scholarly journals Novel inhibitors of the condensing enzymes of the Type II fatty acid synthase of pea (Pisum sativum)

2000 ◽  
Vol 347 (1) ◽  
pp. 205-209 ◽  
Author(s):  
A. Lesley JONES ◽  
Derek HERBERT ◽  
Andrew J. RUTTER ◽  
Jane E. DANCER ◽  
John L. HARWOOD
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Higher Plants ◽  
Acyl Carrier Protein ◽  
Type Ii ◽  
General Activity ◽  
The Individual ◽  
Derivatives Of

The type II fatty acid synthases (FASs) of higher plants (and Escherichia coli) contain three condensing enzymes called β-ketoacyl-ACP synthases (KAS), where ACP is acyl-carrier-protein. We have used novel derivatives of the antibiotic thiolactomycin to inhibit these enzymes. Overall de novo fatty acid biosynthesis was measured using [1-14C]acetate substrate and chloroplast preparations from pea leaves, and [1-14C]laurate was used to distinguish between the effects of the inhibitors on KAS I from those on KAS II. In addition, the activities of these enzymes, together with the short-chain condensing enzyme, KAS III, were measured directly. Six analogues were tested and two, both with extended hydrocarbon side chains, were found to be more effective inhibitors than thiolactomycin. Incubations with chloroplasts and direct assay of the individual condensing enzymes showed that all three compounds inhibited the pea FAS condensing enzymes in the order KAS II > KAS I > KAS III. These results demonstrate the general activity of thiolactomycin and its derivatives against these FAS condensation reactions, and suggest that such compounds will be useful for further detailed studies of inhibition and for use as pharmaceuticals against Type II FASs of pathogens.

Fatty-acid synthase activity and mRNA level in hypertrophic type II cells from silica-treated rats

1994 ◽  
Vol 267 (2) ◽  
pp. L128-L136
Author(s):  
J. Rami ◽  
W. Stenzel ◽  
S. M. Sasic ◽  
C. Puel-M'Rini ◽  
J. P. Besombes ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Mrna Level ◽  
Type Ii Cells ◽  
Mrna Levels ◽  
Type Ii ◽  
Maximum Increase

Silica instillation causes a massive increase in lung surfactant. Two populations of type II pneumocytes can be isolated from rats administered silica by intratracheal injection: type IIA cells similar to type II cells from normal rats and type IIB cells, which are larger and contain elevated levels of surfactant protein A and phospholipid. Activities of choline-phosphate cytidylyltransferase, a rate-regulatory enzyme in phosphatidylcholine biosynthesis, and fatty-acid synthase (FAS) are increased in type IIB cells isolated from rats 14 days after silica injection. In the present study, we examined the increase in FAS and cytidylyltransferase activities in type IIB cells as a function of time after silica administration. FAS activity increased rapidly, was approximately threefold elevated 1 day after silica administration and has reached close to the maximum increase by 3 days. Cytidylyltransferase activity was not increased on day 1, was significantly increased on day 3 but was not maximally increased until day 7. Inhibition of de novo fatty-acid biosynthesis, by in vivo injection of hydroxycitric acid and inclusion of agaric acid in the type II cell culture medium, abolished the increase in cytidylyltransferase activity on day 3 but not FAS and had no effect on activities of two other enzymes of phospholipid synthesis. FAS mRNA levels were not increased in type IIB cells isolated 1-14 days after silica injection. These data show that the increase in FAS activity in type IIB cells is an early response to silica, that it mediates the increase in cytidylyltransferase activity, and that it is not due to enhanced FAS gene expression.

