scholarly journals Dual-localized enzymatic components that constitute the mitochondrial and plastidial fatty acid synthase systems

2020 ◽  
Author(s):  
Xin Guan ◽  
Yozo Okazaki ◽  
Rwisdom Zhang ◽  
Kazuki Saito ◽  
Basil J Nikolau
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Acyl Chain ◽  
Type Ii ◽  
Dual Localization ◽  
Genes Encoding ◽  
Enoyl Acp Reductase ◽  
Identification And Characterization

ABSTRACTWe report the identification and characterization of genes encoding three enzymes that are shared between the mitochondrial and plastidial-localized Type II fatty acid synthase systems (mtFAS and ptFAS, respectively). Two of these enzymes, β-ketoacyl-ACP reductase (pt/mtKR) and enoyl-ACP reductase (pt/mtER) catalyze two of the reactions that constitute the core, 4-reaction cycle of the FAS system, which iteratively elongate the acyl-chain by 2-carbon atoms per cycle. The third enzyme, malonyl-CoA:ACP transacylase (pt/mtMCAT) catalyzes the reaction that loads the mtFAS system with substrate, by malonylating the phosphopantetheinyl cofactor of acyl carrier protein (ACP). GFP-transgenic experiments determined the dual localization of these enzymes, which were validated by the characterization of mutant alleles, which were transgenically rescued by transgenes that were singularly retargeted to either plastids or mitochondria. The singular retargeting of these proteins to plastids rescued the embryo-lethality associated with disruption of the essential ptFAS system, but these rescued plants display phenotypes typical of the lack of mtFAS function. Specifically, these phenotypes include reduced lipoylation of the H subunit of the glycine decarboxylase complex, the hyperaccumulation of glycine, and reduced growth; all these traits are reversible by growing these plants in an elevated CO2 atmosphere, which suppresses mtFAS-associated, photorespiration-dependent chemotypes.

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 Purification and Biochemical Characterization of theMycobacterium tuberculosisβ-Ketoacyl-acyl Carrier Protein Synthases KasA and KasB

2001 ◽  
Vol 276 (50) ◽  
pp. 47029-47037 ◽  
Author(s):  
Merrill L. Schaeffer ◽  
Gautam Agnihotri ◽  
Craig Volker ◽  
Howard Kallender ◽  
Patrick J. Brennan ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Mycolic Acid ◽  
Carrier Protein ◽  
Mycolic Acids ◽  
E Coli ◽  
Long Chain

Mycolic acids are vital components of theMycobacterium tuberculosiscell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents. Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performsde novofatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors. In this study, we present the initial characterization of purified KasA and KasB, two β-ketoacyl-ACP synthase (KAS) enzymes of theM. tuberculosisFASII system. KasA and KasB were expressed inE. coliand purified by affinity chromatography. Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP. Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons. Activity of KasA and KasB increased with use ofM. tuberculosisAcpM, suggesting that structural differences between AcpM andE. coliACP may affect their recognition by the enzymes. Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin. These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery.

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 Candida tropicalis Etr1p andSaccharomyces cerevisiae Ybr026p (Mrf1′p), 2-Enoyl Thioester Reductases Essential for Mitochondrial Respiratory Competence

2001 ◽  
Vol 21 (18) ◽  
pp. 6243-6253 ◽  
Author(s):  
Juha M. Torkko ◽  
Kari T. Koivuranta ◽  
Ilkka J. Miinalainen ◽  
Ahmed I. Yagi ◽  
Werner Schmitz ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Candida Tropicalis ◽  
Acyl Carrier Protein ◽  
Reductase Activity ◽  
Acid Synthesis ◽  
Mutant Phenotype ◽  
Mitochondrial Targeting ◽  
Identification And Characterization

ABSTRACT We report here on the identification and characterization of novel 2-enoyl thioester reductases of fatty acid metabolism, Etr1p fromCandida tropicalis and its homolog Ybr026p (Mrf1′p) fromSaccharomyces cerevisiae. Overexpression of these proteins in S. cerevisiae led to the development of significantly enlarged mitochondria, whereas deletion of the S. cerevisiae YBR026c gene resulted in rudimentary mitochondria with decreased contents of cytochromes and a respiration-deficient phenotype. Immunolocalization and in vivo targeting experiments showed these proteins to be predominantly mitochondrial. Mitochondrial targeting was essential for complementation of the mutant phenotype, since targeting of the reductases to other subcellular locations failed to reestablish respiratory growth. The mutant phenotype was also complemented by a mitochondrially targeted FabI protein from Escherichia coli. FabI represents a nonhomologous 2-enoyl-acyl carrier protein reductase that participates in the last step of the type II fatty acid synthesis. This indicated that 2-enoyl thioester reductase activity was critical for the mitochondrial function. We conclude that Etr1p and Ybr026p are novel 2-enoyl thioester reductases required for respiration and the maintenance of the mitochondrial compartment, putatively acting in mitochondrial synthesis of fatty acids.

scholarly journals Biophysical Characterization of Acyl Carrier Protein Domains from a Polyunsaturated Fatty Acid Synthase

2012 ◽  
Vol 26 (S1) ◽  
Author(s):  
Uldaeliz Trujillo Rodriguez ◽  
Delise Oyola-Robles ◽  
Stefan Arold ◽  
Fernando Alves De Melo ◽  
John E. Ladbury ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Polyunsaturated Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Protein Domains ◽  
Carrier Protein ◽  
Biophysical Characterization

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
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