Induction, identification, and cell-free translation of mRNAs coding for peroxisomal proteins in Candida tropicalis

Peroxisomes have been purified from Candida tropicalis grown on oleic acid and shown to be nearly pure by marker enzyme analysis, electron microscopy, and comparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They contain approximately 20 polypeptides, among which acyl-CoA oxidase, trifunctional hydratase-dehydrogenase-epimerase, and catalase have been identified. Rabbit antisera have been elicited that react with these three proteins. When C. tropicalis is grown on alkanes, a dozen mRNAs are strikingly induced. Nine of the 12 induced mRNAs code for polypeptides that comigrate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with peroxisomal proteins, among which three have been identified immunochemically as the acyl-CoA oxidase, the trifunctional protein, and catalase. These results indicate that some genes coding for peroxisomal proteins are strongly expressed during growth of C. tropicalis on alkanes. The data are consistent with evidence in other species that peroxisomes form by the post-translational incorporation of newly made proteins into pre-existing peroxisomes, generally without proteolytic processing, followed by peroxisome division.     

The carboxyl-terminal tripeptide Ala-Lys-Ile is essential for targeting Candida tropicalis trifunctional enzyme to yeast peroxisomes.

The gene encoding Candida tropicalis peroxisomal trifunctional enzyme, hydratase-dehydrogenase-epimerase (HDE), was expressed in both Candida albicans and Saccharomyces cerevisiae. The cellular location of HDE was determined by subcellular fractionation followed by Western blot analysis of peroxisomal and cytosolic fractions using antiserum specific for HDE. HDE was found to be exclusively targeted to and imported into peroxisomes in both heterologous expression systems. Deletion and mutational analyses were used to determine the regions within HDE which are essential for its targeting to peroxisomes. Deletion of a carboxyl-terminal tripeptide Ala-Lys-Ile completely abolished targeting of HDE to peroxisomes, whereas large internal deletions of HDE (amino acids 38-353 or 395-731) had no effect on HDE targeting to peroxisomes in either yeast. This tripeptide is similar to, but distinct from, other tripeptide peroxisomal targeting sequences (PTSs) as identified in peroxisomal firefly luciferase and four mammalian peroxisomal proteins. Substitutions within the carboxyl-terminal tripeptide (Ala----Gly and Lys----Gln) supported targeting of HDE to peroxisomes of C. albicans but not of S. cerevisiae. This is the first detailed analysis of the peroxisomal targeting signal in a yeast peroxisomal protein.     

Efficient association of in vitro translation products with purified stable Candida tropicalis peroxisomes.

Newly synthesized peroxisomal proteins enter preexisting peroxisomes posttranslationally in vivo, generally without proteolytic processing. An efficient reconstitution of this process in vitro together with cloned DNAs for peroxisomal proteins would make possible investigation of the molecular information that targets proteins to peroxisomes. We have previously reported the isolation of clones for Candida tropicalis peroxisomal proteins; here we describe the association (and possible import) of peroxisomal proteins with peroxisomes in vitro. C. tropicalis was grown in a medium containing Brij 35, resulting in the induction of a moderate number of medium-sized peroxisomes. These peroxisomes, isolated in a sucrose gradient, had a catalase latency of 54% and were sufficiently stable to be concentrated and used in an import assay. The reticulocyte lysate translation products of total RNA from oleate-grown cells were incubated with the peroxisomes at 26 degrees C in the presence of 50 mM KCl, protease inhibitors, 0.5 M sucrose, 2.5 mM MOPS (morpholinepropanesulfonic acid) (pH 7.2), and 0.5 mM EDTA. Ten major translation products (which could be immunoprecipitated with antiserum against peroxisomal protein) became progressively associated with the peroxisomes during the first 30 min of incubation (some up to approximately 70%). These include acyl coenzyme A oxidase and the trifunctional protein hydratase-dehydrogenase-epimerase. This association did not occur at 4 degrees C nor did it occur if the peroxisomes were replaced with mitochondria.     

Acyl-CoA oxidase contains two targeting sequences each of which can mediate protein import into peroxisomes.

