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Molecular Characterization of a Glyoxysomal Long Chain Acyl-CoA Oxidase That Is Synthesized as a Precursor of Higher Molecular Mass in Pumpkin

1998; Elsevier BV; Volume: 273; Issue: 14 Linguagem: Inglês

10.1074/jbc.273.14.8301

1083-351X

Hiroshi Hayashi, Luigi De Bellis, Katsushi Yamaguchi, Akira Kato, Makoto Hayashi, Mikio Nishimura,

Biotin and Related Studies

A cDNA clone for pumpkin acyl-CoA oxidase (EC1.3.3.6; ACOX) was isolated from a λgt11 cDNA library constructed from poly(A)+ RNA extracted from etiolated cotyledons. The inserted cDNA clone contains 2313 nucleotides and encodes a polypeptide of 690 amino acids. Analysis of the amino-terminal sequence of the protein indicates that the pumpkin acyl-CoA oxidase protein is synthesized as a larger precursor containing a cleavable amino-terminal presequence of 45 amino acids. This presequence shows high similarity to the typical peroxisomal targeting signal (PTS2). Western blot analysis following cell fractionation in a sucrose gradient revealed that ACOX is localized in glyoxysomes. A partial purification of ACOX from etiolated pumpkin cotyledons indicated that the ACOX cDNA codes for a long chain acyl-CoA oxidase. The amount of ACOX increased and reached to the maximum activity by day 5 of germination but decreased about 4-fold on the following days during the subsequent microbody transition from glyoxysomes to leaf peroxisomes. By contrast, the amount of mRNA was already high at day 1 of germination, increased by about 30% at day 3, and faded completely by day 7. These data indicated that the expression pattern of ACOX was very similar to that of the glyoxysomal enzyme 3-ketoacyl-CoA thiolase, another marker enzyme of the β-oxidation spiral, during germination and suggested that the expression of each enzyme of β-oxidation is coordinately regulated. A cDNA clone for pumpkin acyl-CoA oxidase (EC1.3.3.6; ACOX) was isolated from a λgt11 cDNA library constructed from poly(A)+ RNA extracted from etiolated cotyledons. The inserted cDNA clone contains 2313 nucleotides and encodes a polypeptide of 690 amino acids. Analysis of the amino-terminal sequence of the protein indicates that the pumpkin acyl-CoA oxidase protein is synthesized as a larger precursor containing a cleavable amino-terminal presequence of 45 amino acids. This presequence shows high similarity to the typical peroxisomal targeting signal (PTS2). Western blot analysis following cell fractionation in a sucrose gradient revealed that ACOX is localized in glyoxysomes. A partial purification of ACOX from etiolated pumpkin cotyledons indicated that the ACOX cDNA codes for a long chain acyl-CoA oxidase. The amount of ACOX increased and reached to the maximum activity by day 5 of germination but decreased about 4-fold on the following days during the subsequent microbody transition from glyoxysomes to leaf peroxisomes. By contrast, the amount of mRNA was already high at day 1 of germination, increased by about 30% at day 3, and faded completely by day 7. These data indicated that the expression pattern of ACOX was very similar to that of the glyoxysomal enzyme 3-ketoacyl-CoA thiolase, another marker enzyme of the β-oxidation spiral, during germination and suggested that the expression of each enzyme of β-oxidation is coordinately regulated. There are at least three types of microbodies in higher plants (glyoxysomes, leaf peroxisomes, and unspecialized microbodies) that are distinguishable by their enzyme complements (1Beevers H. Annu. Rev. Plant Physiol. 1979; 30: 159-193Crossref Google Scholar, 2Huang A.H.C. Trelease R.N. Moore T.S. Plant Peroxisome. Academic Press, New York1983Google Scholar). During the postgerminative growth of pumpkin seedlings and upon exposure to light, etiolated cotyledons turn green; at the same time, a functional transition from glyoxysomes to leaf peroxisomes occurs (3Titus D.E. Becker W.M. J. Cell Biol. 1985; 101: 1288-1299Crossref PubMed Scopus (107) Google Scholar, 4Nishimura M. Yamaguchi J. Mori H. Akazawa T. Yokota S. Plant Physiol. 