Enhanced fatty acid biosynthesis and normal surfactant secretion in hypertrophic rat type II cells

1991 ◽  
Vol 260 (6) ◽  
pp. L577-L585 ◽  
Author(s):  
J. Rami ◽  
S. M. Sasic ◽  
S. A. Rooney
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Enzyme Activation ◽  
Type Ii Cells ◽  
Type Ii ◽  
Acid Biosynthesis ◽  
Surfactant Secretion ◽  
Phosphatidylcholine Secretion

Silica instillation causes lung surfactant accumulation as well as hyperplasia and hypertrophy of type II pneumocytes. Two populations of type II cells can be isolated from silica-treated rats: type IIA, which are similar to type II cells from normal animals and type IIB, which are larger and have a higher rate of phosphatidylcholine biosynthesis. We have compared fatty acid biosynthesis and phosphatidylcholine secretion in types IIA and IIB cells and in type II cells from control rats. The cells were isolated by elastase digestion and panning on immunoglobulin G-coated plates and fractionated into types IIA and IIB by centrifugal elutriation. Type IIB cells contained more phospholipid and had an enhanced rate of [3H]choline incorporation into phosphatidylcholine. The activity of choline-phosphate cytidylyltransferase was elevated in the type IIB cells and the extent of the increase was diminished when phosphatidylglycerol was included in the assay, suggesting that the enhanced activity was due to enzyme activation rather than protein synthesis. The basal rate of phosphatidylcholine secretion was the same in all three groups as was the response to a variety of secretagogues. Incorporation of [3H]acetate into fatty acids was elevated in type IIB cells and the activity of fatty acid synthase was eightfold greater than in control cells. These data show that de novo fatty acid biosynthesis is increased in hypertrophic type II cells and that surfactant secretion is not elevated.

scholarly journals Reaction mechanism of recombinant 3-oxoacyl-(acyl-carrier-protein) synthase III from Cuphea wrightii embryo, a fatty acid synthase type II condensing enzyme

1999 ◽  
Vol 345 (1) ◽  
pp. 153-160 ◽  
Author(s):  
Amine ABBADI ◽  
Monika BRUMMEL ◽  
Burkhardt S. SCHüTT ◽  
Mary B. SLABAUGH ◽  
Ricardo SCHUCH ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Binding Sites ◽  
Fatty Acid Synthase ◽  
Catalytic Mechanism ◽  
Higher Plants ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Type Ii ◽  
Acetyl Coa ◽  
Condensing Enzyme

A unique feature of fatty acid synthase (FAS) type II of higher plants and bacteria is 3-oxoacyl-[acyl-carrier-protein (ACP)] synthase III (KAS III), which catalyses the committing condensing reaction. Working with KAS IIIs from Cuphea seeds we obtained kinetic evidence that KAS III catalysis follows a Ping-Pong mechanism and that these enzymes have substrate-binding sites for acetyl-CoA and malonyl-ACP. It was the aim of the present study to identify these binding sites and to elucidate the catalytic mechanism of recombinant Cuphea wrightii KAS III, which we expressed in Escherichia coli. We engineered mutants, which allowed us to dissect the condensing reaction into three stages, i.e. formation of acyl-enzyme, decarboxylation of malonyl-ACP, and final Claisen condensation. Incubation of recombinant enzyme with [1-14C]acetyl-CoA-labelled Cys111, and the replacement of this residue by Ala and Ser resulted in loss of overall condensing activity. The Cys111Ser mutant, however, still was able to bind acetyl-CoA and to catalyse subsequent binding and decarboxylation of malonyl-ACP to acetyl-ACP. We replaced His261 with Ala and Arg and found that the former lost activity, whereas the latter retained overall condensing activity, which indicated a general-base action of His261. Double mutants Cys111Ser/His261Ala and Cys111Ser/His261Arg were not able to catalyse overall condensation, but the double mutant containing Arg induced decarboxylation of [2-14C]malonyl-ACP, a reaction indicating the role of His261 in general-acid catalysis. Finally, alanine scanning revealed the involvement of Arg150 and Arg306 in KAS III catalysis. The results offer for the first time a detailed mechanism for a condensing reaction catalysed by a FAS type II condensing enzyme.

scholarly journals Identification of active site residues implies a two-step catalytic mechanism for acyl-ACP thioesterase