Acyl-CoA oxidase is a major induced enzyme in peroxisomes of Candida tropicalis grown on fatty acids. The gene, POX4, encoding acyl-CoA oxidase was expressed in vitro, and the resulting polypeptide was imported into purified peroxisomes in a temperature-dependent fashion. Plasmids containing fragments of POX4 were prepared, expressed and the polypeptides tested for import into peroxisomes. We identified two regions of acyl-CoA oxidase (amino acids 1-118 and 309-427) that contained information that specifically targeted fragments of acyl-CoA oxidase to peroxisomes. The corresponding regions of the gene were fused to cDNA encoding the cytosolic enzyme dihydrofolate reductase (DHFR), and the expressed fusion proteins were likewise imported into peroxisomes. DHFR itself neither bound to, nor was imported into peroxisomes. Thus, there are at least two regions of peroxisomal targeting information in the acyl-CoA oxidase gene.     

Crystal structure of the bifunctional dihydroneopterin aldolase/6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Streptococcus pneumoniae

The enzymes dihydroneopterin aldolase (DHNA) and 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyse two consecutive steps in the biosynthesis of folic acid. Neither of these enzymes has a counterpart in mammals, and they have therefore been suggested as ideal targets for antimicrobial drugs. Some of the enzymes within the folate pathway can occur as bi- or trifunctional complexes in bacteria and parasites, but the way in which bifunctional DHNA-HPPK enzymes are assembled is unclear. Here, we report the determination of the structure at 2.9 A resolution of the DHNA-HPPK (SulD) bifunctional enzyme complex from the respiratory pathogen Streptococcus pneumoniae. In the crystal, DHNA is assembled as a core octamer, with 422 point group symmetry, although the enzyme is active as a tetramer in solution. Individual HPPK monomers are arranged at the ends of the DHNA octamer, making relatively few contacts with the DHNA domain, but more extensive interactions with adjacent HPPK domains. As a result, the structure forms an elongated cylinder, with the HPPK domains forming two tetramers at each end. The active sites of both enzymes face outward, and there is no clear channel between them that could be used for channelling substrates. The HPPK-HPPK interface accounts for about one-third of the total area between adjacent monomers in SulD, and has levels of surface complementarity comparable to that of the DHNA-DHNA interfaces. There is no "linker" polypeptide between DHNA and HPPK, reducing the conformational flexibility of the HPPK domain relative to the DHNA domain. The implications for the organisation of bi- and trifunctional enzyme complexes within the folate biosynthesis pathway are discussed.     

Conserved Alternative Splicing of Arabidopsis Transthyretin-Like Determines Protein Localization and S-Allantoin Synthesis in Peroxisomes

S-allantoin, a major ureide compound, is produced in plant peroxisomes from oxidized purines. Sequence evidence suggested that the Transthyretin-like (TTL) protein, which interacts with brassinosteroid receptors, may act as a bifunctional enzyme in the synthesis of S-allantoin. Here, we show that recombinant TTL from Arabidopsis thaliana catalyzes two enzymatic reactions leading to the stereoselective formation of S-allantoin, hydrolysis of hydroxyisourate through a C-terminal Urah domain, and decarboxylation of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline through an N-terminal Urad domain. We found that two different mRNAs are produced from the TTL gene through alternative use of two splice acceptor sites. The corresponding proteins differ in the presence (TTL¹⁻) and the absence (TTL²⁻) of a rare internal peroxisomal targeting signal (PTS2). The two proteins have similar catalytic activity in vitro but different in vivo localization: TTL¹⁻ localizes in peroxisomes, whereas TTL²⁻ localizes in the cytosol. Similar splice variants are present in monocots and dicots. TTL originated in green algae through a Urad-Urah fusion, which entrapped an N-terminal PTS2 between the two domains. The presence of this gene in all Viridiplantae indicates that S-allantoin biosynthesis has general significance in plant nitrogen metabolism, while conservation of alternative splicing suggests that this mechanism has general implications in the regulation of the ureide pathway in flowering plants.     