1986; 81: 313-316Crossref PubMed Google Scholar). In fat-storing seeds of plants such as pumpkin, lipid bodies are present in seed cells that store triacylglycerols, which are subsequently converted to fatty acids by lipase. Fatty acids represent the main energy and carbon sources for germinating seedlings. In glyoxysomes, fatty acids are degraded to acetyl-CoA via the β-oxidation pathway, and acetyl-CoA is metabolized by the glyoxylate cycle bypassing the decarboxylating steps of the Krebs cycle. We have shown previously that the expression of glyoxysomal enzymes and leaf peroxisomal enzymes are regulated not only at the transcriptional level but also at the posttranscriptional level during the microbody transition (5Mori H. Takeda-Yoshikawa Y. Hara-Nishimura I. Nishimura M. Eur. J. Biochem. 1991; 197: 331-336Crossref PubMed Scopus (68) Google Scholar, 6Tsugeki R. Hara-Nishimura I. Mori H. Nishimura M. Plant Cell Physiol. 1993; 34: 51-57PubMed Google Scholar). The gene expressions of the enzymes of the β-oxidation and glyoxylate cycles seem to be coordinately regulated. In a recent paper, we reported the nucleotide and deduced amino acid sequences of the cDNA for 3-ketoacyl-CoA thiolase (7Kato A. Hayashi M. Takeuchi Y. Nishimura M. Plant Mol. Biol. 1996; 22: 843-852Crossref Scopus (77) Google Scholar). The time course for thiolase mRNA and thiolase levels during germination and postgerminative growth implied that the regulation of expression of this enzyme is similar to that of glyoxylate cycle enzymes, e.g. malate synthase (8Mori H. Nishimura M. FEBS Lett. 1989; 244: 163-166Crossref Scopus (37) Google Scholar) and citrate synthase (9Kato A. Hayashi M. Mori H. Nishimura M. Plant Mol. Biol. 1995; 27: 377-390Crossref PubMed Scopus (64) Google Scholar). The glyoxysomal β-oxidation spiral consists of three different proteins: acyl-CoA oxidase (ACOX), 1The abbreviations used are: ACOX, acyl-CoA oxidase; PTS, peroxisomal targeting signal; thiolase, 3-ketoacyl-CoA thiolase; PRISCOX, pristanoyl-CoA oxidase; MOPS, 3-(N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: ACOX, acyl-CoA oxidase; PTS, peroxisomal targeting signal; thiolase, 3-ketoacyl-CoA thiolase; PRISCOX, pristanoyl-CoA oxidase; MOPS, 3-(N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis. enoyl-CoA hydratase/3-hydroxy acyl-CoA dehydrogenase (bifunctional protein), and 3-ketoacyl-CoA thiolase (thiolase). ACOX converts acyl-CoA into trans-2-enoyl-CoA in the first step of the β-oxidation spiral and corresponds to the acyl-CoA dehydrogenase present in mitochondria of mammalian cells. Both enzymes are flavoproteins. Some plant ACOXs have been purified and characterized (10Kirsch T. Löffler H.-G. Kindl H. J. Biol. Chem. 1986; 261: 8570-8575Abstract Full Text PDF PubMed Google Scholar, 11Hooks M.A. Bode K. Couée I. Biochem. J. 1996; 320: 607-614Crossref PubMed Scopus (32) Google Scholar) and have been shown to have different substrate specificities (for long, medium, and short chain acyl-CoAs, respectively). To further investigate the β-oxidation enzymes at the molecular level, we cloned a cDNA coding for a long chain ACOX, which is localized in glyoxysomes. Here, we report the nucleotide and deduced amino acid sequences of the cDNA. Developmental changes in the level of mRNA and protein were also determined in pumpkin cotyledons during seed germination and subsequent postgerminative growth.DISCUSSIONIn the present study, we report the cDNA sequence of a pumpkin glyoxysomal long chain ACOX in addition to the cDNA sequence of a previously reported Phalaenopsis ACOX (21Do Y.Y. Huang P.L. Arch. Biochem. Biophys. 1997; 344: 295-300Crossref PubMed Scopus (15) Google Scholar).The present results clearly show that the protein encoded by this gene is a plant long chain ACOX. The deduced amino acid sequences of pumpkin and Phalaenopsis cDNA sequences have an identity of 76%, indicating that the Phalaenopsis cDNA also codes for a long chain ACOX. Comparing other ACOXs, the best identity (30%) is obtained for the rat pristanoyl-CoA oxidase, which acts on 2-methyl-branched CoA-esters and straight long chain acyl-CoAs (22Van Veldhoven P.P. Van Rompuy J.C.T. Fransen M. De Béthune B. Mannaerts G.P. Eur. J. Biochem. 