Document Detail

Characterization of Pseudomonas aeruginosa enoyl-acyl carrier protein reductase (FabI): a target for the antimicrobial triclosan and its role in acylated homoserine lactone synthesis.
MedLine Citation:
PMID:  10464225     Owner:  NLM     Status:  MEDLINE    
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.
T T Hoang; H P Schweizer
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Journal of bacteriology     Volume:  181     ISSN:  0021-9193     ISO Abbreviation:  J. Bacteriol.     Publication Date:  1999 Sep 
Date Detail:
Created Date:  1999-10-07     Completed Date:  1999-10-07     Revised Date:  2013-04-17    
Medline Journal Info:
Nlm Unique ID:  2985120R     Medline TA:  J Bacteriol     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  5489-97     Citation Subset:  IM    
Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523, USA.
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MeSH Terms
4-Butyrolactone / analogs & derivatives*,  biosynthesis
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
Reg. No./Substance:
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 Reductase (NADH)

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