Proteome analysis of Arabidopsis leaf peroxisomes reveals novel targeting peptides, metabolic pathways, and defense mechanisms

We have established a protocol for the isolation of highly purified peroxisomes from mature Arabidopsis thaliana leaves and analyzed the proteome by complementary gel-based and gel-free approaches. Seventy-eight nonredundant proteins were identified, of which 42 novel proteins had previously not been associated with plant peroxisomes. Seventeen novel proteins carried predicted peroxisomal targeting signals (PTS) type 1 or type 2; 11 proteins contained PTS-related peptides. Peroxisome targeting was supported for many novel proteins by in silico analyses and confirmed for 11 representative full-length fusion proteins by fluorescence microscopy. The targeting function of predicted and unpredicted signals was investigated and SSL>, SSI>, and ASL> were established as novel functional PTS1 peptides. In contrast with the generally accepted confinement of PTS2 peptides to the N-terminal domain, the bifunctional transthyretin-like protein was demonstrated to carry internally a functional PTS2. The novel enzymes include numerous enoyl-CoA hydratases, short-chain dehydrogenases, and several enzymes involved in NADP and glutathione metabolism. Seven proteins, including beta-glucosidases and myrosinases, support the currently emerging evidence for an important role of leaf peroxisomes in defense against pathogens and herbivores. The data provide new insights into the biology of plant peroxisomes and improve the prediction accuracy of peroxisome-targeted proteins from genome sequences.     

Heterologous expression and purification of active human phosphoribosylglycinamide formyltransferase as a single domain

We report here for the first time that the GART domain of the human trifunctional enzyme possessing GARS, AIRS, and GART activities can be expressed independently inEscherichia coli at high levels as a stable protein with enzymatic characteristics comparable to those of native trifunctional protein. Human trifunctional enzyme is involved inde novo purine biosynthesis, and has long been recognized as a target for antineoplastic intervention. The GART domain was expressed inE. coli under the control of bacteriophage T7 promotor and isolated by a three-step chromatographic procedure. Two residues, Asp 951 and His 915, were shown to be catalytically crucial by site-directed mutagenesis and subsequent characterization of purified mutant proteins. The active monofunctional GART protein produced inE. coli can serve as a valuable substitute of trifunctional enzyme for structural and functional studies which have been until now hindered because of insufficient quantity, instability, and size of the trifunctional GART protein.     

Homology between hydroxybutyryl and hydroxyacyl coenzyme A dehydrogenase enzymes from Clostridium acetobutylicum fermentation and vertebrate fatty acid beta-oxidation pathways.

The enzymes NAD-dependent beta-hydroxybutyryl coenzyme A dehydrogenase (BHBD) and 3-hydroxyacetyl coenzyme A (3-hydroxyacyl-CoA) dehydrogenase are part of the central fermentation pathways for butyrate and butanol production in the gram-positive anaerobic bacterium Clostridium acetobutylicum and for the beta oxidation of fatty acids in eucaryotes, respectively. The C. acetobutylicum hbd gene encoding a bacterial BHBD was cloned, expressed, and sequenced in Escherichia coli. The deduced primary amino acid sequence of the C. acetobutylicum BHBD showed 45.9% similarity with the equivalent mitochondrial fatty acid beta-oxidation enzyme and 38.4% similarity with the 3-hydroxyacyl-CoA dehydrogenase part of the bifunctional enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase from rat peroxisomes. The pig mitochondrial 3-hydroxyacyl-CoA dehydrogenase showed 31.7% similarity with the 3-hydroxyacyl-CoA dehydrogenase part of the bifunctional enzyme from rat peroxisomes. The phylogenetic relationship between these enzymes supports a common evolutionary origin for the fatty acid beta-oxidation pathways of vertebrate mitochondria and peroxisomes and the bacterial fermentation pathway.     

Import of human bifunctional enzyme into peroxisomes of human hepatoma cells in vitro.

A polypeptide containing the carboxyl-terminal fragment of human peroxisomal enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme was synthesized in vitro from its cDNA clone. This expression polypeptide was transported into purified rat liver peroxisomes. When the expression polypeptide was incubated with postnuclear supernatant fractions of human hepatoma cells and analyzed by Nycodenz gradient SDS-PAGE and fluorography, it was imported specifically into peroxisomes as indicated by its resistance to proteinase K degradation. A deletion of the last nine amino acid residues at the carboxyl-terminus of this polypeptide prevents its peroxisomal import. A tripeptide sequence, SKL, located at the carboxyl-terminus of human bifunctional enzyme appears to be the targeting signal for the peroxisomal importation of bifunctional enzyme in human cells.     