2018 ◽  
Vol 475 (23) ◽  
pp. 3861-3873 ◽  
Author(s):  
Fuyuan Jing ◽  
Marna D. Yandeau-Nelson ◽  
Basil J. Nikolau
Keyword(s):  
Fatty Acid ◽  
Sequence Alignment ◽  
Fatty Acid Synthase ◽  
Catalytic Mechanism ◽  
Fatty Acid Biosynthesis ◽  
Tertiary Structure ◽  
Acyl Carrier Protein ◽  
Cocos Nucifera ◽  
Catalytic Residue ◽  
Thioester Bond

In plants and bacteria that use a Type II fatty acid synthase, isozymes of acyl-acyl carrier protein (ACP) thioesterase (TE) hydrolyze the thioester bond of acyl-ACPs, terminating the process of fatty acid biosynthesis. These TEs are therefore critical in determining the fatty acid profiles produced by these organisms. Past characterizations of a limited number of plant-sourced acyl-ACP TEs have suggested a thiol-based, papain-like catalytic mechanism, involving a triad of Cys, His, and Asn residues. In the present study, the sequence alignment of 1019 plant and bacterial acyl-ACP TEs revealed that the previously proposed Cys catalytic residue is not universally conserved and therefore may not be a catalytic residue. Systematic mutagenesis of this residue to either Ser or Ala in three plant acyl-ACP TEs, CvFatB1 and CvFatB2 from Cuphea viscosissima and CnFatB2 from Cocos nucifera, resulted in enzymatically active variants, demonstrating that this Cys residue (Cys348 in CvFatB2) is not catalytic. In contrast, the multiple sequence alignment, together with the structure modeling of CvFatB2, suggests that the highly conserved Asp309 and Glu347, in addition to previously proposed Asn311 and His313, may be involved in catalysis. The substantial loss of catalytic competence associated with site-directed mutants at these positions confirmed the involvement of these residues in catalysis. By comparing the structures of acyl-ACP TE and the Pseudomonas 4-hydroxybenzoyl-CoA TE, both of which fold in the same hotdog tertiary structure and catalyze the hydrolysis reaction of thioester bond, we have proposed a two-step catalytic mechanism for acyl-ACP TE that involves an enzyme-bound anhydride intermediate.

scholarly journals Identification of a Novel Mycobacterial 3-Hydroxyacyl-Thioester Dehydratase, HtdZ (Rv0130), by Functional Complementation in Yeast

2008 ◽  
Vol 190 (11) ◽  
pp. 4088-4090 ◽  
Author(s):  
Aner Gurvitz ◽  
J. Kalervo Hiltunen ◽  
Alexander J. Kastaniotis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mycobacterium Tuberculosis ◽  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Acid Synthesis ◽  
Functional Complementation ◽  
Type Ii ◽  
Mutant Cells ◽  
Dehydratase Activity

ABSTRACT We report on the identification of Mycobacterium tuberculosis HtdZ (Rv0130), representing a novel 3-hydroxyacyl-thioester dehydratase. HtdZ was picked up by the functional complementation of Saccharomyces cerevisiae htd2Δ cells lacking the dehydratase of mitochondrial type II fatty acid synthase. Mutant cells expressing HtdZ contained dehydratase activity, recovered their respiratory ability, and partially restored de novo lipoic acid synthesis.

