| Rapid and flexible biochemical assays for evaluating 4'-phosphopantetheinyl transferase activity. | |
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MedLine Citation:
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PMID: 21466506 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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Phosphopantetheinyl transferases (PPTases) are of great interest due to their essential roles in activating fatty acid, polyketide, and non-ribosomal peptide synthetase enzymes for both primary and secondary metabolism, as well as an increasing number of biotechnological applications. However, existing techniques for PPTase characterisation and development are unwieldy and technically challenging. To address this we have developed the indigoidine-synthesising non-ribosomal peptide synthetase BpsA as a reporter for PPTase activity. Simple co-transformation allows rapid assessment of the ability of a PPTase candidate to activate BpsA in vivo. Kinetic parameters with respect to either CoA or BpsA as variable substrate can then be derived in vitro by continuously measuring the rate of indigoidine synthesis as the PPTase progressively converts BpsA from apo to holo-form. Subsequently a competition assay, in which BpsA and purified carrier proteins compete for a limited pool of CoA, enables elucidation of kinetic parameters for a PPTase with those carrier proteins. We used this system to conduct a rapid characterisation of three different PPTase enzymes: Sfp of Bacillus subtilis ATCC6633, PcpS of P. aeruginosa PAO1, and the putative PPTase PP1183 of P. putida KT2440. We also demonstrate the utility of this system for discovery and characterisation of PPTase inhibitors. |
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Authors:
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Jeremy G Owen; Janine N Copp; David F Ackerley |
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Publication Detail:
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Type: JOURNAL ARTICLE Date: 2011-4-6 |
Journal Detail:
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Title: The Biochemical journal Volume: - ISSN: 1470-8728 ISO Abbreviation: - Publication Date: 2011 Apr |
Date Detail:
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Created Date: 2011-4-6 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 2984726R Medline TA: Biochem J Country: - |
Other Details:
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Languages: ENG Pagination: - Citation Subset: - |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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