Plant peroxisomes,reactive oxygen metabolism and nitric oxide

In plant cells, as in most eukaryotic organisms, peroxisomes are probably the major sites of intracellular H2O2 production, as a result of their essentially oxidative type of metabolism. Like mitochondria and chloroplasts, peroxisomes also produce superoxide radicals (O2*-) and there are, at least, two sites of superoxide generation: one in the organelle matrix, the generating system being xanthine oxidase, and another site in the peroxisomal membranes dependent on NAD(P)H. In peroxisomal membranes, three integral polypeptides (PMPs) with molecular masses of 18, 29, and 32 kDa have been shown to generate O2*- radicals. Besides catalase, several antioxidative systems have been demonstrated in plant peroxisomes, including different superoxide dismutases, the four enzymes of the ascorbate-glutathione cycle plus ascorbate and glutathione, and three NADP-dependent dehydrogenases. A CuZn-SOD and two Mn-SODs have been purified and characterized from different types of plant peroxisomes. The presence of the enzyme nitric oxide synthase (NOS) and its reaction product, nitric oxide (NO*), has been recently demonstrated in plant peroxisomes. Different experimental evidence has suggested that peroxisomes have a ROS-mediated cellular function in leaf senescence and in stress situations induced by xenobiotics and heavy metals. Peroxisomes could also have a role in plant cells as a source of signal molecules like NO*, O2*- radicals, H2O2, and possibly S-nitrosoglutathione (GSNO). It seems reasonable to think that a signal molecule-producing function similar to that postulated for plant peroxisomes could also be performed by human, animal and yeast peroxisomes, where research on oxy radicals, antioxidants and nitric oxide is less advanced than in plant peroxisomes.     

The large subunit of the pig heart mitochondrial membrane-bound β-oxidation complex is a long-chain enoyl-CoA hydratase: 3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme

The subunit locations of the component enzymes of the pig heart trifunctional mitochondrial β-oxidation complex are suggested by analyzing the primary structure of the large subunit of this membrane-bound multienzyme complex [Yang S.-Y.et al. (1994) Biochem. biophys. Res. Commun. 198, 431–437] with those of the subunits of the E. coli fatty acid oxidation complex and the corresponding mitochondrial matrix β-oxidation enzymes. Long-chain enoyl-CoA hydratase and long-chain 3-hydroxyacyl-CoA dehydrogenase are located in the amino-terminal and the central regions of the 79 kDa polypeptide, respectively, whereas the long-chain 3-ketoacyl-CoA thiolase is associated with the 46 kDa subunit of this complex. The pig heart mitochondrial bifunctional β-oxidation enzyme is more homologous to the large subunit of the prokaryotic fatty acid oxidation complex than to the peroxisomal trifunctional β-oxidation enzyme. The evolutionary trees of 3-hydroxyacyl-CoA dehydrogenases and enoyl-CoA hydratases suggest that the mitochondrial inner membrane-bound bifunctional β-oxidation enzyme and the corresponding matrix monofunctional β-oxidation enzymes are more remotely related to each other than to their corresponding prokaryotic enzymes, and that the genes of E. coli multifunctional fatty acid oxidation protein and pig heart mitochondrial bifunctional β-oxidation enzyme diverged after the appearance of eukaryotic cells.     

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.     

Novel Subtype of Peroxisomal Acyl-CoA Oxidase Deficiency and Bifunctional Enzyme Deficiency with Detectable Enzyme Protein: Identification by Means of Complementation Analysis

We describe four infants with a novel subtype of an isolated deficiency of one of the peroxisomal β-oxidation enzymes with detectable enzyme protein. The patients showed characteristic clinical and biochemical abnormalities, including hypotonia, psychomotor retardation, hepatomegaly, typical facial appearance, accumulation of very-long-chain fatty acids, and decreased lignoceric acid oxidation. However, β-oxidation enzyme proteins were detected by immunoblot analyses, and large peroxisomes were identified by immunofluorescence staining. In order to identify the underlying defect in these patients, complementation analysis was introduced using fibroblasts from these patients and patients with an established deficiency of either acyl-CoA oxidase or bifunctional enzyme, as identified by immunoblotting. In the complementing combinations, fused cells showed increased lignoceric acid oxidation, resistance against 1-pyrene dodecanoic acid/UV selection, and normalization of the size and the distribution of peroxisomes. The results indicate that two patients with a more severe clinical course were suffering from bifunctional enzyme deficiency and that the other two infants, who were siblings and had a less severe clinical presentation, were the first patients with acyl-CoA oxidase deficiency with detectable enzyme protein.     

