Identification and quantitation of acyl thioesters by thin-layer chromatography of hydroxamic acid derivatives

325

380

Section: Materials-sources Of Supplies Are As Follows: [mentioning

confidence: 99%

“…Synthase I (FabB) is required for a critical step in the elongation of unsaturated fatty acids. Mutants (fabB) lacking synthase I activity require supplementation with exogenous unsaturated fatty acids to support growth (7,8). Synthase II (FabF) controls the temperaturedependent regulation of fatty acid composition (9,10).…”

mentioning

confidence: 99%

Inhibition of β-Ketoacyl-Acyl Carrier Protein Synthases by Thiolactomycin and Cerulenin

Price¹,

Choi

Heath

et al. 2001

325

380

“…Hydroxylamine Treatment of Membranes and Cytosolic FactorMembranes and cytosolic factor were subjected to treatment with hydroxylamine at pH 6.5, to determine if either one contained a sensitive thioester linkage necessary for the observed activity (47). To this end, 0.35 mg of membrane protein or 0.14 mg of cytosolic factor, fractionated through the Centricon-50 step, were each treated with 1.2 M hydroxylamine-hydrochloride (derived from a 3.08 M stock solution and titrated to pH 6.5 with 10 M NaOH) in a final volume of 50 l. These reactions were incubated at room temperature for 2 h. As controls, identical samples were incubated at room temperature without hydroxylamine for 2 h. Then, all samples, including the untreated incubated ones, were exchanged twice with 0.5 ml of 50 mM HEPES, pH 7.5, using Centricon-30 devices, to remove the excess hydroxylamine prior to the acylation assays (described above).…”

Section: -[Lauroyl]-[4ј-mentioning

confidence: 99%

A Special Acyl Carrier Protein for Transferring Long Hydroxylated Fatty Acids to Lipid A in Rhizobium

Brozek

Carlson

Raetz

1996

Lipid A, the hydrophobic anchor of lipopolysaccharides in the outer membranes of Gram-negative bacteria, varies in structure among different Rhizobiaceae. The Rhizobium meliloti lipid A backbone, like that of Escherichia coli, is a ␤1-6-linked glucosamine disaccharide that is phosphorylated at positions 1 and 4. Rhizobium leguminosarum lipid A lacks both phosphates, but contains aminogluconate in place of the proximal glucosamine 1-phosphate, and galacturonic acid instead of the 4-phosphate. A peculiar feature of the lipid As of all Rhizobiaceae is acylation with 27-hydroxyoctacosanoic acid, a long hydroxylated fatty acid not found in E. coli. We now describe an in vitro system, consisting of a membrane enzyme and a cytosolic acyl donor from R. leguminosarum, that transfers 27-hydroxyoctacosanoic acid to (Kdo) 2 -lipid IV A , a key lipid A precursor common to both E. coli and R. leguminosarum. The 27-hydroxyoctacosanoic acid moiety was detected in the lipid product by mass spectrometry. The membrane enzyme required the presence of Kdo residues in the acceptor substrate for activity. The cytosolic acyl donor was purified from wild-type R. leguminosarum using the acylation of (Kdo) 2 -[4-32 P]-lipid IV A as the assay. Amino-terminal sequencing of the purified acyl donor revealed an exact 19-amino acid match with a partially sequenced gene (orf*) of R. leguminosarum. Orf * contains the consensus sequence, DSLD, for attachment of 4-phosphopantetheine. When the entire orf * gene was sequenced, it was found to encode a protein of 92 amino acids. Orf * is a new kind of acyl carrier protein because it is only ϳ25% identical both to the constitutive acyl carrier protein (AcpP) and to the inducible acyl carrier protein (NodF) of R. leguminosarum. Mass spectrometry of purified active Orf * confirmed the presence of 4-phosphopantetheine and 27-hydroxyoctacosanoic acid in the major species. Smaller mass peaks indicative of Orf * acylation with hydroxylated 20, 22, 24, and 26 carbon fatty acids were also observed. Given the specialized function of Orf * in lipid A acylation, we suggest the new designation AcpXL.