1994; 222: 795-801Crossref PubMed Scopus (39) Google Scholar). Mammalian peroxisomes contain three ACOX isozymes that are not capable of oxidizing acyl-chain CoA esters of less than 8 carbons. In mammalian cells, the β-oxidation of short chain fatty acids is accomplished in mitochondria, in which acyl-CoA dehydrogenases act instead of ACOXs. Three peroxisomal mammalian ACOXs have been identified: PRISCOX (30% identity), palmitoyl-CoA oxidase (28% identity), which reacts with CoA esters of very long, long, and medium chain fatty acids (28Osumi T. Hashimoto T. Biochem. Biophys. Res. Commun. 1978; 83: 479-485Crossref PubMed Scopus (181) Google Scholar), and trihydroxycoprostanoyl-CoA oxidase (29% identity), which oxidizes the CoA esters of the bile acid intermediates dihydroxycoprostanic acid and trihydroxycoprostanic acid (29Van Veldhoven P.P. Vanhove G. Asselberghs S. Eyssen H.J. Mannaerts G.P. J. Biol. Chem. 1992; 267: 20065-20074Abstract Full Text PDF PubMed Google Scholar). On the contrary, plant peroxisomes seem to contain ACOXs that are active on short, medium, and long chain acyl-CoAs (11Hooks M.A. Bode K. Couée I. Biochem. J. 1996; 320: 607-614Crossref PubMed Scopus (32) Google Scholar) and are able to perform a complete β-oxidation of fatty acids to acetyl-CoA (2Huang A.H.C. Trelease R.N. Moore T.S. Plant Peroxisome. Academic Press, New York1983Google Scholar). Three plant ACOX isozymes have previously been purified and characterized. One is from cucumber cotyledons that is active on long and medium chain acyl-CoAs and that is a homodimer with subunits of 72 kDa (10Kirsch T. Löffler H.-G. Kindl H. J. Biol. Chem. 1986; 261: 8570-8575Abstract Full Text PDF PubMed Google Scholar). The other two are from maize and are active on medium and short chain acyl-CoAs, respectively (11Hooks M.A. Bode K. Couée I. Biochem. J. 1996; 320: 607-614Crossref PubMed Scopus (32) Google Scholar). The former is a monomeric enzyme of 62 kDa, and the latter is a homotetrameric enzyme of 15 kDa. Three different genes seem to code for the three ACOX isoforms, as they have different subunit molecular weights (11Hooks M.A. Bode K. Couée I. Biochem. J. 1996; 320: 607-614Crossref PubMed Scopus (32) Google Scholar). The report by Hooks et al. (11Hooks M.A. Bode K. Couée I. Biochem. J. 1996; 320: 607-614Crossref PubMed Scopus (32) Google Scholar) was the first to imply the presence of a short chain ACOX in eukaryotic cells. Mammalian ACOX isoforms, nevertheless, show slightly different substrate preferences and seem to have very similar subunit molecular weights of about 75 kDa. Therefore, only the plant long chain ACOX should share common ancestral genes with the mammalian ACOXs.In the present study, we were able to correlate the sequence of the isolated ACOX clone with a long chain specific ACOX by applying an antiserum against the expressed ACOX/histidine-tagged fusion protein. This antiserum recognized only long chain and medium chain ACOX activity and not short chain ACOX activity when pumpkin enzymes were separated by hydrophobic interaction chromatography (Fig. 4). The immunoreactive band corresponded to a molecular mass of 73 kDa in accordance with the calculated molecular mass of mature pumpkin long chain ACOX (72,414 Da) and with the previous report of 72 kDa for the cucumber long chain ACOX (10Kirsch T. Löffler H.-G. Kindl H. J. Biol. Chem. 1986; 261: 8570-8575Abstract Full Text PDF PubMed Google Scholar).The levels of ACOX mRNA do not seem to be greatly controlled by light. The ACOX protein that built up during the initial 5 days of germination disappeared during the transition from glyoxysomes to leaf peroxisomes upon exposure of the seedlings to light. Similar patterns have previously been observed for malate synthase (8Mori H. Nishimura M. FEBS Lett. 1989; 244: 163-166Crossref Scopus (37) Google Scholar) and citrate synthase (9Kato A. Hayashi M. Mori H. Nishimura M. Plant Mol. Biol. 1995; 27: 377-390Crossref PubMed Scopus (64) Google Scholar). The appearance and disappearance of the mRNAs preceded the change in the ACOX protein during the microbody transition. Thus, the ACOX levels seem to be determined at both the translational and posttranslational levels.It is worth noting that pumpkin glyoxysomal long chain ACOX proteins are synthesized as larger precursors containing a cleavable amino-terminal presequence, namely PTS2 (27Kato A. Hayashi M. Kondo M. Nishimura M. Plant Cell. 1996; 8: 1601-1611PubMed Google Scholar, 30Subramani S. J. Biol. Chem. 1996; 271: 32483-32486Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), as in the case for some other plant peroxisomal proteins, such as malate dehydrogenase (23Gietl C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5773-5777Crossref PubMed Scopus (97) Google Scholar, 24Kato A. Takeda-Yoshikawa Y. Hayashi M. Kondo M. Hara-Nishimura I. Nishimura M. Plant Cell Physiol. 