Import of alcohol oxidase into peroxisomes of Saccharomyces cerevisiae.

Saccharomyces cerevisiae is unable to grow on methanol because it lacks the enzymes required for its metabolism. To study the possibility of whether or not the methanol oxidation pathway of Hansenula polymorpha can be transferred to S. cerevisiae, the gene coding for alcohol oxidase, a peroxisomal homo-octameric flavoprotein, was introduced into S. cerevisiae. Transformed cells contain varying amounts of alcohol oxidase depending on the plasmid used. Immunocytochemical experiments indicate that the protein is imported into peroxisomes, whether organelle proliferation is induced or not. Cells lack alcohol oxidase activity however, and only the monomeric, non-functional, form of the protein is found. These findings indicate that the H. polymorpha peroxisomal targeting signal of alcohol oxidase is recognized in S. cerevisiae and protein monomers are imported.     

Suramin prevents import of acyl-CoA oxidase into rat liver peroxisomes.

The inhibitory effect of suramin on the import of [35S]acyl-CoA oxidase into purified rat liver peroxisomes was investigated in vitro. The import of acyl-CoA oxidase was inhibited completely by 10 microM suramin, whilst the latency of catalase remained unchanged. The important value decreased 60% by pretreatment of peroxisomes with 10 microM suramin, but it did not decrease by pretreatment of translation products. Polysulfonate compounds which have two clusters of negative charges, such as Cibacron blue F3GA and Trypan blue, as well as suramin, inhibited the import, whilst mono- and disulfonate compounds did not.     

Acyl-CoA oxidase from Candida tropicalis

The preparation of a highly purified acyl-CoA oxidase from the cell extract of an n-alkane-utilizing yeast, Candida tropicalis, is described. It can be crystallized from ammonium sulfate solutions without an increase in specific activity, and is homogeneous on ultracentrifuge and disc electrophoresis. The enzyme is an octamer with approximately a 600,000 molecular weight, and has an isoelectric point of 5.5. It exhibits a typical flavoprotein spectrum with absorption maxima at 277, 365 and 445 nm, and contains 8 mol of FAD per mol of enzyme. The enzyme catalyzes the stoichiometric conversion of palmitoyl-CoA and O₂ into 2-hexadecenoyl-CoA and H₂O₂. It oxidizes acyl-CoAs with carbon chain lengths of 4 to 20, and is most active toward lauroyl-CoA, but acetyl- and succinyl-CoAs are not oxidized. The enzyme is sulfhydryl dependent and is inactivated by silver and mercury compounds.     

Acyl-CoA oxidase from Candida tropicalis

Acyl coenzyme A oxidase (acyl-CoA oxidase) has been isolated in good yield from Candida tropicalis pK 233 grown on n-alkanes. Gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measurement of flavin content suggest that the oxidase is an octamer of Mr 75 000 subunits each containing one flavin. The oxidase yields the red semiquinone form on dithionite or photochemical reduction, slowly forms an N-5 adduct with 0.16 M sulfite at pH 7.4, and is rapidly reduced by borohydride, forming the 3,4-dihydroflavin isomer. The red flavosemiquinone is only kinetically stabilized with respect to disproportionation in the free enzyme but is thermodynamically stabilized on binding enoyl-CoA derivatives. The enzyme is reduced by butyryl-, octanoyl-, and palmitoyl-CoA without formation of prominent long-wavelength bands. Acyl-CoA oxidase and the acyl-CoA dehydrogenases share many similarities in their interaction with CoA derivatives. For example, both enzymes stabilize the anionic radical on binding enoyl-CoA derivatives, both dehydrogenate 2-oxoheptadecyldethio-CoA but cannot utilize S-heptadecyl-CoA, both form long-wavelength bands with CoA persulfide species, and both enzymes are attacked by the suicide substrates 3,4-pentadienoyl-CoA and (methylene-cyclopropyl)acetyl-CoA at the flavin prosthetic group.     

