Two Modes of Regulation of the Fatty Acid Elongase ELOVL6 by the 3-Ketoacyl-CoA Reductase KAR in the Fatty Acid Elongation Cycle

Fatty acids (FAs) are diverse molecules, and such diversity is important for lipids to exert their functions under several environmental conditions. FA elongation occurs at the endoplasmic reticulum and produces a variety of FA species; the FA elongation cycle consists of four distinct enzyme reactions. For this cycle to be driven efficiently, there must exist coordinated regulation of protein components of the FA elongation machinery. However, such regulation is poorly understood. In the present study, we performed biochemical analyses using the FA elongase ELOVL6 and the 3-ketoacyl-CoA reductase KAR, which catalyze the first and second steps of the FA elongation cycle, respectively. In vitro FA elongation assays using membrane fractions demonstrated that ELOVL6 activity was enhanced similar to 10-fold in the presence of NADPH, although ELOVL6 itself did not require NADPH for its catalysis. On the other hand, KAR does use NADPH as a reductant in its enzyme reaction. Activity of purified ELOVL6 was enhanced by similar to 3-fold in the presence of KAR. This effect was KAR enzyme activity-independent, since it was observed in the absence of NADPH and in the KAR mutant. However, ELOVL6 enzyme activity was further enhanced in a KAR enzyme activity-dependent manner. Therefore, KAR regulates ELOVL6 via two modes. In the first mode, KAR may induce conformational changes in ELOVL6 to become structure that can undergo catalysis. In the second mode, conversion of 3-ketoacyl-CoA to 3-hydroxyacyl-CoA by KAR may facilitate release of the product from the presumed ELOVL6-KAR complex

Long-chain bases of sphingolipids are transported into cells via the acyl-CoA synthetases

Transport of dietary lipids into small-intestinal epithelial cells is pathologically and nutritionally important. However, lipid uptake remains an almost unexplored research area. Although we know that long-chain bases (LCBs), constituents of sphingolipids, can enter into cells efficiently, the molecular mechanism of LCB uptake is completely unclear. Here, we found that the yeast acyl-CoA synthetases (ACSs) Faa1 and Faa4 are redundantly involved in LCB uptake. In addition to fatty acid-activating activity, transporter activity toward long-chain fatty acids (LCFAs) has been suggested for ACSs. Both LCB and LCFA transports were largely impaired in faa1. faa4. cells. Furthermore, LCB and LCFA uptakes were mutually competitive. However, the energy dependency was different for their transports. Sodium azide/2-deoxy-D-glucose treatment inhibited import of LCFA but not that of LCB. Furthermore, the ATP-AMP motif mutation FAA1 S271A largely impaired the metabolic activity and LCFA uptake, while leaving LCB import unaffected. These results indicate that only LCFA transport requires ATP. Since ACSs do not metabolize LCBs as substrates, Faa1 and Faa4 are likely directly involved in LCB transport. Furthermore, we revealed that ACSs are also involved in LCB transport in mammalian cells. Thus, our findings provide strong support for the hypothesis that ACSs directly transport LCFAs

Mouse aldehyde dehydrogenase ALDH3B2 is localized to lipid droplets via two C-terminal tryptophan residues and lipid modification

Aldehyde dehydrogenases (ALDHs) catalyse the conversion of toxic aldehydes into non-toxic carboxylic acids. Of the 21 ALDHs in mice, it is the ALDH3 family members (ALDH3A1, ALDH3A2, ALDH3B1, ALDH3B2 and ALDH3B3) that are responsible for the removal of lipid-derived aldehydes. However, ALDH3B2 and ALDH3B3 have yet to be characterized. In the present study, we examined the enzyme activity, tissue distribution and subcellular localization of ALDH3B2 and ALDH3B3. Both were found to exhibit broad substrate preferences from medium to long-chain aldehydes, resembling ALDH3A2 and ALDH3B1. Although ALDH3B2 and ALDH3B3 share extremely high sequence similarity, their localizations differ, with ALDH3B2 found in lipid droplets and ALDH3B3 localized to the plasma membrane. Both were modified by prenylation at their C-termini; this modification greatly influenced their membrane localization and enzymatic activity towards hexadecanal. We found that their C-terminal regions, particularly the two tryptophan residues (Trp(462) and Trp(469)) of ALDH3B2 and the two arginine residues (Arg(462) and Arg(463)) of ALDH3B3, were important for the determination of their specific localization. Abnormal quantity and perhaps quality of lipid droplets are implicated in several metabolic diseases. We speculate that ALDH3B2 acts to remove lipid-derived aldehydes in lipid droplets generated via oxidative stress as a quality control mechanism

Identification of residues important for the catalysis, structure maintenance, and substrate specificity of yeast 3-hydroxyacyl-CoA dehydratase Phs1

Yeast Phs1 is a 3-hydroxyacyl-CoA dehydratase involved in very long-chain fatty acid elongation. In the present study, we biochemically characterized Phs1 mutants with Ala-substitution at each of seven highly conserved amino-acid residues. All mutants exhibited reduced Phs1 activity. The E60A, Q79A, and R141A mutants were sensitive to digitonin, indicative of their reduced structural integrity. The fatty acid elongation cycle was greatly inhibited in the R83A, R141A, and G152A mutant membranes. The enzyme kinetics study implicated the direct involvement of the Arg83 and Gly152 residues in the catalytic process. The E60A mutation was found to affect the substrate specificity. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved

Biochemical characterization of the very long-chain fatty acid elongase ELOVL7

Very long-chain fatty acids (VLCFAs) have a variety of physiological functions and are related to numerous disorders. The key step of VLCFA elongation is catalyzed by members of the elongase family, ELOVLs. Mammals have seven ELOVLs (ELOVL1-7), yet none of them has been purified and analyzed. In the presented study we purified ELOVL7 and measured its activity by reconstituting it into proteoliposomes. Purified ELOVL7 exhibited high activity toward acyl-CoAs with C18 carbon chain length. The calculated Km values toward C18: 3-CoA(n-3) and malonyl-CoA were both in the μM range. We also found that progression of the VLCFA cycle enhances ELOVL7 activity

Determination of kinetic parameters of ELOVL6.