1998; 39: 186-195Crossref PubMed Scopus (45) Google Scholar), citrate synthase (9Kato A. Hayashi M. Mori H. Nishimura M. Plant Mol. Biol. 1995; 27: 377-390Crossref PubMed Scopus (64) Google Scholar), and thiolase (7Kato A. Hayashi M. Takeuchi Y. Nishimura M. Plant Mol. Biol. 1996; 22: 843-852Crossref Scopus (77) Google Scholar, 25Preisig-Müller R. Kindle H. Plant Mol. Biol. 1993; 22: 59-66Crossref PubMed Scopus (58) Google Scholar). In all cloned mammalian ACOXs, a carboxyl-terminal signal (PTS1) is present, but there is no PTS2 signal (31Gould S.J. Keller G.-A.A. Subramani S. J. Cell Biol. 1987; 105: 2923-2931Crossref PubMed Scopus (359) Google Scholar). This indicates that the plant ACOX import mechanism differs from the mammalian one. It has been suggested that ACOX is a key enzyme of β-oxidation because it can control and regulate the flux of acyl-CoAs at the first step of the β-oxidation spiral (32Kindl H. Stumpf P.K. Conn E.E. The Biochemistry of Plants. 9. Academic Press, New York1987: 31-52Google Scholar). Particularly, the long chain acyl-CoA oxidase may represent a regulatory point considering the fact that most fatty acids of plant storage lipids are long chain molecules. In conclusion, this type of control mechanism could tightly regulate the long chain ACOX (as the first step of the β-oxidation cascade), or it could be involved in a coordinate or differential regulation of the expression of the three ACOX enzymes in plant tissues (11Hooks M.A. Bode K. Couée I. Biochem. J. 1996; 320: 607-614Crossref PubMed Scopus (32) Google Scholar). To verify such a hypothesis, the cloning and an expression analysis of the two other ACOXs will be necessary. There are at least three types of microbodies in higher plants (glyoxysomes, leaf peroxisomes, and unspecialized microbodies) that are distinguishable by their enzyme complements (1Beevers H. Annu. Rev. Plant Physiol. 1979; 30: 159-193Crossref Google Scholar, 2Huang A.H.C. Trelease R.N. Moore T.S. Plant Peroxisome. Academic Press, New York1983Google Scholar). During the postgerminative growth of pumpkin seedlings and upon exposure to light, etiolated cotyledons turn green; at the same time, a functional transition from glyoxysomes to leaf peroxisomes occurs (3Titus D.E. Becker W.M. J. Cell Biol. 1985; 101: 1288-1299Crossref PubMed Scopus (107) Google Scholar, 4Nishimura M. Yamaguchi J. Mori H. Akazawa T. Yokota S. Plant Physiol. 1986; 81: 313-316Crossref PubMed Google Scholar). In fat-storing seeds of plants such as pumpkin, lipid bodies are present in seed cells that store triacylglycerols, which are subsequently converted to fatty acids by lipase. Fatty acids represent the main energy and carbon sources for germinating seedlings. In glyoxysomes, fatty acids are degraded to acetyl-CoA via the β-oxidation pathway, and acetyl-CoA is metabolized by the glyoxylate cycle bypassing the decarboxylating steps of the Krebs cycle. We have shown previously that the expression of glyoxysomal enzymes and leaf peroxisomal enzymes are regulated not only at the transcriptional level but also at the posttranscriptional level during the microbody transition (5Mori H. Takeda-Yoshikawa Y. Hara-Nishimura I. Nishimura M. Eur. J. Biochem. 1991; 197: 331-336Crossref PubMed Scopus (68) Google Scholar, 6Tsugeki R. Hara-Nishimura I. Mori H. Nishimura M. Plant Cell Physiol. 