The primary structure of a peroxisomal fatty acyl-CoA oxidase from the yeast Candida tropicalis pK233

We report the isolation and nucleotide (nt) sequence determination of a gene encoding peroxisomal fatty acyl-CoA oxidase (AOx) from the yeast Candida tropicalis pK233. The AOx gene contains no intervening sequences and has a single open reading frame of 2127 nt encoding a protein of 708 amino acids (aa), not including the initiator methionine. The Mr of the protein is 79,155. Codon utilization in the gene is not random, with 87.4% of the aa specified by 25 principal codons. The principal codons used in the expression of AOx in C. tropicalis are similar to those used in highly expressed genes of Saccharomyces cerevisiae. The AOx protein shows a 94.2% homology with POX4 protein of C. tropicalis. One stretch of 36 aa shows no homology between the two proteins.     

The reductive half-reaction in acyl-CoA oxidase from Candida tropicalis: interaction with acyl-CoA analogues and an unusual thioesterase activity

A series of acyl-CoA analogues has been used to probe the substrate binding site and reductive half-reaction of acyl-CoA oxidase from the alkane utilizing yeast Candida tropicalis. Alkyl-SCoA thioethers, from octyl- to hexadecyl-SCoA, bind to the oxidase with progressively larger spectral perturbation of the flavin chromophore and with an incremental binding energy of about 260 cal/methylene group. The hydrocarbon binding subsite for acyl-CoA oxidase appears extensive and only weakly hydrophobic. CoA binding per se appears to contribute about 2.8 kcal to the observed binding energy. A number of acyl-CoA analogues such as 3-thia-acyl-, 3-oxa-acyl-, trans-3-enoyl-, and 3-keto-acyl-CoA derivatives form charge transfer complexes with the oxidase, but these long wavelength bands are both less pronounced and much less stable than those encountered with the acyl-CoA dehydrogenases. This instability reflects an intrinsic thioesterase activity of the oxidase which is observed with those ligands forming enolate to oxidized flavin charge-transfer complexes, but not with normal substrates such as palmitoyl-CoA. Chemical precedent suggests that these enzyme-bound enolates eliminate CoA via a ketene intermediate. The differences in behavior between acyl-CoA oxidase and dehydrogenase toward the ligands used in this work are discussed in terms of the need to exclude oxygen from productive encounters with substrate-reduced dehydrogenase.     

Absence of DNA in peroxisomes of Candida tropicalis.

Yeast peroxisomes were purified to near homogeneity from cells of Candida tropicalis grown on oleic acid for the purpose of examining the possible presence of DNA in this organelle. The purification procedure includes the effective conversion of cells to spheroplasts with Zymolyase and sodium sulfite and the separation of the organelles at extremely low ionic strength. The mitochondrial contamination was less than 1%, based on several criteria, and the yield of peroxisomes was about 40%. The purified peroxisomal fraction contained a very small amount of DNA, which yielded restriction fragments indistinguishable from those of mitochondrial DNA. The absence of DNA in peroxisomes was also supported by cesium chloride density gradient centrifugation of the organelles lysed with a detergent, staining of the organelles with a fluorescent dye specific to DNA, and labeling of the DNA with [3H]adenine.     

A novel acyl-CoA oxidase that can oxidize short-chain acyl-CoA in plant peroxisomes

