Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.184 (LasR)
 897 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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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PMID:Characterization of Pseudomonas aeruginosa enoyl-acyl carrier protein reductase (FabI): a target for the antimicrobial triclosan and its role in acylated homoserine lactone synthesis. 1046 25

Purification of proteins from Escherichia coli under native conditions is often hampered by inclusion-body formation after overexpression from T7 promoter-based expression vectors. This is probably due to the relatively high copy number of the ColE1-based expression vectors. To circumvent these problems, the low-copy-number pViet and pNam expression vectors were constructed. These vectors contain the pSC101 origin of replication and allow the expression of oligohistidine and intein chitin-binding domain fusion proteins, respectively. Since pViet and pNam do not replicate in E. coli B strains, an E. coli K-12 host strain [SA1503(DE3)] was constructed. This strain is defective in the Lon and OmpT proteases and allows IPTG-inducible expression of recombinant proteins from the T7 promoter. The new vectors were successfully tested by purification of three very insoluble proteins (RmlD, LasI and RhlI) under non-denaturing conditions, and all three proteins retained enzymatic activity. The purified hexahistidine (His6)-tagged Pseudomonas aeruginosa RhlI protein was subjected to more detailed analyses, which indicated that (1) only butyryl-acyl carrier protein (ACP) and S-adenosylmethionine (SAM) were required for synthesis of N-butyryl-L-homoserine lactone; (2) when present at physiological concentrations, butyryl-coenzyme A and NADPH were not substrates for RhlI; (3) RhlI was able to synthesize N-hexanoyl-L-homoserine lactone from hexanoyl-ACP and SAM; (4) RhlI was able to direct synthesis of N-butyryl-L-homoserine lactone from crotonyl-ACP in a reaction coupled to purified P. aeruginosa FabI (enoyl-ACP reductase).
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PMID:Construction and use of low-copy number T7 expression vectors for purification of problem proteins: purification of mycobacterium tuberculosis RmlD and pseudomonas aeruginosa LasI and RhlI proteins, and functional analysis of purified RhlI. 1052 60

The two acyl-homoserine lactones (AHLs) N-(butyryl)-L-homoserine lactone and N-[3-oxododecanoyl]-L-homoserine lactone (3-oxo-C(12)-HSL) are required for quorum sensing in Pseudomonas aeruginosa. These AHLs derive their invariant lactone rings from S-adenosylmethionine and their variable acyl chains from the cellular acyl-acyl carrier protein (ACP) pool. This reaction is catalysed by specific AHL synthases, which exhibit acyl chain specificity. Culture supernatants of P. aeruginosa contain multiple 3-oxo-AHLs that differ in their acyl chain lengths but their physiological role, if any, remains unknown. An in vitro fatty acid-3-oxo-AHL synthesis system was established utilizing purified P. aeruginosa Fab proteins, ACP and the LasI 3-oxo-AHL synthase. In the presence of excess protein, substrates and cofactors, this system produced almost exclusively 3-oxo-C(12)-HSL. When the beta-ketoacyl-ACP reductase (FabG) catalysed step was made rate-limiting by switching from the preferred NADPH cofactor to NADH, increased levels of short chain 3-oxo-AHLs were produced, presumably because shorter-chain ketoacyl-ACPs accumulated and thus became LasI substrates. Consistent with these in vitro observations, a fabG(Ts) mutant produced increased amounts of 3-oxo-AHLs in vivo. Thus, in vitro and in vivo evidence indicated that modulation of FabG activity of the fatty acid biosynthetic pathway may determine the acyl chain lengths of these 3-oxo-AHLs and that the LasI 3-oxo-AHL synthase is sufficient for their synthesis.
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PMID:Beta-ketoacyl acyl carrier protein reductase (FabG) activity of the fatty acid biosynthetic pathway is a determining factor of 3-oxo-homoserine lactone acyl chain lengths. 1248 Aug 88