1993; 34: 51-57PubMed Google Scholar). The gene expressions of the enzymes of the β-oxidation and glyoxylate cycles seem to be coordinately regulated. In a recent paper, we reported the nucleotide and deduced amino acid sequences of the cDNA for 3-ketoacyl-CoA thiolase (7Kato A. Hayashi M. Takeuchi Y. Nishimura M. Plant Mol. Biol. 1996; 22: 843-852Crossref Scopus (77) Google Scholar). The time course for thiolase mRNA and thiolase levels during germination and postgerminative growth implied that the regulation of expression of this enzyme is similar to that of glyoxylate cycle enzymes, e.g. malate synthase (8Mori H. Nishimura M. FEBS Lett. 1989; 244: 163-166Crossref Scopus (37) Google Scholar) and citrate synthase (9Kato A. Hayashi M. Mori H. Nishimura M. Plant Mol. Biol. 1995; 27: 377-390Crossref PubMed Scopus (64) Google Scholar). The glyoxysomal β-oxidation spiral consists of three different proteins: acyl-CoA oxidase (ACOX), 1The abbreviations used are: ACOX, acyl-CoA oxidase; PTS, peroxisomal targeting signal; thiolase, 3-ketoacyl-CoA thiolase; PRISCOX, pristanoyl-CoA oxidase; MOPS, 3-(N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: ACOX, acyl-CoA oxidase; PTS, peroxisomal targeting signal; thiolase, 3-ketoacyl-CoA thiolase; PRISCOX, pristanoyl-CoA oxidase; MOPS, 3-(N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis. enoyl-CoA hydratase/3-hydroxy acyl-CoA dehydrogenase (bifunctional protein), and 3-ketoacyl-CoA thiolase (thiolase). ACOX converts acyl-CoA into trans-2-enoyl-CoA in the first step of the β-oxidation spiral and corresponds to the acyl-CoA dehydrogenase present in mitochondria of mammalian cells. Both enzymes are flavoproteins. Some plant ACOXs have been purified and characterized (10Kirsch T. Löffler H.-G. Kindl H. J. Biol. Chem. 1986; 261: 8570-8575Abstract Full Text PDF PubMed Google Scholar, 11Hooks M.A. Bode K. Couée I. Biochem. J. 1996; 320: 607-614Crossref PubMed Scopus (32) Google Scholar) and have been shown to have different substrate specificities (for long, medium, and short chain acyl-CoAs, respectively). To further investigate the β-oxidation enzymes at the molecular level, we cloned a cDNA coding for a long chain ACOX, which is localized in glyoxysomes. Here, we report the nucleotide and deduced amino acid sequences of the cDNA. Developmental changes in the level of mRNA and protein were also determined in pumpkin cotyledons during seed germination and subsequent postgerminative growth. DISCUSSIONIn the present study, we report the cDNA sequence of a pumpkin glyoxysomal long chain ACOX in addition to the cDNA sequence of a previously reported Phalaenopsis ACOX (21Do Y.Y. Huang P.L. Arch. Biochem. Biophys. 1997; 344: 295-300Crossref PubMed Scopus (15) Google Scholar).The present results clearly show that the protein encoded by this gene is a plant long chain ACOX. The deduced amino acid sequences of pumpkin and Phalaenopsis cDNA sequences have an identity of 76%, indicating that the Phalaenopsis cDNA also codes for a long chain ACOX. Comparing other ACOXs, the best identity (30%) is obtained for the rat pristanoyl-CoA oxidase, which acts on 2-methyl-branched CoA-esters and straight long chain acyl-CoAs (22Van Veldhoven P.P. Van Rompuy J.C.T. Fransen M. De Béthune B. Mannaerts G.P. Eur. J. Biochem. 1994; 222: 795-801Crossref PubMed Scopus (39) Google Scholar). Mammalian peroxisomes contain three ACOX isozymes that are not capable of oxidizing acyl-chain CoA esters of less than 8 carbons. In mammalian cells, the β-oxidation of short chain fatty acids is accomplished in mitochondria, in which acyl-CoA dehydrogenases act instead of ACOXs. Three peroxisomal mammalian ACOXs have been identified: PRISCOX (30% identity), palmitoyl-CoA oxidase (28% identity), which reacts with CoA esters of very long, long, and medium chain fatty acids (28Osumi T. Hashimoto T. Biochem. Biophys. Res. Commun. 1978; 83: 479-485Crossref PubMed Scopus (181) Google Scholar), and trihydroxycoprostanoyl-CoA oxidase (29% identity), which oxidizes the CoA esters of the bile acid intermediates dihydroxycoprostanic acid and trihydroxycoprostanic acid (29Van Veldhoven P.P. Vanhove G. Asselberghs S. Eyssen H.J. Mannaerts G.P. J. Biol. Chem. 1992; 267: 20065-20074Abstract Full Text PDF PubMed Google Scholar). On the contrary, plant peroxisomes seem to contain ACOXs that are active on short, medium, and long chain acyl-CoAs (11Hooks M.A. Bode K. Couée I. Biochem. J. 1996; 320: 607-614Crossref PubMed Scopus (32) Google Scholar) and are able to perform a complete β-oxidation of fatty acids to acetyl-CoA (2Huang A.H.C. Trelease R.N. Moore T.S. Plant Peroxisome. Academic Press, New York1983Google Scholar). Three plant ACOX isozymes have previously been purified and characterized. One is from cucumber cotyledons that is active on long and medium chain acyl-CoAs and that is a homodimer with subunits of 72 kDa (10Kirsch T. Löffler H.