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

2000 ◽  
Vol 345 (1) ◽  
pp. 153 ◽  
Author(s):  
Amine ABBADI ◽  
Monika BRUMMEL ◽  
Burkhardt S. SCHÜTT ◽  
Mary B. SLABAUGH ◽  
Ricardo SCHUCH ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Reaction Mechanism ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Type Ii ◽  
Condensing Enzyme

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.

scholarly journals Depletion of Mitochondrial Acyl Carrier Protein in Bloodstream-Form Trypanosoma brucei Causes a Kinetoplast Segregation Defect

2011 ◽  
Vol 10 (3) ◽  
pp. 286-292 ◽  
Author(s):  
April M. Clayton ◽  
Jennifer L. Guler ◽  
Megan L. Povelones ◽  
Eva Gluenz ◽  
Keith Gull ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Trypanosoma Brucei ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Phospholipid Composition ◽  
Conditional Knockout ◽  
Bloodstream Form ◽  
Carrier Protein ◽  
Type Ii ◽  
Kinetoplast Dna

ABSTRACT Like other eukaryotes, trypanosomes have an essential type II fatty acid synthase in their mitochondrion. We have investigated the function of this synthase in bloodstream-form parasites by studying the effect of a conditional knockout of acyl carrier protein (ACP), a key player in this fatty acid synthase pathway. We found that ACP depletion not only caused small changes in cellular phospholipids but also, surprisingly, caused changes in the kinetoplast. This structure, which contains the mitochondrial genome in the form of a giant network of several thousand interlocked DNA rings (kinetoplast DNA [kDNA]), became larger in some cells and smaller or absent in others. We observed the same pattern in isolated networks viewed by either fluorescence or electron microscopy. We found that the changes in kDNA size were not due to the disruption of replication but, instead, to a defect in segregation. kDNA segregation is mediated by the tripartite attachment complex (TAC), and we hypothesize that one of the TAC components, a differentiated region of the mitochondrial double membrane, has an altered phospholipid composition when ACP is depleted. We further speculate that this compositional change affects TAC function, and thus kDNA segregation.

β-Ketoacyl-acyl carrier protein synthases in the regulation of fatty acid synthase activity in higher plants — An overview

Lipid / Fett ◽  
1997 ◽  
Vol 99 (8) ◽  
pp. 278-281 ◽  
Author(s):  
Ricardo Schuch ◽  
Elke Winter ◽  
Fritzi Maike Brück ◽  
Monika Brummel ◽  
Friedrich Spener
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Higher Plants ◽  
Acyl Carrier Protein ◽  
Carrier Protein

scholarly journals Structural comparison of Acinetobacter baumannii β-ketoacyl-acyl carrier protein reductases in fatty acid and aryl polyene biosynthesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Woo Cheol Lee ◽  
Sungjae Choi ◽  
Ahjin Jang ◽  
Kkabi Son ◽  
Yangmee Kim
Keyword(s):  
Fatty Acid ◽  
Acinetobacter Baumannii ◽  
Gene Cluster ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Gene Clusters ◽  
Carrier Protein ◽  
Aryl Group ◽  
Visible Absorption

AbstractSome Gram-negative bacteria harbor lipids with aryl polyene (APE) moieties. Biosynthesis gene clusters (BGCs) for APE biosynthesis exhibit striking similarities with fatty acid synthase (FAS) genes. Despite their broad distribution among pathogenic and symbiotic bacteria, the detailed roles of the metabolic products of APE gene clusters are unclear. Here, we determined the crystal structures of the β-ketoacyl-acyl carrier protein (ACP) reductase ApeQ produced by an APE gene cluster from clinically isolated virulent Acinetobacter baumannii in two states (bound and unbound to NADPH). An in vitro visible absorption spectrum assay of the APE polyene moiety revealed that the β-ketoacyl-ACP reductase FabG from the A. baumannii FAS gene cluster cannot be substituted for ApeQ in APE biosynthesis. Comparison with the FabG structure exhibited distinct surface electrostatic potential profiles for ApeQ, suggesting a positively charged arginine patch as the cognate ACP-binding site. Binding modeling for the aryl group predicted that Leu185 (Phe183 in FabG) in ApeQ is responsible for 4-benzoyl moiety recognition. Isothermal titration and arginine patch mutagenesis experiments corroborated these results. These structure–function insights of a unique reductase in the APE BGC in comparison with FAS provide new directions for elucidating host–pathogen interaction mechanisms and novel antibiotics discovery.

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.

Protein interactions of fatty acid synthase II

2000 ◽  
Vol 28 (6) ◽  
pp. 615-616 ◽  
Author(s):  
G. Honeyman ◽  
T. Fawcett
Keyword(s):  
Fatty Acid ◽  
Protein Interactions ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Hybrid Approach ◽  
Carrier Protein ◽  
Yeast Two Hybrid ◽  
Two Hybrid

We have used a yeast two-hybrid approach to detect direct protein interactions between fatty acid synthase components. Enoyl-acyl carrier protein (ACP) reductase was found to interact with stearoyl-ACP desaturase and acyl-ACP thioesterase, but none of these proteins interacted with ACP in the yeast nucleus.

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