Product diversity and regulation of type II fatty acid synthases

2004 ◽  
Vol 82 (1) ◽  
pp. 145-155 ◽  
Author(s):  
Ying-Jie Lu ◽  
Yong-Mei Zhang ◽  
Charles O Rock
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Unsaturated Fatty Acids ◽  
Fatty Acid Biosynthesis ◽  
Product Distribution ◽  
Type Ii ◽  
Human Pathogens ◽  
Gram Positive ◽  
Basic Set ◽  
Fas Ii

Fatty acid biosynthesis is catalyzed in most bacteria by a group of highly conserved proteins known as the type II fatty acid synthase (FAS II) system. FAS II has been extensively studied in the Escherichia coli model system, and the recent explosion of bioinformatic information has accelerated the investigation of the pathway in other organisms, mostly important human pathogens. All FAS II systems possess a basic set of enzymes for the initiation and elongation of acyl chains. This review focuses on the variations on this basic theme that give rise to the diversity of products produced by the pathway. These include multiple mechanisms to generate unsaturated fatty acids and the accessory components required for branched-chain fatty acid synthesis in Gram-positive bacteria. Most of the known mechanisms that regulate product distribution of the pathway arise from the fundamental biochemical properties of the expressed enzymes. However, newly identified transcriptional factors in bacterial fatty acid biosynthetic pathways are a fertile field for new investigation into the genetic control of the FAS II system. Much more work is needed to define the role of these factors and the mechanisms that regulate their DNA binding capability, but there appear to be fundamental differences in how the expression of the pathway genes is controlled in Gram-negative and in Gram-positive bacteria.Key words: fatty acid synthase, bacteria.

scholarly journals β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) Is a Determining Factor in Branched-Chain Fatty Acid Biosynthesis

2000 ◽  
Vol 182 (2) ◽  
pp. 365-370 ◽  
Author(s):  
Keum-Hwa Choi ◽  
Richard J. Heath ◽  
Charles O. Rock
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Chain Fatty Acid ◽  
Carrier Protein ◽  
Acid Biosynthesis ◽  
Biochemical Basis ◽  
E Coli ◽  
Branched Chain

ABSTRACT A universal set of genes encodes the components of the dissociated, type II, fatty acid synthase system that is responsible for producing the multitude of fatty acid structures found in bacterial membranes. We examined the biochemical basis for the production of branched-chain fatty acids by gram-positive bacteria. Two genes that were predicted to encode homologs of the β-ketoacyl-acyl carrier protein synthase III of Escherichia coli (eFabH) were identified in theBacillus subtilis genome. Their protein products were expressed, purified, and biochemically characterized. Both B. subtilis FabH homologs, bFabH1 and bFabH2, carried out the initial condensation reaction of fatty acid biosynthesis with acetyl-coenzyme A (acetyl-CoA) as a primer, although they possessed lower specific activities than eFabH. bFabH1 and bFabH2 also utilized iso- and anteiso-branched-chain acyl-CoA primers as substrates. eFabH was not able to accept these CoA thioesters. Reconstitution of a complete round of fatty acid synthesis in vitro with purified E. coli proteins showed that eFabH was the only E. colienzyme incapable of using branched-chain substrates. Expression of either bFabH1 or bFabH2 in E. coli resulted in the appearance of a branched-chain 17-carbon fatty acid. Thus, the substrate specificity of FabH is an important determinant of branched-chain fatty acid production.

scholarly journals Elucidation of transient protein-protein interactions within carrier protein-dependent biosynthesis

2020 ◽  
Author(s):  
Michael Burkart ◽  
Thomas Bartholow ◽  
Terra Sztain ◽  
Ashay Patel ◽  
D Lee ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Protein Interactions ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Acyl Carrier Protein ◽  
Protein Docking ◽  
Carrier Protein ◽  
Protein Protein Interactions ◽  
Acid Biosynthesis ◽  
E Coli

Abstract Fatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB in Escherichia coli. This work reveals the molecular basis of six discrete binding events responsible for E. coli FAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways. ONE SENTENCE SUMMARY: Through a combination of structural and computational analysis, a comparative evaluation of protein-protein interactions in de novo fatty acid biosynthesis in E. coli is performed.

scholarly journals 1,2-Dithiole-3-Ones as Potent Inhibitors of the Bacterial 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH)