Short-chain acyl-CoA oxidases are beta-oxidation enzymes that are active on short-chain acyl-CoAs and that appear to be present in higher plant peroxisomes and absent in mammalian peroxisomes. Therefore, plant peroxisomes are capable of performing complete beta-oxidation of acyl-CoA chains, whereas mammalian peroxisomes can perform beta-oxidation of only those acyl-CoA chains that are larger than octanoyl-CoA (C8). In this report, we have shown that a novel acyl-CoA oxidase can oxidize short-chain acyl-CoA in plant peroxisomes. A peroxisomal short-chain acyl-CoA oxidase from Arabidopsis was purified following the expression of the Arabidopsis cDNA in a baculovirus expression system. The purified enzyme was active on butyryl-CoA (C4), hexanoyl-CoA (C6), and octanoyl-CoA (C8). Cell fractionation and immunocytochemical analysis revealed that the short-chain acyl-CoA oxidase is localized in peroxisomes. The expression pattern of the short-chain acyl-CoA oxidase was similar to that of peroxisomal 3-ketoacyl-CoA thiolase, a marker enzyme of fatty acid beta-oxidation, during post-germinative growth. Although the molecular structure and amino acid sequence of the enzyme are similar to those of mammalian mitochondrial acyl-CoA dehydrogenase, the purified enzyme has no activity as acyl-CoA dehydrogenase. These results indicate that the short-chain acyl-CoA oxidases function in fatty acid beta-oxidation in plant peroxisomes, and that by the cooperative action of long- and short-chain acyl-CoA oxidases, plant peroxisomes are capable of performing the complete beta-oxidation of acyl-CoA.     

Translocation of acyl-CoA oxidase into peroxisomes requires ATP hydrolysis but not a membrane potential

An efficient system for the import of newly synthesized proteins into highly purified rat liver peroxisomes was reconstituted in vitro. 35S- Labeled acyl-CoA oxidase (AOx) was incorporated into peroxisomes in a proteinase K-resistant fashion. This import was specific (did not occur with mitochondria) and was dependent on temperature, time, and peroxisome concentration. Under optimal conditions approximately 30% of [35S]AOx became proteinase resistant. The import of AOx into peroxisomes could be dissociated into two steps: (a) binding occurred at 0 degrees C in the absence of ATP; (b) translocation occurred only at 26 degrees C and required the hydrolysis of ATP. GTP would not substitute for ATP and translocation was not inhibited by carbonylcyanide-m-chlorophenylhydrazone, valinomycin, or other ionophores.     

Import of proteins into peroxisomes.

Peroxisomes are organelles that confine an important set of enzymes within their single membrane boundaries. In man, a wide variety of genetic disorders is caused by loss of peroxisome function. In the most severe cases, the clinical phenotype indicates that abnormalities begin to appear during embryological development. In less severe cases, the quality of life of adults is affected. Research on yeast model systems has contributed to a better understanding of peroxisome formation and maintenance. This framework of knowledge has made it possible to understand the molecular basis of most of the peroxisome biogenesis disorders. Interestingly, most peroxisome biogenesis disorders are caused by a failure to target peroxisomal proteins to the organellar matrix or membrane, which classifies them as protein targeting diseases. Here we review recent fundamental research on peroxisomal protein targeting and discuss a few burning questions in the field concerning the origin of peroxisomes.     

The carboxy-terminal end of glycolate oxidase directs a foreign protein into tobacco leaf peroxisomes.

The carboxy-terminal residues of several peroxisomal proteins were shown to act as a peroxisomal targetting signal. This study was conducted to test whether the C-terminus of glycolate oxidase, a key enzyme in the glycolate metabolism pathway, is functioning as a targetting signal that directs proteins into plant leaf peroxisomes. A chimeric gene coding for a fusion protein composed of the C-terminal-truncated beta-glucuronidase, a synthetic linker of four amino acids and the last six C-terminal amino acids of glycolate oxidase, was constructed. Transformation of tobacco plants with the chimeric gene resulted in expression of beta-glucuronidase enzymic activity. About 50% of the transgenic beta-glucuronidase activity was localized to the peroxisomes. The results indicate that the six C-terminal amino acid residues of glycolate oxidase act as a targetting signal that is recognized by leaf peroxisomes.     

Production of adipic acid by short- and long-chain fatty acid acyl-CoA oxidase engineered in yeast Candida tropicalis

Bioprocess and Biosystems Engineering - In this study, to produce adipic acid, mutant strains of Candida tropicalis KCTC 7212 deficient of AOX genes encoding acyl-CoA oxidases which are important...     

