| Characterization of HmqF, a Protein Involved in the Biosynthesis of Unsaturated Quinolones Produced by Burkholderia thailandensis. | |
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MedLine Citation:
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PMID: 22320268 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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The opportunistic pathogen Burkholderia thailandensis produces a number of structurally similar unsaturated quinolones involved in quorum sensing. However little is known about the biosynthesis of these unsaturated quinolones. In this study we have characterized the starting point in biosynthesis of unsaturated quinolone molecules produced in Burkholderia thailandensis. We have shown by using in vitro enzymology and liquid chromatography/mass spectrometry that the protein HmqF is involved in the biosynthesis of unsaturated quinolones produced by Burkholderia thailandensis. HmqF consists of three domains; an adenylation domain (A domain), a dehydrogenase domain (DH domain), and an acyl carrier domain (ACP). The three domains (A, DH and ACP) were cloned and expressed individually in E.coli and their reactivity was studied using HPLC/MS based assays. Our in vitro studies show that the A domain catalyzes ATP-dependent activation of medium chain (C6-C14) fatty acids without activation by CoA. Results from competitions assays are consistent with decanoic acid as the preferred substrate. Incubation of the ACP domain with 4'-phosphopantetheine transferase and CoA led to the formation of phosphopantetheinylated-ACP (Ppant-ACP). In Ppant ejection assay using MS/MS a mass was detected consistent with the mass of a cyclic variant of dephosphorylated Ppant. We further demonstrated that Ppant-ACP could be loaded with medium chain fatty acids in the presence of ATP and A domain. MS analysis was consistent with the formation of Ppant-ACP thiol esters of the fatty acids. MS/MS Ppant ejection experiments confirmed the loss of 2H in samples of fatty acids loaded-Ppant-ACP in the presence of DH domain. HPLC analysis of benzyl amide ligation products allowed us to conclude that dehydrogenation produced trans-β,γ-unsaturation in the fatty acid chains. Our results are in good agreement with naturally observed quinolone molecules produced by B. thailandensis, which predominately produce 9 carbon trans-β,γ-unsaturated alkyl chain quinolone molecules. |
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Authors:
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Aparna Agarwal; Caroline Kahyaoglu; Darren Bernard Hansen |
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Publication Detail:
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Type: JOURNAL ARTICLE Date: 2012-2-8 |
Journal Detail:
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Title: Biochemistry Volume: - ISSN: 1520-4995 ISO Abbreviation: - Publication Date: 2012 Feb |
Date Detail:
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Created Date: 2012-2-10 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 0370623 Medline TA: Biochemistry 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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