-G. Kindl H. J. Biol. Chem. 1986; 261: 8570-8575Abstract Full Text PDF PubMed Google Scholar). The other two are from maize and are active on medium and short chain acyl-CoAs, respectively (11Hooks M.A. Bode K. Couée I. Biochem. J. 1996; 320: 607-614Crossref PubMed Scopus (32) Google Scholar). The former is a monomeric enzyme of 62 kDa, and the latter is a homotetrameric enzyme of 15 kDa. Three different genes seem to code for the three ACOX isoforms, as they have different subunit molecular weights (11Hooks M.A. Bode K. Couée I. Biochem. J. 1996; 320: 607-614Crossref PubMed Scopus (32) Google Scholar). The report by Hooks et al. (11Hooks M.A. Bode K. Couée I. Biochem. J. 1996; 320: 607-614Crossref PubMed Scopus (32) Google Scholar) was the first to imply the presence of a short chain ACOX in eukaryotic cells. Mammalian ACOX isoforms, nevertheless, show slightly different substrate preferences and seem to have very similar subunit molecular weights of about 75 kDa. Therefore, only the plant long chain ACOX should share common ancestral genes with the mammalian ACOXs.In the present study, we were able to correlate the sequence of the isolated ACOX clone with a long chain specific ACOX by applying an antiserum against the expressed ACOX/histidine-tagged fusion protein. This antiserum recognized only long chain and medium chain ACOX activity and not short chain ACOX activity when pumpkin enzymes were separated by hydrophobic interaction chromatography (Fig. 4). The immunoreactive band corresponded to a molecular mass of 73 kDa in accordance with the calculated molecular mass of mature pumpkin long chain ACOX (72,414 Da) and with the previous report of 72 kDa for the cucumber long chain ACOX (10Kirsch T. Löffler H.-G. Kindl H. J. Biol. Chem. 1986; 261: 8570-8575Abstract Full Text PDF PubMed Google Scholar).The levels of ACOX mRNA do not seem to be greatly controlled by light. The ACOX protein that built up during the initial 5 days of germination disappeared during the transition from glyoxysomes to leaf peroxisomes upon exposure of the seedlings to light. Similar patterns have previously been observed for malate synthase (8Mori H. Nishimura M. FEBS Lett. 1989; 244: 163-166Crossref Scopus (37) Google Scholar) and citrate synthase (9Kato A. Hayashi M. Mori H. Nishimura M. Plant Mol. Biol. 1995; 27: 377-390Crossref PubMed Scopus (64) Google Scholar). The appearance and disappearance of the mRNAs preceded the change in the ACOX protein during the microbody transition. Thus, the ACOX levels seem to be determined at both the translational and posttranslational levels.It is worth noting that pumpkin glyoxysomal long chain ACOX proteins are synthesized as larger precursors containing a cleavable amino-terminal presequence, namely PTS2 (27Kato A. Hayashi M. Kondo M. Nishimura M. Plant Cell. 1996; 8: 1601-1611PubMed Google Scholar, 30Subramani S. J. Biol. Chem. 1996; 271: 32483-32486Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), as in the case for some other plant peroxisomal proteins, such as malate dehydrogenase (23Gietl C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5773-5777Crossref PubMed Scopus (97) Google Scholar, 24Kato A. Takeda-Yoshikawa Y. Hayashi M. Kondo M. Hara-Nishimura I. Nishimura M. Plant Cell Physiol. 1998; 39: 186-195Crossref PubMed Scopus (45) Google Scholar), citrate synthase (9Kato A. Hayashi M. Mori H. Nishimura M. Plant Mol. Biol. 1995; 27: 377-390Crossref PubMed Scopus (64) Google Scholar), and thiolase (7Kato A. Hayashi M. Takeuchi Y. Nishimura M. Plant Mol. Biol. 1996; 22: 843-852Crossref Scopus (77) Google Scholar, 25Preisig-Müller R. Kindle H. Plant Mol. Biol. 1993; 22: 59-66Crossref PubMed Scopus (58) Google Scholar). In all cloned mammalian ACOXs, a carboxyl-terminal signal (PTS1) is present, but there is no PTS2 signal (31Gould S.J. Keller G.