Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Jan 1;321(Pt 1):21–28. doi: 10.1042/bj3210021

Peroxisomal multifunctional enzyme of beta-oxidation metabolizing D-3-hydroxyacyl-CoA esters in rat liver: molecular cloning, expression and characterization.

Y M Qin 1, M H Poutanen 1, H M Helander 1, A P Kvist 1, K M Siivari 1, W Schmitz 1, E Conzelmann 1, U Hellman 1, J K Hiltunen 1
PMCID: PMC1218032  PMID: 9003397

Abstract

In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enzyme (MFE) named perMFE-II. The purified 2-enoyl-CoA hydratase 2 with an M(r) of 31500 from rat liver [Malila, Siivari, Mäkelä, Jalonen, Latipää, Kunau and Hiltunen (1993) J. Biol. Chem. 268, 21578-21585] was subjected to tryptic fragmentation and the resulting peptides were isolated and sequenced. Surprisingly, the full-length cDNA, amplified by PCR, had an open reading frame of 2205 bp encoding a polypeptide with a predicted M(r) of 79,331 and contained a potential peroxisomal targeting signal in the C-terminus (Ala-Lys-Leu). The sequenced peptide fragments of hydratase 2 gave a full match in the middle portion of the cDNA-derived amino acid sequence. The predicted amino acid sequence showed a high degree of similarity with pig 17 beta-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal beta-oxidation. Recombinant perMFE-II (produced in Pichia pastoris) had 2-enoyl-CoA hydratase 2 and D-specific 3-hydroxyacyl-CoA dehydrogenase activities and was catalytically active with several straight-chain trans-2-enoyl-CoA, 2-methyltetradecenoyl-CoA and pristenoyl-CoA esters. The results showed that in addition to an earlier described multifunctional isomerase-hydratase-dehydrogenase enzyme from rat liver peroxisomes (perMFE-I), another MFE exists in rat liver peroxisomes. They both catalyse sequential hydratase and dehydrogenase reactions of beta-oxidation but through reciprocal stereochemical courses.

Full Text

The Full Text of this article is available as a PDF (911.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adamski J., Husen B., Marks F., Jungblut P. W. Purification and properties of oestradiol 17 beta-dehydrogenase extracted from cytoplasmic vesicles of porcine endometrial cells. Biochem J. 1992 Dec 1;288(Pt 2):375–381. doi: 10.1042/bj2880375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baker M. E. A common ancestor for Candida tropicalis and dehydrogenases that synthesize antibiotics and steroids. FASEB J. 1990 Sep;4(12):3028–3032. doi: 10.1096/fasebj.4.12.2394320. [DOI] [PubMed] [Google Scholar]
  3. Baker M. E. Protochlorophyllide reductase is homologous to human carbonyl reductase and pig 20 beta-hydroxysteroid dehydrogenase. Biochem J. 1994 Jun 1;300(Pt 2):605–607. doi: 10.1042/bj3000605b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen L. S., Jin S. J., Tserng K. Y. Purification and mechanism of delta 3,delta 5-t-2,t-4-dienoyl-CoA isomerase from rat liver. Biochemistry. 1994 Aug 30;33(34):10527–10534. doi: 10.1021/bi00200a039. [DOI] [PubMed] [Google Scholar]
  5. Christiansen R. Z., Christiansen E. N., Bremer J. The stimulation of erucate metabolism in isolated rat hepatocytes by rapeseed oil and hydrogenated marine oil-containing diets. Biochim Biophys Acta. 1979 Jun 21;573(3):417–429. doi: 10.1016/0005-2760(79)90216-9. [DOI] [PubMed] [Google Scholar]