Post-translational import of fatty acyl-CoA oxidase and catalase into peroxisomes of rat liver in vitro

Total polysomal RNA of rat liver was translated in vitro in a rabbit reticulocyte lysate system. The translation products were mixed with a postnuclear supernatant fraction of rat liver and incubated post-translationally at 26 degrees C for 15-60 min. The import assay mixture was separated into a particulate fraction and supernatant by centrifugation, both of which were analyzed by immunoprecipitation with a goat antibody against rat liver peroxisomal proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. One peroxisomal translation product (Mr 72,000) appeared in the particulate fraction, was partly proteinase K-resistant, and addition of detergents prior to proteolysis abolished this resistance. In isopycnic centrifugation of the uptake assay mixture, the protease-resistant 35S-polypeptide of Mr 72,000 cosedimented with the peroxisomes. This translation product was identified immunochemically as fatty acyl-CoA oxidase; both before and after import it was indistinguishable in size from subunit A of the purified enzyme by prolonged sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the cell-free translation products were incubated with highly purified peroxisomes, 35S-catalase entered peroxisomes (by the criterion of protease resistance), and its entry was stimulated by the addition of a high speed supernatant (cytosolic) fraction of rat liver. These results demonstrate the post-translational import into peroxisomes in vitro of at least two cell-free translation products.     

Import of stably folded proteins into peroxisomes.

By virtue of their synthesis in the cytoplasm, proteins destined for import into peroxisomes are obliged to traverse the single membrane of this organelle. Because the targeting signal for most peroxisomal matrix proteins is a carboxy-terminal tripeptide sequence (SKL or its variants), these proteins must remain import competent until their translation is complete. We sought to determine whether stably folded proteins were substrates for peroxisomal import. Prefolded proteins stabilized with disulfide bonds and chemical cross-linkers were shown to be substrates for peroxisomal import, as were mature folded and disulfide-bonded IgG molecules containing the peroxisomal targeting signal. In addition, colloidal gold particles conjugated to proteins bearing the peroxisomal targeting signal were translocated into the peroxisomal matrix. These results support the concept that proteins may fold in the mammalian cytosol, before their import into the peroxisome, and that protein unfolding is not a prerequisite for peroxisomal import.     

Presence of three acyl-CoA oxidases in rat liver peroxisomes. An inducible fatty acyl-CoA oxidase, a noninducible fatty acyl-CoA oxidase, and a noninducible trihydroxycoprostanoyl-CoA oxidase

Mammalian liver peroxisomes are capable of beta-oxidizing a variety of substrates including very long chain fatty acids and the side chains of the bile acid intermediates di- and trihydroxycoprostanic acid. The first enzyme of peroxisomal beta-oxidation is acyl-CoA oxidase. It remains unknown whether peroxisomes possess one or several acyl-CoA oxidases. Peroxisomal oxidases from rat liver were partially purified by (NH4)2SO4 precipitation and heat treatment, and the preparation was subjected to chromatofocusing, chromatography on hydroxylapatite and dye affinity matrices, and gel filtration. The column eluates were assayed for palmitoyl-CoA and trihydroxycoprostanoyl-CoA oxidase activities and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results revealed the presence of three acyl-CoA oxidases: 1) a fatty acyl-CoA oxidase with a pI of 8.3 and an apparent molecular mass of 145 kDa. The enzyme consisted mainly of 52- and 22.5-kDa subunits and could be induced by clofibrate treatment; 2) a noninducible fatty acyl-CoA oxidase with a pI of 7.1 and an apparent molecular mass of 427 kDa. It consisted mainly, if not exclusively, of one polypeptide component of 71 kDa; and 3) a noninducile trihydroxycoprostanoyl-CoA oxidase with a pI of 7.1 and an apparent molecular mass of 139 kDa. It consisted mainly, if not exclusively, of one polypeptide component of 69 kDa. Our findings are probably related to the recent discovery of two species of acyl-CoA oxidase mRNA in rat liver (Miyazawa, S., Hayashi, H., Hijikata, M., Ishii, N., Furata, S., Kagamiyama, H., Osumi, T., and Hashimoto, T. (1987) J. Biol. Chem. 262, 8131-8137) and they probably also explain why in human peroxisomal beta-oxidation defects an accumulation of very long chain fatty acids is not always accompanied by an excretion of bile acid intermediates and vice versa.     