2004 ◽  
Vol 48 (8) ◽  
pp. 3093-3102 ◽  
Author(s):  
Xin He ◽  
Anne McElwee Reeve ◽  
Umesh R. Desai ◽  
Glen E. Kellogg ◽  
Kevin A. Reynolds
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Enzyme Inhibitor ◽  
Acyl Carrier Protein ◽  
Phenyl Group ◽  
Initial Step ◽  
Structural Features ◽  
Inhibitor Complex

ABSTRACT The enzyme FabH catalyzes the initial step of fatty acid biosynthesis via a type II dissociated fatty acid synthase. The pivotal role of this essential enzyme, combined with its unique structural features and ubiquitous occurrence in bacteria, has made it an attractive new target for the development of antibacterial and antiparasitic compounds. We have searched the National Cancer Institute database for compounds bearing structural similarities to thiolactomycin, a natural product which exhibits a weak activity against FabH. This search has yielded several substituted 1,2-dithiole-3-ones that are potent inhibitors of FabH from both Escherichia coli (ecFabH) and Staphylococcus aureus (saFabH). The most potent inhibitor was 4,5-dichloro-1,2-dithiole-3-one, which had 50% inhibitory concentration (IC50) values of 2 μM (ecFabH) and 0.16 μM (saFabH). The corresponding 3-thione analog exhibited comparable activities. Analogs in which the 4-chloro substituent was replaced with a phenyl group were also potent inhibitors, albeit somewhat less effectively (IC50 values of 5.7 and 0.98 μM for ecFabH and saFabH, respectively). All of the 5-chlorinated inhibitors were most effective when they were preincubated with FabH in the absence of substrates. The resulting enzyme-inhibitor complex did not readily regain activity after excess inhibitor was removed, suggesting that a slow dissociation occurs. In stark contrast, a series of inhibitors in which the 5-chloro substituent was replaced with the isosteric and isoelectronic trifluoromethyl group were poorer inhibitors (IC50 values typically ranging from 25 to >100 μM for both ecFabH and saFabH), did not require a preincubation period for maximal activity, and generated an enzyme-inhibitor complex which readily dissociated. Possible modes of binding of 5-chloro-1,2-dithiole-3-ones and 5-chloro-1,2-dithiole-3-thiones with FabH which account for the role of the 5-chloro substituent were considered.

Mechanistic diversity and regulation of Type II fatty acid synthesis

2002 ◽  
Vol 30 (6) ◽  
pp. 1050-1055 ◽  
Author(s):  
H. Marrakchi ◽  
Y.-M. Zhang ◽  
C. O. Rock
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Unsaturated Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Specific Interaction ◽  
Acid Synthesis ◽  
Protein Crystal ◽  
Type Ii ◽  
Acid Biosynthesis

Fatty acid biosynthesis is catalysed in most bacteria by a group of highly conserved proteins known as the Type II fatty acid synthase (FAS) system. The Type II system organization is distinct from its mammalian counterpart and offers several unique sites for selective inhibition by antibacterial agents. There has been remarkable progress in the understanding of the genetics, biochemistry and regulation of Type II FASs. One important advance is the discovery of the interaction between the fatty acid degradation regulator, FadR, and the fatty acid biosynthesis regulator, FabR, in the transcriptional control of unsaturated fatty acid synthesis in Escherichia coli. The availability of genomic sequences and high-resolution protein crystal structures has expanded our understanding of Type II FASs beyond the E. coli model system to a number of pathogens. The molecular diversity among the pathway enzymes is illustrated by the discovery of a new type of enoyl-reductase in Streptococcus pneumoniae [enoyl-acyl carrier protein (ACP) reductase II, FabK], the presence of two enoyl-reductases in Bacillus subtilis (enoyl-ACP reductases I and III, FabI and FabL), and the use of a new mechanism for unsaturated fatty acid formation in S. pneumoniae (trans-2-cis-3-enoyl-ACP isomerase, FabM). The solution structure of ACP from Mycobacterium tuberculosis revealed features common to all ACPs, but its extended C-terminal domain may reflect a specific interaction with very-long-chain intermediates.

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