  6. Cooper T. G., Beevers H. Beta oxidation in glyoxysomes from castor bean endosperm. J Biol Chem. 1969 Jul 10;244(13):3514–3520. [PubMed] [Google Scholar]
  7. DE DUVE C., PRESSMAN B. C., GIANETTO R., WATTIAUX R., APPELMANS F. Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem J. 1955 Aug;60(4):604–617. doi: 10.1042/bj0600604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Filppula S. A., Sormunen R. T., Hartig A., Kunau W. H., Hiltunen J. K. Changing stereochemistry for a metabolic pathway in vivo. Experiments with the peroxisomal beta-oxidation in yeast. J Biol Chem. 1995 Nov 17;270(46):27453–27457. doi: 10.1074/jbc.270.46.27453. [DOI] [PubMed] [Google Scholar]
  9. Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gubler U., Hoffman B. J. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  11. Hellman U., Wernstedt C., Góez J., Heldin C. H. Improvement of an "In-Gel" digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal Biochem. 1995 Jan 1;224(1):451–455. doi: 10.1006/abio.1995.1070. [DOI] [PubMed] [Google Scholar]
  12. Hiltunen J. K., Filppula S. A., Häyrinen H. M., Koivuranta K. T., Hakkola E. H. Peroxisomal beta-oxidation of polyunsaturated fatty acids. Biochimie. 1993;75(3-4):175–182. doi: 10.1016/0300-9084(93)90075-4. [DOI] [PubMed] [Google Scholar]
  13. Hiltunen J. K., Kunau W. H. Epimerization of 3-hydroxyacyl-CoA esters as an auxiliary reaction in the beta-oxidation of unsaturated fatty acids. Prog Clin Biol Res. 1990;321:265–272. [PubMed] [Google Scholar]
  14. Hiltunen J. K., Kärki T., Hassinen I. E., Osmundsen H. beta-Oxidation of polyunsaturated fatty acids by rat liver peroxisomes. A role for 2,4-dienoyl-coenzyme A reductase in peroxisomal beta-oxidation. J Biol Chem. 1986 Dec 15;261(35):16484–16493. [PubMed] [Google Scholar]
  15. Hiltunen J. K., Palosaari P. M., Kunau W. H. Epimerization of 3-hydroxyacyl-CoA esters in rat liver. Involvement of two 2-enoyl-CoA hydratases. J Biol Chem. 1989 Aug 15;264(23):13536–13540. [PubMed] [Google Scholar]
  16. Hiltunen J. K., Wenzel B., Beyer A., Erdmann R., Fosså A., Kunau W. H. Peroxisomal multifunctional beta-oxidation protein of Saccharomyces cerevisiae. Molecular analysis of the fox2 gene and gene product. J Biol Chem. 1992 Apr 5;267(10):6646–6653. [PubMed] [Google Scholar]
  17. Huang X. On global sequence alignment. Comput Appl Biosci. 1994 Jun;10(3):227–235. doi: 10.1093/bioinformatics/10.3.227. [DOI] [PubMed] [Google Scholar]
  18. Kunau W. H., Bühne S., de la Garza M., Kionka C., Mateblowski M., Schultz-Borchard U., Thieringer R. Comparative enzymology of beta-oxidation. Biochem Soc Trans. 1988 Jun;16(3):418–420. doi: 10.1042/bst0160418. [DOI] [PubMed] [Google Scholar]
  19. Lazarow P. B., De Duve C. A fatty acyl-CoA oxidizing system in rat liver peroxisomes; enhancement by clofibrate, a hypolipidemic drug. Proc Natl Acad Sci U S A. 1976 Jun;73(6):2043–2046. doi: 10.1073/pnas.73.6.2043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Leenders F., Adamski J., Husen B., Thole H. H., Jungblut P. W. Molecular cloning and amino acid sequence of the porcine 17 beta-estradiol dehydrogenase. Eur J Biochem. 1994 May 15;222(1):221–227. doi: 10.1111/j.1432-1033.1994.tb18860.x. [DOI] [PubMed] [Google Scholar]