-A.A. Subramani S. J. Cell Biol. 1987; 105: 2923-2931Crossref PubMed Scopus (359) Google Scholar). This indicates that the plant ACOX import mechanism differs from the mammalian one. It has been suggested that ACOX is a key enzyme of β-oxidation because it can control and regulate the flux of acyl-CoAs at the first step of the β-oxidation spiral (32Kindl H. Stumpf P.K. Conn E.E. The Biochemistry of Plants. 9. Academic Press, New York1987: 31-52Google Scholar). Particularly, the long chain acyl-CoA oxidase may represent a regulatory point considering the fact that most fatty acids of plant storage lipids are long chain molecules. In conclusion, this type of control mechanism could tightly regulate the long chain ACOX (as the first step of the β-oxidation cascade), or it could be involved in a coordinate or differential regulation of the expression of the three ACOX enzymes in plant tissues (11Hooks M.A. Bode K. Couée I. Biochem. J. 1996; 320: 607-614Crossref PubMed Scopus (32) Google Scholar). To verify such a hypothesis, the cloning and an expression analysis of the two other ACOXs will be necessary. In the present study, we report the cDNA sequence of a pumpkin glyoxysomal long chain ACOX in addition to the cDNA sequence of a previously reported Phalaenopsis ACOX (21Do Y.Y. Huang P.L. Arch. Biochem. Biophys. 1997; 344: 295-300Crossref PubMed Scopus (15) Google Scholar). The present results clearly show that the protein encoded by this gene is a plant long chain ACOX. The deduced amino acid sequences of pumpkin and Phalaenopsis cDNA sequences have an identity of 76%, indicating that the Phalaenopsis cDNA also codes for a long chain ACOX. Comparing other ACOXs, the best identity (30%) is obtained for the rat pristanoyl-CoA oxidase, which acts on 2-methyl-branched CoA-esters and straight long chain acyl-CoAs (22Van Veldhoven P.P. Van Rompuy J.C.T. Fransen M. De Béthune B. Mannaerts G.P. Eur. J. Biochem. 1994; 222: 795-801Crossref PubMed Scopus (39) Google Scholar). Mammalian peroxisomes contain three ACOX isozymes that are not capable of oxidizing acyl-chain CoA esters of less than 8 carbons. In mammalian cells, the β-oxidation of short chain fatty acids is accomplished in mitochondria, in which acyl-CoA dehydrogenases act instead of ACOXs. Three peroxisomal mammalian ACOXs have been identified: PRISCOX (30% identity), palmitoyl-CoA oxidase (28% identity), which reacts with CoA esters of very long, long, and medium chain fatty acids (28Osumi T. Hashimoto T. Biochem. Biophys. Res. Commun. 1978; 83: 479-485Crossref PubMed Scopus (181) Google Scholar), and trihydroxycoprostanoyl-CoA oxidase (29% identity), which oxidizes the CoA esters of the bile acid intermediates dihydroxycoprostanic acid and trihydroxycoprostanic acid (29Van Veldhoven P.P. Vanhove G. Asselberghs S. Eyssen H.J. Mannaerts G.P. J. Biol. Chem. 1992; 267: 20065-20074Abstract Full Text PDF PubMed Google Scholar). On the contrary, plant peroxisomes seem to contain ACOXs that are active on short, medium, and long chain acyl-CoAs (11Hooks M.A. Bode K. Couée I. Biochem. J. 1996; 320: 607-614Crossref PubMed Scopus (32) Google Scholar) and are able to perform a complete β-oxidation of fatty acids to acetyl-CoA (2Huang A.H.C. Trelease R.N. Moore T.S. Plant Peroxisome. Academic Press, New York1983Google Scholar). Three plant ACOX isozymes have previously been purified and characterized. One is from cucumber cotyledons that is active on long and medium chain acyl-CoAs and that is a homodimer with subunits of 72 kDa (10Kirsch T. Löffler H.-G. Kindl H. J. Biol. Chem. 1986; 261: 8570-8575Abstract Full Text PDF PubMed Google Scholar). The other two are from maize and are active on medium and short chain acyl-CoAs, respectively (11Hooks M.A. Bode K. Couée I. Biochem. J. 1996; 320: 607-614Crossref PubMed Scopus (32) Google Scholar). The former is a monomeric enzyme of 62 kDa, and the latter is a homotetrameric enzyme of 15 kDa. Three different genes seem to code for the three ACOX isoforms, as they have different subunit molecular weights (11Hooks M.A. Bode K. Couée I. Biochem. J. 1996; 320: 607-614Crossref PubMed Scopus (32) Google Scholar). The report by Hooks et al. (11Hooks M.A. Bode K. Couée I. Biochem. J. 1996; 320: 607-614Crossref PubMed Scopus (32) Google Scholar) was the first to imply the presence of a short chain ACOX in eukaryotic cells. Mammalian ACOX