Inducible long chain alcohol oxidase from alkane-grown Candida tropicalis

Summary The oxidation of primary aliphatic alcohols by microsomal membrane fractions of alkane grown Candida tropicalis was shown to be due to the action of an inducible alcohol oxidase with a wide substrate specificity towards aliphatic alcohols. Stoichiometric studies showed that NADH production, in the presence of fatty alcohols, was due to the activity of an inducible fatty aldehyde dehydrogenase. The oxidase activity could be measured directly by hydrogen peroxide production via a peroxidase and a chromogenic redox indicator.     

Immunocytochemical localization of urate oxidase, fatty acyl-CoA oxidase, and catalase in bovine kidney peroxisomes

We investigated the localization of urate oxidase, peroxisomal fatty acyl-CoA oxidase, and catalase in bovine kidney by immunoblot analysis and protein A-gold immunocytochemistry, using the respective polyclonal monospecific antibodies raised against the enzymes purified from rat liver. By immunoblot analysis, these three proteins were detected in bovine kidney and bovine liver homogenates. Subcellular localization of these three enzymes in kidney was ascertained by protein A-gold immunocytochemical staining of Lowicryl K4M-embedded tissue. Peroxisomes in bovine kidney cortical epithelium possessed crystalloid cores or nucleoids, which were found to be the exclusive sites of urate oxidase localization. The limiting membrane, the marginal plate, and the matrix of renal peroxisomes were negative for urate oxidase staining. In contrast, catalase and fatty acyl-CoA oxidase were found in the peroxisome matrix. These results demonstrate that, unlike rat kidney peroxisomes which lack urate oxidase, peroxisomes of bovine kidney contain this enzyme as well as peroxisomal fatty acyl-CoA oxidase.     

Immunochemically distinct NADP-linked isocitrate dehydrogenase isozymes in mitochondria and peroxisomes of Candida tropicalis

Although peroxisomal localization of NADP-linked isocitrate dehydrogenase (Idp) was first demonstrated in Candida tropicalis, the mitochondrial isozyme has not been found in this yeast. Here we report that the presence of mitochondrial Idp in the yeast was demonstrated by screening for its gene with a DNA probe containing conserved sequences of Idps from various organisms. The nucleotide sequence of the gene (CtIDP1) revealed a 1,290-bp open reading frame corresponding to a 430-amino-acid protein with a high similarity to previously reported Idps. Overexpression of CtIDP1 in Saccharomyces cerevisiae gave a high intracellular Idp activity, and the purified recombinant Idp was shown to be a homodimer with a subunit molecular mass of approximately 44 kDa, different from that of peroxisomal Idp (45 kDa) previously purified from C. tropicalis. Western blot analysis of the subcellular fractions from acetate-grown C. tropicalis with polyclonal antibodies raised against the recombinant CtIdp1 showed that the CtIdp1 in C. tropicalis was localized in mitochondria but not in peroxisomes. Similar levels of CtIDP1 mRNA and its protein product were detected in cells grown on glucose, acetate, and n-alkane, although a slight decrease was observed in n-alkane-grown cells. From these results, CtIdp1 was demonstrated to be mitochondrial Idp. The properties of mitochondrial Idp and peroxisomal Idp isozymes were proven to be similar, but they were immunochemically distinct, suggesting the presence of another gene responsible for peroxisomal Idp in C. tropicalis. Received: 11 March 1997 / Accepted: 24 June 1997