P-hydroxyacyl-CoA

The next step in fatty acid degradation is the addition of a water molecule to the double bond of the enoyl CoA hydration), with formation of p-hydroxyacyl CoA. 

Biosynthesis occurs from 3-hydroxybutyryl-CoA. Some bacteria incorporate other P-hydroxyac-ids into the polymer.f Apparently various hydroxy-acyl-CoAs can be diverted from the P oxidation pathway to polymer synthesis, and synthases that will accept a variety of P-hydroxyacyl-CoA substrates have been isolated. h i More than 80 different hydroxyacyl groups can be incorporated into the polymer.1 A bacterially produced copolymer of P-... 

Conjugate addition of water (1) is followed by a proton shift (2) to give the p-hydroxyacyl CoA product. 

H20 adds across the double bond, and a p-hydroxyacyl CoA is formed. —Enzyme enoyl CoA hydratase... 

Enzyme L-3-hydroxyacyI CoA dehydrogenase (which is specific for the L-isomer of the P-hydroxyacyl CoA)... 

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). 

II cleaves acyl-CoAs of >6 carbons and P-hydroxyacyl-CoAs, but is unable to cleave acyl-pantetheine thioesters. The physiological function of thioesterases I and II is unknown. Null mutants have been constructed in both tesA and tesB, and the double-mutant strain generated. None of these strains has an observable growth phenotype, indicating that neither protein is essential. However, the tesAB double-null mutant still retains about 10% of the total wild-type thioesterase activity, indicating the existence of a third unidentified thioesterase in E. coli. 

P-Hydroxyacyl-CoA dehydrase catalyzes the third reaction in chain elongation and involves dehydration of 3-hydroxyacyl-CoA to enoyl-CoA (Fig. 1). The gene that encodes this enzyme has not been cloned although the enzymatic activity has been described in microsomal preparations. In contrast to the membrane-bound elongation activities that have a cytosolic orientation, the P-hydroxyacyl-CoA dehydrase activity is embedded within the ER membrane (J. Bemert, 1979). 

A second oxidation reaction is catalyzed by P-hydroxyacyl-CoA dehydrogenase, an NAD -dependent enzyme. The product is a p-ketoacyl-GoA. 

Vallejo-Cordoba, B., Mazorra-Manzano, M. A., and Gonzalez-Cordova, A. R, Determination of P-hydroxyacyl CoA-dehydrogenase activity in meat by electrophoretically mediated microanalysis, J. Cap. Elec. Micro. Tech., 8, 81, 2003. 

Fig. 5. Kinetics of rat liver microsomal p-hydroxyacyl-CoA dehydrase using p-hydroxy-16.0-CoA with intact microsomes ( ) or after treating microsomes with deoxycholate (o). This preparation was then centrifuged at 100,000 g for 1 hour and supernatant (p) and pellet (A) were assayed. From Bernert and Sprecher (1979a).

The enzyme P-hydroxyacyl-CoA-dehydrogenase (HADH, EC 1.1.1.35) is also suitable for the detection of frozen meat or fish. In the oxidation of fatty acids, HADH catalyzes the reaction shown in Formula 12.29. This enzyme is bound to the iimer membrane of mitochondria and is liberated in the freeze/thaw process. Its activity can then be measured in the issuing sap with acetoacetyl CoA or with the artificial substrate N-acetylacetoacetylcysteamine. 

Feeney, R.E., Yin Yeh Antifreeze proteins from fish bloods. Adv. Protein Chem. 32, 191 (1978) Fernandez, M., Mano, S., Garcia de Fernando, G.D., Ordonez, J.A., Hoz, L. Use of P-hydroxyacyl-CoA-dehydrogenase (HADH) activity to differentiate frozen from unfrozen fish and shellfish. Eur. Food Res. Technol. 209, 205 (1999)... 

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