<p>(A and B) Purified 3xFLAG-ELOVL6 proteins (250 ng) were reconstituted into proteoliposomes and incubated with the indicated concentrations of C16∶0-CoA and 27.3 µM [¹⁴C]malonyl-CoA (1.5 µCi/ml) (A) or 8 µM C16∶0-CoA and the indicated concentrations of [¹⁴C]malonyl-CoA (B) for 90 min at 37°C. After termination of the reactions, lipids were saponified, acidified, extracted, and separated by normal-phase TLC, followed by detection and quantification with a bioimaging analyzer BAS-2500. The obtained values were expressed in Michaelis–Menten plots and Lineweaver–Burk plots (insets).</p

KAR stimulates ELOVL6 enzyme activity in KAR enzyme activity-independent and -dependent manners.

<p>(A) The yeast TatY56 (<i>ayr1</i>Δ<i>::natNT2 YBR159W-FLAG-AID</i>) cells harboring the pWK40 (2xHA vector), pTN41(<i>2xHA-YBR159w</i>), pTN42 (<i>2xHA-KAR</i>), pTN44 (<i>2xHA-KAR Y202A/K206A</i>), pTN45 (<i>2xHA-KAR S189A</i>), or pTN46 (<i>2xHA-KAR N161A</i>) plasmid were treated with 0.5 mM 3-indolacetic acid for 2 h. Total membrane proteins (20 µg) prepared from them were incubated with 20 µM C16∶0-CoA, 73 µM malonyl-CoA, and 27 µM [¹⁴C]malonyl-CoA (0.075 µCi) for 30 min at 37°C in the presence of 1 mM NADPH. After termination of the reactions, lipids were saponified, acidified, extracted, converted to methyl ester forms, separated by reverse-phase TLC, and detected by a bioimaging analyzer BAS-2500. (B) Total membrane fractions prepared from HEK 293T cells transfected with the pCE-puro 3xFLAG-KAR or pCE-puro 3xFLAG-KAR S189A plasmid were solubilized with 2% Triton X-100, and subjected to affinity-purification using anti-FLAG M2 agarose affinity beads. Purified proteins (1 µg) were separated by SDS-PAGE and stained with Coomassie brilliant blue. (C) Purified 3xFLAG-KAR proteins (15 ng) were treated with Endo H or PNGase F and subjected to immunoblotting using an anti-FLAG M2 antibody. (D-F) Purified 3xFLAG-ELOVL6 (10 ng) and/or 3xFLAG-KAR proteins of either wild type or S189A mutant (20 ng) were reconstituted into proteoliposomes and subjected to immunoblotting using an anti-FLAG M2 antibody (D) or to an <i>in vitro</i> FA elongation assay (E and F). (E) Proteoliposomes were incubated with 8 µM C16∶0-CoA and 27.3 µM [¹⁴C]malonyl-CoA (1.5 µCi/ml) for 90 min at 37°C in the presence or absence of 1 mM NADPH. After termination of the reactions, lipids were saponified, acidified, extracted, and separated by normal-phase TLC, followed by detection using a bioimaging analyzer BAS-2500. keto, 3-keto-FA; *keto, a by-product of 3-ketoacyl-CoA; hydroxy, 3-hydroxy-FA; **, <i>trans</i>-2-enoyl-FA produced through non-enzymatic conversion or produced by contaminated 3-hydroxyacyl-CoA dehydratase. (F) Values presented are the sum of the reaction products in (E) and represent the mean ± S.D. from three independent experiments. Statistically significant differences are indicated (**p<0.01; <i>t</i>-test).</p

Substrate specificity, plasma membrane localization, and lipid modification of the aldehyde dehydrogenase ALDH3B1

The accumulation of reactive aldehydes is implicated in the development of several disorders. Aldehyde dehydrogenases (ALDHs) detoxify aldehydes by oxidizing them to the corresponding carboxylic acids. Among the 19 human ALDHs, ALDH3A2 is the only known ALDH that catalyzes the oxidation of long-chain fatty aldehydes including C16 aldehydes (hexadecanal and trans-2-hexadecenal) generated through sphingolipid metabolism. In the present study, we have identified that ALDH3B1 is also active in vitro toward C16 aldehydes and demonstrated that overexpression of ALDH3B1 restores the sphingolipid metabolism in the ALDH3A2-deficient cells. In addition, we have determined that ALDH3B1 is localized in the plasma membrane through its C-terminal dual lipidation (palmitoylation and prenylation) and shown that the prenylation is required particularly for the activity toward hexadecanal. Since knockdown of ALDH3B1 does not cause further impairment of the sphingolipid metabolism in the ALDH3A2-deficient cells, the likely physiological function of ALDH3B1 is to oxidize lipid-derived aldehydes generated in the plasma membrane and not to be involved in the sphingolipid metabolism in the endoplasmic reticulum. (C) 2013 Elsevier B.V. All rights reserved