Substrate specificity and stereoselectivity of fatty alcohol oxidase from the yeast Candida maltosa

Summary In Candida maltosa and other alkene-utilizing yeasts a membrane-bound fatty alcohol oxidase (FAOD) is induced by growth on n-alkenes. The oxidation of 1-alkanols to the corresponding aldehydes is accompanied by the stoichiometric consumption of 1 mol O₂ and formation of 1 mol hydrogen peroxide (H₂O₂). The FAOD of C. maltosa shows a broad substrate specificity. It catalyses the oxidation of 1-alkanols (C₄ to C₂₂), with a maximal activity of 1.85 gmmol H₂O₂/ min × mg protein for 1-octanol, as well as the transformation of 2-alkanols (C₈ to C₁₆) to ketones. Other compounds as ,-alkenediols, -hydroxypalmitic acid, phenylalkanols and terpene alcohols are substrates for the enzyme, although mostly with decreased activities. The oxidation of the racemic 2-alkanols by the FAOD proceeds with very high stereoselectivity for the R(–)-enatiomers only, leaving the S(+)-2-alkanol untouched. Offprint requests to: S. Mauersberger     

The nucleotide sequence of complementary DNA and the deduced amino acid sequence of peroxisomal catalase of the yeast Candida tropicalis pK233

We report the isolation and nucleotide (nt) sequence determination of cDNA encoding peroxisomal catalase (Cat) from the yeast Candida tropicalis pK233. The catalase cDNA (Cat) has a single open reading frame (ORF) of 1455 nt, encoding a protein of 484 amino acids (aa), not including the initiator methionine. The Mr of the protein is 54767. Codon use in the gene is not random, with 90.9% of the aa specified by 25 principal codons. The principal codons used in the expression of Cat in C. tropicalis are similar to those used in the expression of the fatty acyl-CoA oxidase gene of C. tropicalis and of highly expressed genes in Saccharomyces cerevisiae. Cat shows 48.0%, 49.7%, and 48.3% aa identity with human, bovine, and rat catalases, respectively, and 44.3% aa identity with catalase T of S. cerevisiae. The 3 aa of bovine liver catalase previously postulated to participate in catalysis and 79.5% of those aa in the immediate environment of hemin, the prosthetic group of catalase, are conserved in Cat of C. tropicalis.     

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.     

Degradation of long-chain n-alkanes by the yeast Candida maltosa

Summary Microsomal membrane fractions of the yeast Candida maltosa were investigated with respect to their ability to catalyse the oxidation of n-alkanes, fatty alcohols and fatty acids. Analysis of intermediates of n-hexadecane oxidation led to the conclusion that monoterminal attack was predominant, whereas diterminal oxidation proceeded as a minor reaction. The oxidation of long-chain primary alcohols to the corresponding aldehydes occurred without addition of nicotinamide adenine dinucleotide (phosphate) [NAD(P)⁺] and was accompanied by stoichiometric oxygen consumption and hydrogen peroxide production, suggesting that an alcohol oxidase instead of an NAD(P)⁺-requiring alcohol dehydrogenase catalysed these reactions. As shown for n-hexadecane, the hydroxylation of palmitic acid was found to be carbon monoxide-dependent, indicating involvement of a cytochrome P-450 system, as in the case of n-alkane hydroxylation.     

New yeast vectors containing the autonomously replicating sequences from Candida maltosa genome

Two different DNA sequences from the yeast Candida maltosa confer the ability to replicate autonomously to the yeast integrative vector pLD700 on which they are cloned. The recombinant plasmids pLD701 and pLD702 with autonomously replicating sequences (ARS) from Candida maltosa and LEU2 gene from Saccharomyces cerevisiae transform the auxotrophic strain S. cerevisiae DC5 with the efficiency 3-5 x 10(3) per microgram of DNA. Like other yeast vectors harbouring ARS, these plasmids are not stable in yeast cells. Restriction and hybridization analyses have revealed the pLD701 plasmid to contain ARS from chromosomal DNA of C. maltosa. Plasmid pLD701 appears to be a useful vector for yeast transformation.     

