Desaturation of polyunsaturated fatty acids in Mucor circinelloides and the involvement of a novel membrane‐bound malic enzyme

Abstract

1. The component fatty acids of the endogenous phospholipids of microsomal preparations of Mucor, when shaken at 30°C increased in both chain length and in degree of unsaturation. The net effect was the production of y-linolenic acid which, over 2 h, increased from 17% to 32% of total fatty acids present. No further significant changes occurred after t h s time.2. The major site for desaturation/elongation reactions was at the sn-2 position of PtdIns. PtdCho and PtdEtn were not implicated.3. Of numerous metabolites and cofactors added to the microsomes, only malate could prolong the elongation/desaturation reactions for up to 6 h. This effect was shown to be due to a membraneassociated malic enzyme [malate dehydrogenase (decarboxylating) NADP'] with the NADPH produced being used in fatty-acid desaturation.4. Kinetic analysis of cytosolic and microsomal enzymes [both in 0.1 % (mass/vol.) Chaps] could not distinguish between them. However, when the microsomal malic enzyme was dialysed to remove Chaps, it lost 90% of activity, although the cytosolic malic enzyme lost only 20% activity.5. The structural analogue of malate, tartronic acid, which is an inhibitor of malic enzyme, also inhibited the malate-induced stimulation of fatty-acyl group desaturation and elongation in the microsomal membranes.6. It is concluded that two distinct malic enzymes exist, one soluble and one membrane bound, with similar active sites. Both have different roles in the production of NADPH, for lipid metabolism. The former will produce NADPH for fatty-acid biosynthesis whilst the latter produces NADPH for fatty-acid desaturation.The desaturation of fatty acids beyond oleate [18: 1 (9)] is considered to occur in plants and microorganisms with fattyacyl groups attached to phospholipids and thus is a membrane(microsoma1)-associated activity in eukaryotic cells [l-61. The desaturation of oleate to linolenate [I 8 : 2 (9, 12)], and thence to either a-o r y-linoleate [18:3 (9, 12, 15) or 18:3 (6, 9, 12) respectively] and other similar desaturations. occurs by separate desaturases (in the above examples they would be referred to, respectively, as the 412, A15 or A6 desaturases, respectively). These desaturases require both molecular oxygen and NADPH: -CH2-CH2-

Targeted Enhancement of Glutamate-to-γ-Aminobutyrate Conversion in Arabidopsis Seeds Affects Carbon-Nitrogen Balance and Storage Reserves in a Development-Dependent Manner

“…It has previously been suggested that malate is the preferred substrate for fatty acid elongation not requiring ATP or a reductant (Smith et al, 1992;Pleite et al, 2005), which are limited in desiccating seeds. Confirming the findings of Smith et al (1992), Kendrick and Ratledge (1992) showed that a structural analog of malate, tartronic acid, which is an inhibitor of malic enzyme, could inhibit the malate-induced stimulation of fatty acyl group desaturation and elongation in the microsomal membranes of Mucor circinelloides. In our study, an impaired TCA cycle was also reflected in the alteration of genes associated with mitochondrial electron transport and particularly in the up-regulation of alternative oxidase genes.…”

Malic enzyme: the controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation

“…Simultaneous with the substantial increase in lipid accumulation was a slight but definite overall increase in the GLA content of the lipid, indicating that the increased activity of ME was probably also generating NAPDH for fatty acid desaturases (see Kendrick & Ratledge, 1992). Thus, increased ME activity led not only to an increased lipid content of the cells but also to an increased fatty acid desaturase activity.…”

“…alpina and Mc. circinelloides were maintained on solidified Kendrick medium (Kendrick & Ratledge, 1992) at 30 uC and stored at 4 uC. Mc.…”

“…This uniqueness would also seem to apply to the formation of unsaturated fatty acids (Kendrick & Ratledge, 1992), which also requires NADPH and, being a membrane-associated activity, clearly needs a mechanism by which the highly water-soluble cofactor can be supplied to the fatty acid desaturases contained within the membranes of the endoplasmic reticulum. ME evidently fulfils this second role in addition to its role in fatty acid biosynthesis, as we have shown here.…”

Malic Enzyme is a Major Source of NADPH for Lipid Accumulation by Aspergillus Nidulans

“…Some authors report that its main function is in the metabolism of pyruvate (McCullough & Roberts, 1974;Zink, 1972;Zink & Katz, 1973) whilst others have presented evidence that malic enzyme is also a major source of NADPH for de nouo lipid biosynthesis and desaturation (Evans & Ratledge, 1985 ;Kendrick & Ratledge, 1992a). In this communication the importance of malic enzyme with respect to lipid metabolism in Aspergillus nidulans has been investigated.…”