Fig. 7.1 The basic Kennedy pathway for glycerolipid biosynthesis in animals and plants.
acyl specificities. Thus, in animals and for the extra-chloroplastic (endoplasmic reticulum) acyl-transferases in plants, saturated fatty acids (e.g. palmitate) are preferred for esterification at position 1, whereas unsaturated fatty acids (e.g. oleate) are attached to the 2-hydroxyl. In chloroplasts, by contrast, the acyltransferases that utilize acyl-ACP typically place a saturated or unsaturated acid at position 1, while palmitate is preferred at position 2. Therefore, in plants, the subcellular origin of the backbone of glycerolipids can be deduced from the fatty acid distribution (Section 184.108.40.206).
Glycerol 3-phosphate acyltransferase (GPAT) catalyses the first, committed, step in glycerolipid assembly. In mammals, two isoforms (present in mitochondria and the endoplasmic reticulum) have been reported and can be distinguished by their relative sensitivity to N-ethylmaleimide. In most tissues the mitochondrial activity is relatively minor (about 10% total) but, in liver, it is as active as the endoplasmic reticulum isoform. Furthermore, the liver mitochondrial enzyme is under both nutritional and hormonal control. This property allowed the first isolation of a cDNA for a mammalian enzyme involved in glycerophospholipid synthesis when the cDNA for mouse GPAT was isolated by Sul and coworkers using differential screening. The cDNA was shown to code for the mitochondrial isoform by transfecting cell cultures and measuring an increase in acyltransferase activity in mitochondria, but not in the endoplasmic reticulum. When the deduced amino acid sequence of the mouse GPAT was compared to other acyl-transferases a conserved arginine residue was identified that might be useful for binding the negatively charged substrates glycerol 3-phosphate or acyl-CoA. The identification of a conserved arginine agreed with the finding that arginine-modifying agents such as phenylglyoxal inhibited the enzyme.
Lysophosphatidate acyltransferase (LPAT) in most eukaryotic organisms has a high specificity for unsaturated fatty acids. Indeed, in plant seeds the high specificity of this enzyme has proved to be an obstacle for the engineering of 'designer' oils in transgenic plants where the accumulation of unusual fatty acids at position 2 is severely restricted. However, as noted above in plants, a second isoform of the enzyme is present in the chloroplasts that uses acyl-ACP substrates and prefers palmi-toyl-ACP rather than oleoyl-ACP as substrate.
LPAT cDNAs have been cloned from yeast, plants and mammals. In humans, isoforms are present with different tissue distributions. Intrigu-ingly, the human a-isoform is present at very high levels in testes where it has been suggested that it acts to generate phosphatidic acid for signalling purposes (Section 7.11). The acyltransferase is increased substantially (59-fold) when pre-adipo-cytes differentiate into adipocytes, perhaps by phosphorylation under hormonal influences.
Phosphatidate phosphohydrolase catalyses the key dephosphorylation reaction, which yields diacylglycerol (Fig. 7.1) and thus directs carbon away from acidic (anionic) phosphoglyceride synthesis. Two forms of the enzyme have been identified in animals. One is present in the cytosol/ endoplasmic reticulum and its activity is altered by translocation. The cytosolic form is inactive and it is translocated to the endoplasmic reticulum under the influence of fatty acids, fatty acyl-CoAs and phosphatidic acid itself. This makes good sense because a build-up in its substrate (phosphatidate) will activate the enzyme. Moreover, when the enzyme is used for triacylglycerol production (Sections 220.127.116.11 and 3.6.2), the supply of acyl-CoAs (or fatty acids) will stimulate the Kennedy pathway itself.
A second isoform of animal phosphatidate phosphohydrolase is present in the plasma membrane where it is thought to have a role in signal transduction (Section 7.11). Multiple isoforms have also been detected in yeast and in plants, where they again have different subcellular locations.
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