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.

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 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 Multiple Triclosan Targets in Trypanosoma brucei

2004 ◽  
Vol 3 (4) ◽  
pp. 855-861 ◽  
Author(s):  
Kimberly S. Paul ◽  
Cyrus J. Bacchi ◽  
Paul T. Englund
Keyword(s):  
Fatty Acid ◽  
Trypanosoma Brucei ◽  
Fatty Acid Synthesis ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Bloodstream Form ◽  
Type Ii ◽  
Subcellular Compartment ◽  
Genes Encoding ◽  
Enoyl Acp Reductase

ABSTRACT Trypanosoma brucei genes encoding putative fatty acid synthesis enzymes are homologous to those encoding type II enzymes found in bacteria and organelles such as chloroplasts and mitochondria. It was therefore not surprising that triclosan, an inhibitor of type II enoyl-acyl carrier protein (enoyl-ACP) reductase, killed both procyclic forms and bloodstream forms of T. brucei in culture with 50% effective concentrations (EC50s) of 10 and 13 μM, respectively. Triclosan also inhibited cell-free fatty acid synthesis, though much higher concentrations were required (EC50s of 100 to 200 μM). Unexpectedly, 100 μM triclosan did not affect the elongation of [3H]laurate (C12:0) to myristate (C14:0) in cultured bloodstream form parasites, suggesting that triclosan killing of trypanosomes may not be through specific inhibition of enoyl-ACP reductase but through some other mechanism. Interestingly, 100 μM triclosan did reduce the level of incorporation of [3H]myristate into glycosyl phosphatidylinositol species (GPIs). Furthermore, we found that triclosan inhibited fatty acid remodeling in a cell-free assay in the same concentration range required for killing T. brucei in culture. In addition, we found that a similar concentration of triclosan also inhibited the myristate exchange pathway, which resides in a distinct subcellular compartment. However, GPI myristoylation and myristate exchange are specific to the bloodstream form parasite, yet triclosan kills both the bloodstream and procyclic forms. Therefore, triclosan killing may be due to a nonspecific perturbation of subcellular membrane structure leading to dysfunction in sensitive membrane-resident biochemical pathways.

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.

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.

scholarly journals Solution Structure of the Tandem Acyl Carrier Protein Domains from a Polyunsaturated Fatty Acid Synthase Reveals Beads-on-a-String Configuration

PLoS ONE ◽  
2013 ◽  
Vol 8 (2) ◽  
pp. e57859 ◽  
Author(s):  
Uldaeliz Trujillo ◽  
Edwin Vázquez-Rosa ◽  
Delise Oyola-Robles ◽  
Loren J. Stagg ◽  
David A. Vassallo ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Polyunsaturated Fatty Acid ◽  
Solution Structure ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Protein Domains ◽  
Carrier Protein

2-Oxoacyl-[acyl carrier protein] reductase: a component of plant fatty acid synthase

1988 ◽  
Vol 16 (3) ◽  
pp. 392-393 ◽  
Author(s):  
PHILIP S. SHELDON ◽  
RICHARD SAFFORD ◽  
ANTONI R. SLABAS ◽  
ROY G. O. KEKWICK
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Carrier Protein
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