3-Hydroxyacyl-CoA epimerase

Mizugaki, M., Nishimaki, T., Yamamoto, H., Sagi, M. Yamanaka, H. (1982) J. Biochem., 92, 2051-2054, Studies on the metabolism of unsaturated fatty acids. XI. Alterations in the activities of enoyl-CoA hydratase, 3-hydroxyacyl-CoA epimerase and 2,4-dienoyl-CoA reductase in rat liver mitochondria and peroxisomes by clofibrate. 

Fig. 2 Metabolic routes for mcl-PHA biosynthesis. Pseudomonas putida GPol synthesizes PHA through P-oxidation and P. putida KT2440 synthesizes PHA through fatty add de novo synthesis. Special PHA consisting of 4-hydroxyalkanoate, 5- hydroxyalkanoate, or 6-hydroxyalkanoate can be produced by various bacteria when suitable precursors are supplied. 1 acyl-CoA synthetase, 2 acyl-CoA dehydrogenase, 3 enoyl-CoA hydratase, 4 NAD-dependent (5)-3-hydroxyacyl-CoA dehydrogenase, 5 3-ketoacyl-CoA thiolase, 6 (ItFspecific enoyl-CoA hydratase, 7 NADPH-dependent 3-ketoacyl-CoA reducatase, 8 3-hydroxyacyl-CoA epimerase, 9 mcl-PHA polymerase, 10 acetyl-CoA carboxylase, 11 malonyl-CoA-acyl carrier protein (ACP) tiansacylase, 12 3-keto-ACP synthase, 13 3-keto-ACP reductase, 14 3-hydroxyacyl-ACP dehydratase, 15 enoyl-ACP reductase, 16 acyl-ACP thiolase, 17 (l )-3-hydroxyacyl-ACP-CoA transacylase, 18 mcl-PHA polymerase...

P-oxidation pathway This metabolic route generates MCL-PHA precursor molecules from fatty acids. The three key enzymes, which catalyses three key reactions include 3-hydroxyacyl-CoA epimerase that converts (5)-3-hydroxyacyl-CoA into (ff)-3-hydroxyacyl CoA, enoyl-CoA hydratase which converts 2-frans-enoyl-CoA into (if)-3-hydroxyacyl CoA, and finally 3-ketoacyl-CoA reductase which converts 3-ketoacyl-CoA to (R)-3-hydroxyacyl CoA (Huisman et al., 1989). 

Degradation of polyunsaturated fatty acids is likewise by the P-oxidation step but in addition to the need for dodecenoyl-CoA A-isomerase, a further enzymic step (3-hydroxyacyl-CoA epimerase) is required to convert the D-product of enoyl-CoA hydratase into its L-stereoisomer. Thereafter, P-oxidation resumes. 

An epimerase directly converting the l-(+) of 3-hydroxyacyl-CoA into the r-(-) stereoisomer... 

The second pathway involves the production of PHAs via the fatty acid degradation pathway. In this case, the resulting monomers in the polymer chain were similar in structure to the carbon source or shortened by 2, 4 or 6 carbon atoms [29]. In this pathway the fatty acids are first converted to the corresponding acyl-CoA which are then oxidised by the /1-oxidation pathway via enoyl-CoA, (5)-3-hydroxyacyl-CoA and 3-ketoacyl-CoA precursors. Finally, enzymes like the enoyl-CoA hydratase, hydroxyacyl-CoA epimerase, and /3-ketoacyl-CoA reductase connect the /1-oxidation pathway with the medium-chain length PHA biosynthesis through the PHA synthase [30]. 

The second PHA synthesis pathway (pathway II) is related to fatty acid uptake by microorganisms. After fatty acid P-oxidation, acyl-CoA enters the PHA monomer synthesis process. Enzymes including 3-ketoacyl-CoA reductase, epimerase, (I )-enoyI-CoA hydratase/enoyl-CoA hydratase I, acyl-CoA oxidase (putative), and enoyl-CoA hydratase I (putative) were found to be involved in supplying the PHA precursor 3-hydroxyacyl-CoA for PHA synthesis. Pseudomonas putida, Pesudomonas aeruginosa, and A. hydrophila are able to use pathway n to synthesize medium-chain-length (mcl) PHA or copolymers of (/ )-3-hydroxybutyrate (R3HB) and (R)-3-hydroxyhexanoate (PHBHHx). 

Two chemical features are characteristic for the naturally occurring mono- and polyunsaturated fatty acids a) the double bonds possess all-cis configuration, b) the double bonds are isolated by one CHg-group. It is a reasonable assumption that the saturated carboxylic and terminal methyl end of unsaturated acids are degraded by the reaction sequence described in the preceeding chapter. However, oxidation of the cis-/S, y- and cis-od, /S-unsaturated acyl-CoA intermediates requires two additional enzymes a) cis-/S, y-trans-od,/3-enoyl-CoA-isomerase and b) I)(—) hydroxyacyl-CoA epimerase. The isomerase catalyzes the following reaction ... 

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). 

HHx monomers from various fatty acids. The composition of the PHA formed by pseudomonads is highly dependent on the structure of the carbon substrate especially when the PHA precursors are supplied from -oxidation pathway Generally, the chain length of monomers in PHAs is the same as that of carbon sources or shortened by 2, 4, or 6 carbon atoms. The three putative enzymes involved in providing hydroxyacyl-CoA from P-oxidation pathway include hydratase, epimerase and 3-ketoacyl-CoA reductases. 

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