Trans-enoyl-CoA reductases

The results in Table 6 were obtained when both the overall rates of chain elongation and the condensation reaction were measured with 16 0 CoA, 6,9-18 2 CoA and 6,9,12-18 3 CoA as substrates The 8-hydroxy acyl-CoA dehydrase reaction was assayed only with the CoA derivatives of DL-8-hydroxy-stearic acid (8-OH-18 0 CoA) and DL-8-hydroxy-8,11-eicosadienoic acid (8-OH-8,11-20 2 CoA). The 2-trans-enoyl-CoA reductase reaction was assayed with the CoA derivatives of 2-trans-octadecenoic acid (2 trans-18 1 CoA) and 2-trans-8,ll-eicosatrienoic acid (2-trans-8,11-20 3 CoA) All reactions were assayed with the microsomes from rats raised on both a chow diet and a fat-free diet. Both the rates of the condensation reaction and the overall chain elongation reaction were depressed when rats were fed a chow diet These depressed rates of conversion were more pronounced when 16 0 CoA was used as substrate than when 6,9-18 2 CoA or 6,9,12-18 3 CoA were used as substrates. These results support our N-ethylmaleimide inhibition studies and are consistent with the concept that rat liver microsomes contain at least two different condensing enzymes. One condensing enzyme would preferentially utilize saturated substrates while another would act on unsaturated substrates. 

The rates of the 8-hydroxy-acyl-CoA dehydrase reaction were more rapid than found for the condensation or overall chain elongation reactions - i.e. Table 6. The rate of the 8-hydroxy-acyl-CoA dehydrase reaction was the same when 8-OH-18 0 CoA or 8-OH-8,11-20 2 CoA were used as substrates. The rate of this reaction, with both substrates, was the same with microsomes from rats raised on either a chow or a fat-free diet. In a similar way the rates of the 2-trans-enoyl-CoA reductase reaction were the same for both substrates and the rates of this reaction with both substrates were not altered by dietary modification. Again the rate of this reaction with both substrates was much more rapid than found for either the condensation reaction or the overall chain elongation reaction. 

We have also measured the rates of the 8-hydroxyacyl-CoA dehydrase and 2-trans-enoyl-CoA reductase reactions using the CoA derivatives of the appropriate intermediates produced during chain elongation of 5,8-18 2, 7,10-18 2 and 8,11-18 2 (Ludwig S Sprecher, unpublished results). The 8-OH-7,10-20 2 CoA, 6-OH-9,12-20 2 CoA and 3-OH-10,13-20 2 CoA were all dehydrated at about the same rate as was found for B-OH-8,11-20 2 CoA and 3-0H-18 l CoA - i.e. 

Table 6. In a similar way 2-trans-7,10-20 3 CoA, 2-trans-9,12-20 3 CoA and 2-trans-10,13-20 3 CoA were reduced at about the same rate as found for 2-trans-18 l CoA and 2-trans-8,ll-20 3 CoA -i e Table 6 The B-hydroxyacyl-CoA dehydrase and 2-trans-enoyl-CoA reductase reactions are thus not highly substrate specific. Since both of these reactions proceed much more readily than the condensation reaction it might suggest that two or more independent condensing enzymes contribute there products to a common set of enzymes for subsequent reactions. Failure of dietary modification to alter the rates of these two reactions supports this hypothesis. Moreover, we have also shown that the CoA derivative of 3-keto-stearic acid was converted to stearic acid at least four times as rapidly as B-ketostearic acid was produced from 16 0 CoA and malonyl-CoA., The B-keto reductase reaction would thus not be rate limiting in this metabolic process. It thus appears that the rate at which an acid is chain elongated is dictated by the rate of the condensation reaction and that the rate of this reaction is controlled by structural features which are an inherent part of the substrate.

In conclusion, the trans 2-3 enoyl-CoA reductase, one of the proteins constituting the acyl-CoA elongase, has been evidenced in the leek microsomes. The preferred sustrate for this enzyme is the trans 2-3 C20 1-CoA and NADPH is the reductant the more efficient. 

Polyunsaturated fatty acids pose a slightly more complicated situation for the cell. Consider, for example, the case of linoleic acid shown in Figure 24.24. As with oleic acid, /3-oxidation proceeds through three cycles, and enoyl-CoA isomerase converts the cA-A double bond to a trans-b double bond to permit one more round of /3-oxidation. What results this time, however, is a cA-A enoyl-CoA, which is converted normally by acyl-CoA dehydrogenase to a trans-b, cis-b species. This, however, is a poor substrate for the enoyl-CoA hydratase. This problem is solved by 2,4-dienoyl-CoA reductase, the product of which depends on the organism. The mammalian form of this enzyme produces a trans-b enoyl product, as shown in Figure 24.24, which can be converted by an enoyl-CoA isomerase to the trans-b enoyl-CoA, which can then proceed normally through the /3-oxidation pathway. Escherichia coli possesses a... 