  21. Leenders F., Husen B., Thole H. H., Adamski J. The sequence of porcine 80 kDa 17 beta-estradiol dehydrogenase reveals similarities to the short chain alcohol dehydrogenase family, to actin binding motifs and to sterol carrier protein 2. Mol Cell Endocrinol. 1994 Sep;104(2):127–131. doi: 10.1016/0303-7207(94)90114-7. [DOI] [PubMed] [Google Scholar]
  22. Leenders F., Tesdorpf J. G., Markus M., Engel T., Seedorf U., Adamski J. Porcine 80-kDa protein reveals intrinsic 17 beta-hydroxysteroid dehydrogenase, fatty acyl-CoA-hydratase/dehydrogenase, and sterol transfer activities. J Biol Chem. 1996 Mar 8;271(10):5438–5442. doi: 10.1074/jbc.271.10.5438. [DOI] [PubMed] [Google Scholar]
  23. Li J. X., Smeland T. E., Schulz H. D-3-hydroxyacyl coenzyme A dehydratase from rat liver peroxisomes. Purification and characterization of a novel enzyme necessary for the epimerization of 3-hydroxyacyl-CoA thioesters. J Biol Chem. 1990 Aug 15;265(23):13629–13634. [PubMed] [Google Scholar]
  24. Luthria D. L., Baykousheva S. P., Sprecher H. Double bond removal from odd-numbered carbons during peroxisomal beta-oxidation of arachidonic acid requires both 2,4-dienoyl-CoA reductase and delta 3,5,delta 2,4-dienoyl-CoA isomerase. J Biol Chem. 1995 Jun 9;270(23):13771–13776. doi: 10.1074/jbc.270.23.13771. [DOI] [PubMed] [Google Scholar]
  25. Malila L. H., Siivari K. M., Mäkelä M. J., Jalonen J. E., Latipä P. M., Kunau W. H., Hiltunen J. K. Enzymes converting D-3-hydroxyacyl-CoA to trans-2-enoyl-CoA. Microsomal and peroxisomal isoenzymes in rat liver. J Biol Chem. 1993 Oct 15;268(29):21578–21585. [PubMed] [Google Scholar]
  26. Mannaerts G. P., Van Veldhoven P. P. Metabolic pathways in mammalian peroxisomes. Biochimie. 1993;75(3-4):147–158. doi: 10.1016/0300-9084(93)90072-z. [DOI] [PubMed] [Google Scholar]
  27. Novikov D. K., Vanhove G. F., Carchon H., Asselberghs S., Eyssen H. J., Van Veldhoven P. P., Mannaerts G. P. Peroxisomal beta-oxidation. Purification of four novel 3-hydroxyacyl-CoA dehydrogenases from rat liver peroxisomes. J Biol Chem. 1994 Oct 28;269(43):27125–27135. [PubMed] [Google Scholar]
  28. Nuttley W. M., Aitchison J. D., Rachubinski R. A. cDNA cloning and primary structure determination of the peroxisomal trifunctional enzyme hydratase-dehydrogenase-epimerase from the yeast Candida tropicalis pK233. Gene. 1988 Sep 30;69(2):171–180. doi: 10.1016/0378-1119(88)90428-3. [DOI] [PubMed] [Google Scholar]
  29. Osumi T., Hashimoto T. Peroxisomal beta oxidation system of rat liver. Copurification of enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase. Biochem Biophys Res Commun. 1979 Jul 27;89(2):580–584. doi: 10.1016/0006-291x(79)90669-7. [DOI] [PubMed] [Google Scholar]
  30. Palosaari P. M., Hiltunen J. K. Peroxisomal bifunctional protein from rat liver is a trifunctional enzyme possessing 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and delta 3, delta 2-enoyl-CoA isomerase activities. J Biol Chem. 1990 Feb 15;265(5):2446–2449. [PubMed] [Google Scholar]
  31. Persson B., Krook M., Jörnvall H. Characteristics of short-chain alcohol dehydrogenases and related enzymes. Eur J Biochem. 1991 Sep 1;200(2):537–543. doi: 10.1111/j.1432-1033.1991.tb16215.x. [DOI] [PubMed] [Google Scholar]