Regulation of rat hepatic peroxisomal enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme by thyroid hormone.

Rat hepatic t protein that is negatively regulated by thyroid hormone in nuclear globulin extract was characterized by the antibodies. The following evidence indicated that t protein is a peroxisomal enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (bifunctional enzyme). 1. Both proteins had an identical molecular size, and were immunologically indistinguishable from each other. 2. The t protein was abundant in mitochondrial fraction which contained abundant peroxisomes. 3. The amount of the t protein was increased by a peroxisomal proliferator. 4. The activity of the peroxisomal bifunctional enzyme corresponded to the t protein in CM-Sephadex column chromatography. The amount of peroxisomal bifunctional enzyme was increased by thyroidectomy and decreased by 3,5,3'- triiodo-L-thyronine treatment in the whole homogenate of rat liver. These results indicate that the levels of peroxisomal bifunctional enzyme were regulated by thyroid hormone in vivo.     

Mammalian mitochondrial methylenetetrahydrofolate dehydrogenase-cyclohydrolase derived from a trifunctional methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase

We have isolated the cDNA and the gene encoding the murine cytoplasmic methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (DCS). Comparison of these sequences with the 3'-untranslated region of the mitochondrial NAD(+)-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (mt-DC) revealed areas of significant homology. Both exon and intron sequences of the synthetase domain of DCS are homologous to sequences in the untranslated region of mt-DC. A similar comparison between the mt-DC and the DCS sequences of humans as well as Drosophila supports the conclusion that in higher eukaryotes the bifunctional mt-DC replaced a trifunctional precursor through inactivation of the synthetase domain. The mt-DC should be considered in models of one-carbon folate fluxes in mammals.     

The crystal structure of plant sulfite oxidase provides insights into sulfite oxidation in plants and animals

The molybdenum cofactor (Moco) containing sulfite oxidase (SO) from Arabidopsis thaliana has recently been identified and biochemically characterized. The enzyme is found in peroxisomes and believed to detoxify excess sulfite that is produced during sulfur assimilation, or due to air pollution. Plant SO (PSO) is homodimeric and homologous to animal SO, but contains only a single Moco domain without an additional redox center. Here, we present the first crystal structure of a plant Moco enzyme, the apo-state of Arabidopsis SO at 2.6 A resolution. The overall fold and coordination of the Moco are similar to chicken SO (CSO). Comparisons of conserved surface residues and the charge distribution in PSO and CSO reveal major differences near the entrance to both active sites reflecting different electron acceptors. Arg374 has been identified as an important substrate binding residue due to its conformational change when compared to the sulfate bound structure of CSO.     

cDNA cloning and primary structure determination of the peroxisomal trifunctional enzyme hydratase-dehydrogenase-epimerase from the yeast Candida tropicalis pK233

We report the isolation and nucleotide (nt) sequence determination of a cDNA encoding the peroxisomal trifunctional beta-oxidation enzyme hydratase-dehydrogenase-epimerase (HDE) from the yeast Candida tropicalis pK233. Poly(A)+RNA isolated from C. tropicalis cells grown in oleic acid medium was used to construct a cDNA library in lambda gt11. The library was screened with a polyclonal antiserum against HDE. A recombinant was confirmed to encode HDE by hybridization-selection translation and immunoprecipitation. The HDE cDNA (HDE) has a single open reading frame of 2718 nt, encoding a protein of 905 amino acids, not including the initiator methionine. The Mr of the protein is 99,350. A partial gene duplication is believed to have occurred in the evolution of the HDE gene. Codon utilization in the gene is not random, with 86.0% of the amino acids specified by 23 preferentially used codons, a situation similar to that found in genes encoding peroxisomal catalase and the various fatty acyl-CoA oxidases from C. tropicalis. The increase in HDE activity in C. tropicalis cells grown in oleic acid medium as opposed to glucose medium is due, at least in part, to increased HDE-specific mRNA levels.