| Characterization of Pseudomonas aeruginosa enoyl-acyl carrier protein reductase (FabI): a target for the antimicrobial triclosan and its role in acylated homoserine lactone synthesis. | |
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MedLine Citation:
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PMID: 10464225 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The Pseudomonas aeruginosa fabI structural gene, encoding enoyl-acyl carrier protein (ACP) reductase, was cloned and sequenced. Nucleotide sequence analysis revealed that fabI is probably the last gene in a transcriptional unit that includes a gene encoding an ATP-binding protein of an ABC transporter of unknown function. The FabI protein was similar in size and primary sequence to other bacterial enoyl-ACP reductases, and it contained signature motifs for the FAD-dependent pyridine nucleotide reductase and glucose/ribitol dehydrogenase families, respectively. The chromosomal fabI gene was disrupted, and the resulting mutant was viable but possessed only 62% of the total enoyl-ACP reductase activity found in wild-type cell extracts. The fabI-encoded enoyl-ACP reductase activity was NADH dependent and inhibited by triclosan; the residual activity in the fabI mutant was also NADH dependent but not inhibited by triclosan. An polyhistidine-tagged FabI protein was purified and characterized. Purified FabI (i) could use NADH but not NADPH as a cofactor; (ii) used both crotonyl-coenzyme A and crotonyl-ACP as substrates, although it was sixfold more active with crotonyl-ACP; and (iii) was efficiently inhibited by low concentrations of triclosan. A FabI Gly95-to-Val active-site amino acid substitution was generated by site-directed mutagenesis, and the mutant protein was purified. The mutant FabI protein retained normal enoyl-ACP reductase activity but was highly triclosan resistant. When coupled to FabI, purified P. aeruginosa N-butyryl-L-homoserine lactone (C4-HSL) synthase, RhlI, could synthesize C4-HSL from crotonyl-ACP and S-adenosylmethionine. This reaction was NADH dependent and inhibited by triclosan. The levels of C4-HSL and N-(3-oxo)-dodecanoyl-L-homoserine lactones were reduced 50% in a fabI mutant, corroborating the role of FabI in acylated homoserine lactone synthesis in vivo. |
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Authors:
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T T Hoang; H P Schweizer |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Journal of bacteriology Volume: 181 ISSN: 0021-9193 ISO Abbreviation: J. Bacteriol. Publication Date: 1999 Sep |
Date Detail:
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Created Date: 1999-10-07 Completed Date: 1999-10-07 Revised Date: 2013-04-17 |
Medline Journal Info:
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Nlm Unique ID: 2985120R Medline TA: J Bacteriol Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 5489-97 Citation Subset: IM |
Affiliation:
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Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523, USA. |
| Data Bank Information | |
Bank Name/Acc. No.:
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GENBANK/AF104262 |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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4-Butyrolactone
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analogs & derivatives*,
biosynthesis Acylation Anti-Infective Agents, Local / pharmacology* Base Sequence Cloning, Molecular DNA, Bacterial Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) Genes, Bacterial Genome, Bacterial Homoserine / analogs & derivatives*, biosynthesis Molecular Sequence Data Oxidoreductases / genetics, isolation & purification, metabolism, physiology* Pseudomonas aeruginosa / enzymology*, genetics Sequence Analysis, DNA Triclosan / pharmacology* |
| Grant Support | |
ID/Acronym/Agency:
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GM56685/GM/NIGMS NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Anti-Infective Agents, Local; 0/DNA, Bacterial; 0/N-(3-oxododecanoyl)homoserine lactone; 0/N-butyrylhomoserine lactone; 3380-34-5/Triclosan; 498-19-1/Homoserine; 96-48-0/4-Butyrolactone; EC 1.-/Oxidoreductases; EC 1.3.1.9/Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) |
| Comments/Corrections | |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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