“…Thus, FabA catalyzes the essential isomerization reaction necessary to introduce the double bond into the 10-carbon intermediate of the growing acyl chain, and FabB is postulated to be required to elongate one or more of the critical cis unsaturated intermediates (5,22). Accordingly, mutational inactivation of either the fabA or fabB gene results in an unsaturated fatty acid auxotroph phenotype (20,21). The fabA and fabB mutants retain * This work was supported in part by National Institutes of Health Grant GM 34496 (to C. O. R.), Cancer Center Support Grant CA21765, and the American Lebanese Syrian Associated Charities.…”

mentioning

confidence: 99%

“…This enzyme, in addition to performing the dehydration step, has the unique property of isomerizing trans-2-to cis-3-decenoyl-ACP as an essential step in the formation of unsaturated fatty acids in Escherichia coli (17)(18)(19)(20). However, the distribution of FabA is limited to the type II systems found in Gram-negative bacteria that produce unsaturated fatty acids and is always found with its partner, FabB, a condensing enzyme that is also essential for unsaturated fatty acid formation (21). Thus, FabA catalyzes the essential isomerization reaction necessary to introduce the double bond into the 10-carbon intermediate of the growing acyl chain, and FabB is postulated to be required to elongate one or more of the critical cis unsaturated intermediates (5,22).…”

mentioning

confidence: 99%

The Structure of (3R)-Hydroxyacyl-Acyl Carrier Protein Dehydratase (FabZ) from Pseudomonas aeruginosa

Kimber¹,

Martín²,

et al. 2004

126

127

Type II fatty acid biosynthesis systems are essential for membrane formation in bacteria, making the constituent proteins of this pathway attractive targets for antibacterial drug discovery. The third step in the elongation cycle of the type II fatty acid biosynthesis is catalyzed by ␤-hydroxyacyl-(acyl carrier protein) (ACP) dehydratase. There are two isoforms. FabZ, which catalyzes the dehydration of (3R)-hydroxyacyl-ACP to trans-2-acyl-ACP, is a universally expressed component of the bacterial type II system. FabA, the second isoform, as has more limited distribution in nature and, in addition to dehydration, also carries out the isomerization of trans-2-to cis-3-decenoyl-ACP as an essential step in unsaturated fatty acid biosynthesis. We report the structure of FabZ from the important human pathogen Pseudomonas aeruginosa at 2.5 Å of resolution. PaFabZ is a hexamer (trimer of dimers) with the His/Glu catalytic dyad located within a deep, narrow tunnel formed at the dimer interface. Site-directed mutagenesis experiments showed that the obvious differences in the active site residues that distinguish the FabA and FabZ subfamilies of dehydratases do not account for the unique ability of FabA to catalyze isomerization. Because the catalytic machinery of the two enzymes is practically indistinguishable, the structural differences observed in the shape of the substrate binding channels of FabA and FabZ lead us to hypothesize that the different shapes of the tunnels control the conformation and positioning of the bound substrate, allowing FabA, but not FabZ, to catalyze the isomerization reaction.In eubacteria and their endosymbiotic descendants (the plastids of plants and apicomplexan parasites) fatty acids are produced by what is known as the type II fatty acid biosynthetic pathway (1-3). The steps in this pathway are catalyzed by a universal set of enzymes, each encoded by a separate gene, that have been most closely studied in the model organism Escherichia coli (4, 5). The growing acyl chain is shuttled between the pathway enzymes attached to the 4Ј-phosphopantetheine prosthetic group of a dedicated carrier protein, ACP.1 This system contrasts with the type I fatty acid biosynthesis system that exists in metazoans, where multifunctional polypeptide chains (6) encode all activities in chain initiation and elongation. The intermediates in the type I system are shuffled from one catalytic site to another without being released from the complex. In light of the profound differences in these two systems, enzymes of the type II pathway have emerged as attractive targets for the development of novel antimicrobial or antiparasitic agents (2,7,8). The core feature of the type II pathway is the fatty acid elongation cycle, which extends the fatty acid chain by two carbons in each round. There are four steps in the cycle, and the proteins involved in E. coli are 1) condensation of malonyl-ACP with acyl-ACP, catalyzed by the FabB-and FabF-condensing enzymes, 2) reduction of the ␤-keto moiety by the NADPH-dependent FabG re...