FIGURE 24.24 The oxidation pathway for polyunsaturated fatty adds, illustrated for linoleic add. Three cycles of /3-oxidation on linoleoyl-CoA yield the cis-A, d.s-A intermediate, which is converted to a tran.s-A, cis-A intermediate. An additional round of /S-oxi-dation gives d.s-A enoyl-CoA, which is oxidized to the trans-A, d.s-A species by acyl-CoA dehydrogenase. The subsequent action of 2,4-dienoyl-CoA reductase yields the trans-A product, which is converted by enoyl-CoA isomerase to the tran.s-A form. Normal /S-oxida-tion then produces five molecules of acetyl-CoA. 

Polyunsaturated fatty acids are also degraded by [3 oxidation, but the process requires enoyl-CoA isomerase and an additional enzyme, 2,4-dienoyl-CoA reductase (fig. 18.6). For example, the degradation of linoleoyl-CoA (18 2A9-12) begins, like that of oleoyl-CoA, with three rounds of /3 oxidation and results in a A3-cis unsaturated fatty acyl-CoA that is not a substrate for acyl-CoA dehydrogenase. Isomerization of the double bond to the A2-trans position by enoyl-CoA isomerase allows the resumption of... 

Another enzyme, in addition to the isomerase, is required for the oxidation of polyunsaturated fatty acids which have a double bond at an even-numbered carbon atom. In this case the 2,4-dienoyl intermediate resulting from the action of acyl CoA dehydrogenase is acted on by 2,4-dienoyl CoA reductase to form c/s-A3-enoyl CoA (Fig. 4). This is then converted by the isomerase into the trans form which continues down the pathway. These reactions are important since over half the fatty acids of plant and animal lipids are unsaturated (and often polyunsaturated). 

Fig. 5.2. Possible metabolic pathways in facultative anaerobic mitochondria. Shaded boxes show components of the electron-transport chain used during hypoxia, open boxes are components used during aerobiosis, and the hatched boxes (complex I and ATP-synthase) are components used under aerobic as well as anaerobic conditions. ASCT acetate succinate CoA-transferase, C cytochrome c, Cl, CIII and CIV complexes I, III and IV of the respiratory chain, CITR citrate, ECR enoyl-CoA reductase (such as present in Ascaris suum), ETF electron-transfer flavoprotein, ETF RQ OR electron-transfer flavoproteimrhodoquinone oxidoreductase, FRD fumarate reductase, FUM fumarate, MAE malate, OXAC oxaloacetate, PYR pyruvate, RQ rhodoquinone, SDH succinate dehydrogenase, SUCC succinate, Succ-CoA succinyl-CoA, TER trans-2-enoyl-CoA reductase (such as present in E. gracilis), UQ ubiquinone...

Hoffmeister M, Piotrowski M, Nowitzki U, Martin W (2005) Mitochondrial trans-2-enoyl-CoA reductase of wax ester fermentation from Euglena gracilis defines a new family of enzymes involved in lipid synthesis. J Biol Chem 280 4329-4338... 

After one further -oxidation cycle, a 4-cis-enoyl CoA intermediate is formed. It is acted upon by enoyl-CoA dehydrogenase to give 2-trans, 4-cis-dienoyl CoA. Further metabolism of this intermediate proceeds through one cycle of /3-oxidation and requires a second auxiliary enzyme, 2,4-dienoyl-CoA reductase which has high activity in mitochondria. Thus, nine molecules of acetyl-CoA are produced from the oxidation of linoleic acid. 

Two new enzymes are required to handle these situations Enoyl-CoA isomerase (isomerizes a cis-3,4-double bond to a frflns-2,3-double bond), and 2,4-Dienoyl-CoA reductase (reduces the ds-4,5-double bond in the trans-2,3-ds-4,5-dienoyl-CoA derivative formed during feta-oxidation). The resulting products are then broken down by the feta-oxidation enzymes. 

Glutaric acidemia type II is caused by defects in the ETF/ETF-QO proteins. The clinical manifestations of these disorders are similar to medium-chain acyl-CoA dehydrogenase deficiency (discussed later). The double bond formed by the acyl-CoA dehydrogenase has a trans configuration. The double bonds in naturally occurring fatty acids are generally in the cis configuration. The oxidation of unsaturated m-fatty acids requires two auxiliary enzymes, enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase. 

