjects: | Q Science > QD Chemistry | ||||
| Divisions: | Faculty of Science, Engineering and Medicine > Science > Chemistry | ||||
| Series Name: | METHODS IN ENZYMOLOGY | ||||
| Journal or Publication Title: | Methods in Enzymology | ||||
| Publisher: | Academic Press | ||||
| ISSN: | 0076-6879 | ||||
| Official Date: | 2009 | ||||
| Dates: |
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| Volume: | Vol.458 | ||||
| Number of Pages: | 27 | ||||
| Page Range: | pp. 431-457 | ||||
| DOI: | 10.1016/S0076-6879(09)04817-4 | ||||
| Status: | Peer Reviewed | ||||
| Publication Status: | Published | ||||
| Access rights to Published version: | Restricted or Subscription Access |
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