Primary structure and expression of peroxisomal acetylspermidine oxidase in the methylotrophic yeast Candida boidinii

Acetylspermidine oxidase (ASOD) belongs to a family of FAD-containing amine oxidases and catalyzes the oxidation of N-acetylated spermidine in polyamine metabolism. ASOD was purified to apparent homogeneity from cells of the methylotrophic yeast Candida boidinii grown on spermidine as the sole nitrogen source. C. boidinii ASOD catalyzed the oxidation of only N(1)-acetylspermidine. Based on partial amino acid sequences, oligonucleotide primers were designed for polymerase chain reaction, and the ASOD-encoding gene, ASO1, was cloned. The open reading frame encoding ASO1 was 1530 bp long and corresponded to a protein of 509 amino acid residues (calculated molecular mass=57167 Da). ASO1 contained a FAD-binding motif of G-A-G-I-A-G in the N-terminal region and carried an amino acid sequence of -S-K-L at the C-terminal, representing a typical peroxisome targeting signal 1. ASOD was localized in the peroxisomes in overexpressed C. boidinii. To our knowledge, this is the first report on the gene coding for ASOD that can catalyze the oxidation of N-acetylated polyamine as a substrate, from any type of organism.     

Degradation and dehalogenation of monochlorophenols by the phenol-assimilating yeast Candida maltosa

The phenol-assimilating yeast Candida maltosa is able to degrade monochlorophenols but cannot grow on these substrates. 3- and 4-chlorophenol were broken down very rapidly by phenol-grown cells under the formation of 4-chlorocatechol, 5-chloropyrogallol and 4-carboxymethylenebut-2-en-4-olide with concomitant release of chloride.2-Chlorophenol was partially converted into cis,cis-2-chloromuconic acid via 3-chlorocatechol which was also obtained from 3-chlorophenol in low amounts. No further metabolites containing chloride were found.The dehalogenation step in the chlorophenol degradation is the cycloisomerization of the cis,cis-chloromuconic acid to 4-carboxymethylenebut-2-en-4-olide in the ortho fission pathway.Dedicated to prof.Dr. E. Bayer, Tübingen, on the occasion of his 65th birthday.     

Physiological and DNA characterization of Candida maltosa, a hydrocarbon-utilizing yeast.

Selected yeast classified as Candida sake van Uden et Buckley were examined for their physiological, morphological and immunological properties and their DNA relatedness. Candida maltosa Komagata, Nakase et Katsuya is herein recognized as a species separate from C. sake, Candida maltosa was distinguished from C. sake and from C. tropicalis by insignificant DNA reassociation. In addition, C. maltosa was distinguished from C. sake by its higher maximal growth temperature and lower guanine plus cytosine content of its DNA and from C. tropicalis by its failure to utilize soluble starch for growth and its resistance to cycloheximide. The species C. cloacae and C. subtropicalis are placed in synonymy with C. maltosa.     

A soluble acyl-coenzyme A oxidase from the yeast Candida utilis.

trans-2-Enoyl-CoA and two unidentified polar compounds were synthesized from the corresponding long-chain acyl-CoA by a particle-free supernatant fraction obtained from Candida utilis. The enzyme was unreactive toward free fatty acids but desaturated all long-chain acyl CoAs tested (14:0, 16:0, 18:1, 18:2). Molecular oxygen was the only required cofactor. Phenazine methosulfate and 2,6-dichloroindophenol did not replace the requirement for oxygen. The activity was inhibited specifically by NADPH and stimulated by linoleic acid or linolenic acid. The enzyme was not active in log phase cultures, but was detected only in stationary phase cells. Introduction of the trans-2-double bond was confirmed by gas-liquid chromatography, thin-layer chromatography, mass spectrometry, catalytic hydrogenation, oxidative cleavage, and chemical reactivity of the product toward nucleophilic addition.     

Complete nucleotide sequence of cDNA and predicted amino acid sequence of rat acyl-CoA oxidase

cDNA clones for rat acyl-CoA oxidase were isolated. The 3.8-kilobase mRNA sequence of the enzyme was completely covered by two overlapping clones. The composite cDNA sequence consisted of 3741 bases and contained a 1983-base open reading frame which encodes a polypeptide of 661 amino acid residues. Two species of acyl-CoA oxidase cDNA were identified. They differed in their coding nucleotide sequences, only within a small region. They contained the same number of nucleotides and can be translated in a common reading frame. They are 55% and 50% homologous in the above region at the nucleotide and the amino acid levels, respectively. Both types of cDNA were isolated from a library constructed from mRNA of a single rat, thereby suggesting the occurrence of two species of acyl-CoA oxidase in each rat. The amino terminus of the enzyme was determined to be N-acetylmethionine, which corresponds to the initiator methionine, thus confirming the absence of a terminal presequence. We reported previously that a purified preparation of the enzyme contained three polypeptide components, A, B, and C, and suggested that components B and C are produced by a proteolytic cleavage of component A (Osumi, T., Hashimoto, T., and Ui, N. (1980) J. Biochem. (Tokyo) 87, 1735-1746). We located components B and C on the amino- and the carboxyl-terminal sides of component A. Possible functional significances of several stretches of amino acids of the enzyme are discussed, based on the sequence comparison data between rat and yeast acyl-CoA oxidases.     