Oxidation of unsaturated fatty acids requires A -cis-,A -trans-enoyl-CoA isomerase and NADPH-dependent 2,4-dienoyl-CoA reductase, in addition to the enzymes of y3-oxidation. The enoyl-CoA isomerase produces the substrate for the hydration step. The reductase catalyzes the reduction of A -frans-,A -cjs-decadienoyl-CoA to A -rrans-decenoyl-CoA. The latter is isomerized to A -trans-decenoyl isomerase, which is a normal -oxidation intermediate. These reactions are illustrated for oxidation of oleic and linoleic acids in Figures 18-7 and 18-8. 

Another problem arises with the oxidation of polyunsaturated fatty acids. Consider linoleate, a Cx polyunsaturated fatty acid with cis-A and cis-A double bonds (Figure 22.11). The cis-A double bond formed after three rounds of P oxidation is converted into a trans-A" double bond by the aforementioned isomerase. The acyl (ioA produced by another round of P oxidation contains a cis-A double bond. Dehydrogenation of this species by acy] CoA dehydrogenase yields a. 2,4-dienoyl intermediate, which is not a substrate for the next enzyme in the p-oxidation pathway. This impasse is circumvented by 2,4-dienoyl CoA reductase, an enzyme that uses to reduce the 2,4-dienoyl intermediate to trans-A -enoyl CoA. ds-A Enoyl CoA isomerase then converts trans-A -enoyl CoA into the trans-A form, a customary intermediate in the P-oxidation pathway. These catalytic... 

Fig. 8. P-Oxidation of fatty acids in E. coli. Long-chain fatty acids are transported into the cell by FadL and converted to their CoA thioesters by FadD (not shown). The acyl-CoAs are substrates for the (1) acyl-CoA dehydrogenase (YafH) to form a trans-2-enoyl-CoA. The double bond is reduced by (2) rrans-2-enoyl-hydratase (crotonase) activity of FadB. The P-hydroxyacyl-CoA is then a substrate for the NADP -dependent dehydrogenase activity of FadB (3). A thiolase, FadA (4), releases acetyl-CoA from the P-ketoacyl-CoA to form an acyl-CoA for subsequent cycles. (5) Polyunsaturated fatty acyl-CoAs are reduced by the 2,4-dienoyl-CoA reductase (FadH). (6) FadB also catalyzes the isomerization of c/s-unsaturated fatty acids to trans. (7) The epimerase activity of FadB converts O-P-hydroxy thioesters to their L-enantiomers via the /rans-2-enoyl-CoA.

Unsaturated fatty acids can also be degraded by the 3-oxidation pathway. The FadB protein possesses cw-P-enoyl-CoA isomerase activity, which converts cis-3 double bonds to trans-2 (Fig. 8). A 2,4-dienoyl-CoA reductase encoded by fadH is also required for the metabolism of polyunsaturated fatty acids (Fig. 8). This protein is a 73-kDa monomeric, NADP" -dependent, 4Fe-4S flavoprotein. The FadH protein can utilize compounds with either cis or trans double bonds at the 4-position. An epimerase activity of FadB allows for the utilization of D-hydroxy fatty acids. The epimerase is actually a combination of a Z)-P-hydroxyacyl-CoA dehydratase and the crotonase (hydratase) activities, resulting in the conversion of the d to the L enantiomer (Fig. 8). 

Enoyl-CoA Hydratase, a key enzyme in / -oxidation of saturated fatty acids, acts on trans double bonds, but cannot act on the cis double bonds of unsaturated fatty acids. Instead, cells must rely on two additional enzymes, enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase, to complete the oxidation of unsaturated fatty acids. The activity of these enzymes is shown in Figure 18.18. 

It should be noted that enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase only help metabolize dietary unsaturated fatty acids with double bonds in the cis configuration. Dietary fatty acids with trans double bonds are not readily metabolized and may pose a health hazard. 

The most likely deficiency is a lack of 2,4-dienoyl CoA reductase, an enzyme that is essential for the degradation of unsaturated fatty acids with double bonds at even-numbered carbons. Such fatty acids include linoleate (9-ds,12-ds 18 2). Four rounds of oxidation of linoleoyl CoA generate a 10-carbon acyl CoA that contains a trans-A and a cis-A double bond. This intermediate is a substrate for the reductase, which converts the 2,4-dienoyl CoA to ds-A -enoyl CoA. A dehciency of 2,4-dienoyl reductase leads to an accumulation of trans-A, ds-A -decadienoyl CoA molecules in the mitochondrion. The observation that carnitine derivatives of the 2,4-dienoyl CoA are found in blood and urine provides evidence that these molecules accumulate in the mitochondrion and are then attached to carnitine. Formation of carnitine decadienoate allows the acyl molecules to be transported across the inner mitochondrial membrane into the cytosol, and then into the circulation. 

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