isoforms, nevertheless, show slightly different substrate preferences and seem to have very similar subunit molecular weights of about 75 kDa. Therefore, only the plant long chain ACOX should share common ancestral genes with the mammalian ACOXs. In the present study, we were able to correlate the sequence of the isolated ACOX clone with a long chain specific ACOX by applying an antiserum against the expressed ACOX/histidine-tagged fusion protein. This antiserum recognized only long chain and medium chain ACOX activity and not short chain ACOX activity when pumpkin enzymes were separated by hydrophobic interaction chromatography (Fig. 4). The immunoreactive band corresponded to a molecular mass of 73 kDa in accordance with the calculated molecular mass of mature pumpkin long chain ACOX (72,414 Da) and with the previous report of 72 kDa for the cucumber long chain ACOX (10Kirsch T. Löffler H.-G. Kindl H. J. Biol. Chem. 1986; 261: 8570-8575Abstract Full Text PDF PubMed Google Scholar). The levels of ACOX mRNA do not seem to be greatly controlled by light. The ACOX protein that built up during the initial 5 days of germination disappeared during the transition from glyoxysomes to leaf peroxisomes upon exposure of the seedlings to light. Similar patterns have previously been observed for malate synthase (8Mori H. Nishimura M. FEBS Lett. 1989; 244: 163-166Crossref Scopus (37) Google Scholar) and citrate synthase (9Kato A. Hayashi M. Mori H. Nishimura M. Plant Mol. Biol. 1995; 27: 377-390Crossref PubMed Scopus (64) Google Scholar). The appearance and disappearance of the mRNAs preceded the change in the ACOX protein during the microbody transition. Thus, the ACOX levels seem to be determined at both the translational and posttranslational levels. It is worth noting that pumpkin glyoxysomal long chain ACOX proteins are synthesized as larger precursors containing a cleavable amino-terminal presequence, namely PTS2 (27Kato A. Hayashi M. Kondo M. Nishimura M. Plant Cell. 1996; 8: 1601-1611PubMed Google Scholar, 30Subramani S. J. Biol. Chem. 1996; 271: 32483-32486Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), as in the case for some other plant peroxisomal proteins, such as malate dehydrogenase (23Gietl C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5773-5777Crossref PubMed Scopus (97) Google Scholar, 24Kato A. Takeda-Yoshikawa Y. Hayashi M. Kondo M. Hara-Nishimura I. Nishimura M. Plant Cell Physiol. 1998; 39: 186-195Crossref PubMed Scopus (45) Google Scholar), citrate synthase (9Kato A. Hayashi M. Mori H. Nishimura M. Plant Mol. Biol. 1995; 27: 377-390Crossref PubMed Scopus (64) Google Scholar), and thiolase (7Kato A. Hayashi M. Takeuchi Y. Nishimura M. Plant Mol. Biol. 1996; 22: 843-852Crossref Scopus (77) Google Scholar, 25Preisig-Müller R. Kindle H. Plant Mol. Biol. 1993; 22: 59-66Crossref PubMed Scopus (58) Google Scholar). In all cloned mammalian ACOXs, a carboxyl-terminal signal (PTS1) is present, but there is no PTS2 signal (31Gould S.J. Keller G.-A.A. Subramani S. J. Cell Biol. 1987; 105: 2923-2931Crossref PubMed Scopus (359) Google Scholar). This indicates that the plant ACOX import mechanism differs from the mammalian one. It has been suggested that ACOX is a key enzyme of β-oxidation because it can control and regulate the flux of acyl-CoAs at the first step of the β-oxidation spiral (32Kindl H. Stumpf P.K. Conn E.E. The Biochemistry of Plants. 9. Academic Press, New York1987: 31-52Google Scholar). Particularly, the long chain acyl-CoA oxidase may represent a regulatory point considering the fact that most fatty acids of plant storage lipids are long chain molecules. In conclusion, this type of control mechanism could tightly regulate the long chain ACOX (as the first step of the β-oxidation cascade), or it could be involved in a coordinate or differential regulation of the expression of the three ACOX enzymes in plant tissues (11Hooks M.A. Bode K. Couée I. Biochem. J. 1996; 320: 607-614Crossref PubMed Scopus (32) Google Scholar). To verify such a hypothesis, the cloning and an expression analysis of the two other ACOXs will be necessary. We thank Prof. Dr. Claus Schnarrenberger (Free University of Berlin) for stimulating discussions and helpful comments on the manuscript.