Expression of the hydrogen peroxide-generating enzyme fatty acyl CoA oxidase activates NF-kappaB

Peroxisome proliferators are a class of hepatic carcinogens in rodents and have been proposed to act in part by increasing oxidative stress. Fatty acyl CoA oxidase (FAO), which is highly induced by peroxisome proliferators, is the hydrogen peroxide-generating enzyme of the peroxisomal beta-oxidation pathway. We previously showed that the treatment of rats and mice with the peroxisome proliferator ciprofibrate resulted in increased hepatic NF-kappaB activity and suggested that this effect may be secondary to the action of H2O-generating enzymes. To test this possibility directly, we have determined whether transient overexpression of FAO, in the absence of peroxisome proliferators, leads to NF-kappaB activation. Here, we show that FAO overexpression in Cos-1 cells, in the presence of an H2O-generating substrate, can activate a NF-kappaB regulated reporter gene. Electrophoretic mobility shift assays further demonstrated that FAO expression increases nuclear NF-kappaB DNA binding activity in a dose-dependent manner. The antioxidants vitamin E and catalase can inhibit this activation. These results indicate that FAO mediates, at least in part, peroxisome proliferator-induced NF-kappaB activation.     

A comparative immunological investigation of the alkane hydroxylating cytochrome P-450 from the yeast Candida maltosa

The immunological relations of the cytochrome P-450 from the n-alkane utilizing yeast Candida maltosa to cytochrome P-450 forms of other organisms - yeasts, bacteria and mammalia - were investigated using a solid-phase double-antibody radioimmunoassay. Only the microsomal fraction of other n-alkane utilizing yeasts shows a distinct cross-reaction with an antiserum against cytochrome P-450 from Candida maltosa. Neither the tested bacterial nor the mammalian cytochromes P-450 cross-react with the antiserum.     

Physiological and DNA characterization of Candida maltosa,a hydrocarbon-utilizing yeast

Selected yeasts classified as Candida sake van Uden et Buckley were examined for their physiological, morphological and immunological properties and their DNA relatedness. Candida maltosa Komagata, Nakase et Katsuya is herein recognized as a species separate from C. sake. Candida maltosa was distinguished from C. sake and from C. tropicalis by insignificant DNA reassociation. In addition, C. maltosa was distinguished from C. sake by its higher maximal growth temperature and lower guanine plus cytosine content of its DNA and from C. tropicalis by its failure to utilize soluble starch for growth and its resistance to cycloheximide. The species C. cloacae and C. subtropicalis are placed in synonymy with C. maltosa.     

Peroxisomal acyl-coenzyme A oxidase multigene family of the yeast Candida tropicalis; nucleotide sequence of a third gene and its protein product

We have determined the complete nucleotide sequence of gene POX2, which encodes one of the major peroxisomal polypeptides (PXPs) of Candida tropicalis. POX2 is linked to gene POX4, which codes for a subunit (PXP-4) of long-chain acyl-CoA oxidase. Southern blot analysis revealed that POX2 had a significant homology to POX4, and also to gene POX5 which encodes a subunit (PXP-5) of the isozyme of acyl-CoA oxidase. PXP-2, the protein product of POX2, was co-purified with PXP-4 from the isolated peroxisomes. PXP-2 itself was a flavoprotein and likely to form an equimolar complex with PXP-4, although its enzymatic activity was uncertain. POX2 corresponds to a single open reading frame of 724 amino acids and has no introns. The N-terminal sequence and the calculated Mr of the deduced polypeptide were consistent with those of isolated PXP-2. The primary structure was highly homologous to those of PXP-4 and PXP-5 in respect of the amino acid sequence and the hydropathy profile. We conclude that POX2 is a third gene of the peroxisomal acyl-COA oxidase multigene family.     

Isolation and nucleotide sequence of the extracellular acid protease gene (ACP) from the yeast Candida tropicalis

The extracellular acid protease of Candida tropicalis was purified from the supernatant fraction of culture medium containing bovine serum albumin as nitrogen source and the NH2-terminal amino acid (aa) sequence of the protein was determined. The gene for the acid protease (ACP) was isolated using a pool of synthetic oligonucleotides as a probe and a segment of the deduced aa sequence was found to be in agreement with the NH2-terminal aa sequence of the protein. The deduced aa sequence of ACP is similar to the aa sequence of proteases of the pepsin family. The nucleotide sequence of the 5' portion of this gene revealed a coding sequence for a 60 residue propeptide containing two Lys-Arg amino acid pairs that have been identified as sites for peptidase processing of several exported peptides and proteins. The final Lys-Arg site occurs at the junction with the mature extracellular form of the acid protease.