Cryptosporidium parvum Long-Chain Fatty Acid Elongase

Abstract

We report the presence of a new fatty acyl coenzyme A (acyl-CoA) elongation system in Cryptosporidium and the functional characterization of the key enzyme, a single long-chain fatty acid elongase (LCE), in this parasite. This enzyme contains conserved motifs and predicted transmembrane domains characteristic to the elongase family and is placed within the ELO6 family specific for saturated substrates. CpLCE1 gene transcripts are present at all life cycle stages, but the levels are highest in free sporozoites and in stages at 36 h and 60 h postinfection that typically contain free merozoites. Immunostaining revealed localization to the outer surface of sporozoites and to the parasitophorous vacuolar membrane. Recombinant CpLCE1 displayed allosteric kinetics towards malonyl-CoA and palmitoyl-CoA and Michaelis-Menten kinetics towards NADPH. Myristoyl-CoA (C 14:0 ) and palmitoyl-CoA (C 16:0 ) display the highest activity when used as substrates, and only one round of elongation occurs. CpLCE1 is fairly resistant to cerulenin, an inhibitor for both type I and II fatty acid synthases (i.e., maximum inhibitions of 20.5% and 32.7% were observed when C 16:0 and C 14:0 were used as substrates, respectively). These observations ultimately validate the function of CpLCE1.As one of the vital compounds for all organisms, fatty acids of 14 to 18 carbons in length comprise the bulk of cellular fatty acids that serve structural and biological functions, and they are usually the major products of de novo synthesis in most cells. These long-chain fatty acids play important roles in many biological functions such as energy metabolism and membrane structure. There are several metabolic pathways that produce long-chain fatty acids, most notably the type I and type II fatty acid synthases (FASs). The type I enzymes of mammals and fungi are typically cytosolic and composed of multiple enzymes arranged into domains of one or two large polypeptides (31). In contrast, the enzymes of the type II FASs are all located on separate domains and are found in prokaryotes or in eukaryotic organelles of prokaryotic origin (33).Relatively common among eukaryotic organisms are the fatty acid elongase-based systems. These elongase-based systems directly elongate a fatty acyl chain esterfied with CoA (fatty acyl-CoA), which is in contrast to the type I and type II FAS systems, which elongate a fatty acyl chain attached to an acyl carrier protein. The elongase system is comprised of at least four enzymes that are responsible for adding two carbon units to the fatty acyl carboxyl end. The chemistry of this pathway is similar to that used by type I and type II FASs. Fatty acyl elongation begins with the condensation of malonyl-CoA with a fatty acyl-CoA catalyzed by the condensing enzyme LCE (␤-ketoacyl-CoA synthase) (Fig.

Kinetic Flux Profiling Elucidates Two Independent Acetyl-CoA Biosynthetic Pathways in Plasmodium falciparum

“…[5][6][7][8][9][10][11][12][13] C]Glutamine was added to glutamine-free RPMI at a final concentration of 2 mM for [5][6][7][8][9][10][11][12][13] C]glutamine labeling experiments. [6][7][8][9][10][11][12][13] C, 15 N]leucine labeling experiments were performed in complete custom-made RPMI without leucine and supplemented with [6][7][8][9][10][11][12][13] C, 15 N]leucine at the RPMI concentration.…”

“…Steady-state incubations were extracted using the methods described previously (25) with slight modifications. Briefly, infected erythrocytes were transferred to microcentrifuge tubes and spun at 14,000 ϫ g for 30 s. The supernatant was rapidly aspirated, and the cell pellet was extracted with 1 ml of 90% ice-cold methanol (containing the internal standard [4-13 C, 15 N]aspartate). Samples were dried under nitrogen flow and stored at Ϫ80°C until analysis.…”

“…Most of this glucose is metabolized to pyruvate that is subsequently reduced to lactate and excreted (7)(8)(9)(10)(11). However, P. falciparum possess several metabolic pathways that are typically supplied by pyruvate-derived acetyl-CoA including the mitochondrial tricarboxylic acid cycle (TCA), cytosolic fatty acid elongation, and nuclear histone acetylation (12)(13)(14)(15)(16). In most organisms acetyl-CoA is predominantly synthesized through the action of the pyruvate dehydrogenase (PDH) 3 complex.…”

Retinal very long-chain PUFAs: new insights from studies on ELOVL4 protein

“…2 , step 4) generates the elongated fatty acyl (n+2) -CoA ( 114 ). Whether the elongated product is released for use elsewhere in the cell or is retained to undergo another round of elongation depends largely on the specifi c needs of the organism ( 117,(119)(120)(121). Many of the elongases found in cells have overlapping functions ( 124 ), and more than one may act on the same substrate.…”

“…They catalyze the condensation of malonyl-CoA with an acyl (n) -CoA to form the intermediate product called ␤ -ketoacylCoA ( Fig. 2 , step 1) ( 119 ). Reduction of the ␤ -ketoacyl-CoA to ␤ -hydroxyacyl-CoA is mediated by ␤ -ketoacyl reductase in the presence of NAD(P)H ( Fig.…”