  32. Puranen T. J., Poutanen M. H., Peltoketo H. E., Vihko P. T., Vihko R. K. Site-directed mutagenesis of the putative active site of human 17 beta-hydroxysteroid dehydrogenase type 1. Biochem J. 1994 Nov 15;304(Pt 1):289–293. doi: 10.1042/bj3040289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rasmussen J. T., Börchers T., Knudsen J. Comparison of the binding affinities of acyl-CoA-binding protein and fatty-acid-binding protein for long-chain acyl-CoA esters. Biochem J. 1990 Feb 1;265(3):849–855. doi: 10.1042/bj2650849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schmitz W., Fingerhut R., Conzelmann E. Purification and properties of an alpha-methylacyl-CoA racemase from rat liver. Eur J Biochem. 1994 Jun 1;222(2):313–323. doi: 10.1111/j.1432-1033.1994.tb18870.x. [DOI] [PubMed] [Google Scholar]
  35. Seedorf U., Assmann G. Cloning, expression, and nucleotide sequence of rat liver sterol carrier protein 2 cDNAs. J Biol Chem. 1991 Jan 5;266(1):630–636. [PubMed] [Google Scholar]
  36. Seedorf U., Brysch P., Engel T., Schrage K., Assmann G. Sterol carrier protein X is peroxisomal 3-oxoacyl coenzyme A thiolase with intrinsic sterol carrier and lipid transfer activity. J Biol Chem. 1994 Aug 19;269(33):21277–21283. [PubMed] [Google Scholar]
  37. Singh H., Beckman K., Poulos A. Peroxisomal beta-oxidation of branched chain fatty acids in rat liver. Evidence that carnitine palmitoyltransferase I prevents transport of branched chain fatty acids into mitochondria. J Biol Chem. 1994 Apr 1;269(13):9514–9520. [PubMed] [Google Scholar]
  38. Smeland T. E., Li J. X., Chu C. H., Cuebas D., Schulz H. The 3-hydroxyacyl-CoA epimerase activity of rat liver peroxisomes is due to the combined actions of two enoyl-CoA hydratases: a revision of the epimerase-dependent pathway of unsaturated fatty acid oxidation. Biochem Biophys Res Commun. 1989 May 15;160(3):988–992. doi: 10.1016/s0006-291x(89)80098-1. [DOI] [PubMed] [Google Scholar]
  39. Smeland T. E., Nada M., Cuebas D., Schulz H. NADPH-dependent beta-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6673–6677. doi: 10.1073/pnas.89.15.6673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tanaka A., Osumi M., Fukui S. Peroxisomes of alkane-grown yeast: fundamental and practical aspects. Ann N Y Acad Sci. 1982;386:183–199. doi: 10.1111/j.1749-6632.1982.tb21416.x. [DOI] [PubMed] [Google Scholar]
  41. Wanders R. J., van Roermund C. W., Schutgens R. B., Barth P. G., Heymans H. S., van den Bosch H., Tager J. M. The inborn errors of peroxisomal beta-oxidation: a review. J Inherit Metab Dis. 1990;13(1):4–36. doi: 10.1007/BF01799330. [DOI] [PubMed] [Google Scholar]
  42. Wierenga R. K., Drenth J., Schulz G. E. Comparison of the three-dimensional protein and nucleotide structure of the FAD-binding domain of p-hydroxybenzoate hydroxylase with the FAD- as well as NADPH-binding domains of glutathione reductase. J Mol Biol. 1983 Jul 5;167(3):725–739. doi: 10.1016/s0022-2836(83)80106-5. [DOI] [PubMed] [Google Scholar]
  43. de Hoop M. J., Ab G. Import of proteins into peroxisomes and other microbodies. Biochem J. 1992 Sep 15;286(Pt 3):657–669. doi: 10.1042/bj2860657. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES