Alcohol oxidase and catalase in peroxisomes of methanol-grown Candida boidinii.

Microbodies, designated as peroxisomes because of their enzyme complement, have been isolated from methanol-grown cells of Candida boidinii. Spheroplast lysates were separated on non-continuous Ficoll density gradients, resulting in a mitochondrial fraction and a peroxisome fraction. Estimates of purity using the mitochondrial enzyme markers suggested that the contamination of mitochondria in the peroxisome fraction was about 2-3%. As shown by electron microscopy the peroxisomes were 0.4-0.6 mum in diameter and contained crystalloid inclusions. Alcohol oxidase and catalase, which catalyse the oxidation of methanol to formaldehyde in Candida boidinii, could be localized within the peroxisomes. Gel-electrophoretic studies of the peroxisome fraction demonstrated that it contained only two predominant protein bands consistent with alcohol oxidase and catalase. No alcohol oxidase and catalase activity was found in mitochondria.     

Oxidation of formaldehyde by alcohol oxidase of Candida boidinii

A formaldehyde oxidase activity was found in cell-free extracts of methanol-grown yeast Candida boidinii. Loss of alcohol oxidase activity in a mutant, 4₈, led to loss of the formaldehyde oxidase activity, indicating that the same enzyme is probably responsible for both activities. This could be demonstrated with the purified alcohol oxidase which oxidizes, besides lower primary alcohols, formaldehyde to formate. The K _m value for formaldehyde is 5.7 mM. It seems that alcohol oxidase is not implicated in formaldehyde oxidation in vivo.     

Dihydroxyacetone synthase is an abundant constituent of the methanol-induced peroxisome of Candida boidinii

Methylotrophic yeasts induce large peroxisomes when grown on methanol. The recent ability to stabilize and isolate these peroxisomes at pH 5.5 has led to the demonstration that two polypeptides comprise the bulk of the peroxisome of Candida boidinii, alcohol oxidase, and a 79-kDa species, determined by sodium dodecyl sulfate-polyacrylamide electrophoresis (Goodman, J.M., Scott, C.W., Donahue, P.N., and Atherton, J.P. (1984) J. Biol. Chem. 259, 8485-8493). The 79-kDa peroxisomal protein is now identified as dihydroxyacetone synthase, the first enzyme in the assimilatory pathway of formaldehyde utilization. This identification is based on several criteria: The enzyme activity is mainly in a particulate fraction at pH 5.5 but not at pH 8.0. It copurifies with alcohol oxidase and catalase on sucrose gradients. The 79-kDa protein behaves as a 135,000-kDa dimer on gel filtration, similar to the published behavior of the enzyme. The specific activity of dihydroxyacetone synthase in the pure 79-kDa preparation (3.20 units/mg of protein) is close to that reported for the purified enzyme (3.88 units/mg of protein). Antibodies against dihydroxyacetone synthase were used to show that its synthesis, induction, and assembly are similar to that of alcohol oxidase. Neither contains a detectable cleaved leader sequence and both are assembled post-translationally. The localization of dihydroxyacetone synthase to the peroxisome may influence the regulation of the two pathways of formaldehyde utilization and may protect the cell from damage by formaldehyde.     

Oxidation of formaldehyde by alcohol oxidase of Candida boidinii.

A fromaldehyde oxidase activity was found in cellfree extracts of methanol-grown yeast Candida boidinii. Loss of alcohol oxidase activity in a mutant, 48, led to loss of the formaldehyde oxidase activity, indicating that the same enzyme is probably responsible for both activities. This could be demonstrated with the purified alcohol oxidase which oxidizes, besides lower primary alcohols, formaldehyde to formate. The Km value for formaldehyde is 5.7 mM. It seems that alcohol oxidase is not implicated in formaldehyde oxidation in vivo.     

Characterization and high-level production of D-amino acid oxidase in Candida boidinii

D-Amino acid oxidase (DAO, EC 1.4.3.3) from a methylotrophic yeast, Candida boidinii, was produced at a high level under the control of the alcohol oxidase gene promoter in the original host. The enzyme was a peroxisomal and monomeric enzyme, and contained noncovalently-bound FAD as a cofactor. The enzyme was active toward several D-amino acids such as D-Ala, D-Met, and D-Ser. An alcohol oxidase-depleted strain (aod1delta) was found to be a more suitable host for DAO production than the wild-type strain. Several post-translational effects may be responsible for the improvement of the DAO productivity by the aod1delta strain. Finally, an aod1delta strain transformant having multi-copies of an expression plasmid on its chromosome could produce DAO amounting up to 30% of the total soluble proteins.     

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.     

Stereochemistry and mechanism of the methanol oxidase from Candida boidinii (author's transl)]

Under nitrogen atmosphere, methanol oxidase isolated from Candida boidinii catalyzes no hydrogen exchange of ethanol. Reactions with (1R)[1-3H]ethanol or (1S)[1-3H]ethanol occur stereoselectively. The ratio of hydrogen abstraction in the oxidation of ethanol is about 7:1 in favor of the pro R hydrogen atom. The isotope effect kH/k3H is 2 - 2.5 for (1R)[1-3H]ethanol. The isotope effects on Km and V have been determined for four different deuterium-labelled ethanols. The Km for [2H3]methanol is nearly twice that for CH3OH. That is about the same ratio as for ethanol/methanol. For the oxidation of CH3OH and 2H3COH, the ratio of VH/V2H3 = 2.44, while the ratio for methanol/ethanol is only 1.3. It has been conformed that catalase from bovine liver eliminates only the pro R hydrogen atom from ethanol. The isotope effect for the catalase-catalyzed oxidation of (1R)[1-3H]ethanol is 2.1.     

Monomeric alcohol oxidase is preferentially digested by a novel protease from Candida boidinii

A protease activity has been partially purified from peroxisomal matrix fractions of the methylotrophic yeast Candida boidinii. The enzyme migrates as a single peak on a sucrose velocity gradient with an apparent native molecular mass of approximately 80-90 kDa. Activity can be recovered from nonreducing sodium dodecyl sulfate gels as a approximately 20 kDa species, suggesting it is an oligomer. The protein exhibits chymotrypsin-like activity and cleaves the model compound suc-L-L-V-Y-AMC. Additionally, monomers of alcohol oxidase (AO), an abundant protein of C. boidinii peroxisomes, generated in vitro or in pulse-radiolabeled cells, are preferentially sensitive to degradation by the protease. Sensitivity is lost over time in vivo as AO folds and matures into octamers, suggesting that the protease may be involved in these processes.     

The membrane proteins of the methanol-induced peroxisome of Candida boidinii. Initial characterization and generation of monoclonal antibodies

Peroxisomes are massively induced when methylotrophic yeasts are cultured on methanol as the sole carbon and energy source. An analysis of the protein composition of the peroxisomal membrane and the generation of probes against two peroxisomal membrane proteins (PMPs) have been undertaken. Peroxisomes from Candida boidinii were obtained from sucrose gradients as previously described or from a novel one-step purification of the organelle on a Percoll gradient. The protein composition of the membranes from these two preparations was virtually identical. About 10 proteins comprise nearly all of its protein mass. The most prominent proteins have molecular masses of 120, 100, 47, 31-32 (a triplet), and 20 kDa; significant amounts of alcohol oxidase and dihydroxyacetone synthase, the two abundant matrix proteins, also remain associated with the membrane. Glycosylation of the membrane proteins could not be detected. Exposure of the membrane to chaotropes shows that PMPs 100 and 20 are the most easily removable, whereas PMP 47 appears to be the most tightly associated. Mice were injected with peroxisomal membrane, and hybridoma lines were isolated that produced antibody against PMP 20, PMP 47, and dihydroxyacetone synthase. Indirect immunofluorescence with these monoclonal antibodies confirmed that all three proteins are localized to the peroxisomal cluster. Immunoblotting experiments demonstrated that peroxisomal membrane as well as matrix proteins are induced by methanol.     

Antioxidant system within yeast peroxisome. Biochemical and physiological characterization of CbPmp20 in the methylotrophic yeast Candida boidinii

Candida boidinii Pmp20 (CbPmp20), a protein associated with the inner side of peroxisomal membrane, belongs to a recently identified protein family of antioxidant enzymes, the peroxiredoxins, which contain one cysteine residue. Pmp20 homologs containing the putative peroxisome targeting signal type 1 have also been identified in mammals and lower eukaryotes. However, the physiological function of these Pmp20 family proteins has been unclear. In this study, we investigated the biochemical and physiological functions of recombinant CbPmp20 protein in methanol-induced peroxisomes of C. boidinii using the PMP20-deleted strain of C. boidinii (pmp20Delta strain). The His(6)-tagged CbPmp20 fusion protein was found to have glutathione peroxidase activity in vitro toward alkyl hydroperoxides and H(2)O(2). Catalytic activity and dimerization of His(6)-CbPmp20 depended on the only cysteine residue corresponding to Cys(53). The pmp20Delta strain was found to have lost growth ability on methanol as a carbon and energy source. The pmp20Delta growth defect was rescued by CbPmp20, but neither CbPmp20 lacking the peroxisome targeting signal type 1 sequence nor CbPmp20 haboring the C53S mutation retrieved the growth defect. Interestingly, the pmp20Delta strain had a more severe growth defect than the cta1Delta strain, which lacks catalase, another antioxidant enzyme within the peroxisome. During incubation of these strains in methanol medium, the cta1Delta strain accumulated H(2)O(2), whereas the pmp20Delta strain did not. Therefore, it is speculated to be the main function of CbPmp20 is to decompose reactive oxygen species generated at peroxisomal membrane surface, e.g. lipid hydroperoxides, rather than to decompose H(2)O(2). In addition, we detected a physiological level of reduced glutathione in peroxisomal fraction of C. boidinii. These results may indicate a physiological role for CbPmp20 as an antioxidant enzyme within peroxisomes rich in reactive oxygen species.     

Expression of Saccharomyces adenylate kinase gene in Candida boidinii under the regulation of its alcohol oxidase promoter

The methylotrophic yeast, Candida boidinii, was investigated as a new efficient host for heterologous gene expression. The Saccharomyces cerevisiae adenylate kinase gene (ADK1) was used as the first example for heterologous enzyme production in C. boidinii. C. boidinii cells were transformed with plasmids harboring the S. cerevisiae ADK1 gene under the alcohol oxidase (C. boidinii AOD1) promoter. The chromosome-integrant strains produced adenylate kinase protein corresponding to 22%–28% of the total soluble proteins in an enzymatically active form. When the three-copy integrative transformant was grown for 60 h on methanol-glycerol medium in a 1.5-l jar fermentor, adenylate kinase was produced intracellularly with a yield of up to 2 g/l culture medium. As the expression of the S. cerevisiae ADK1 in C. boidinii was under similar regulation to that of the C. boidinii AOD1, the previously cloned 1.7-kb AOD1 promoter fragment was proved to harbor sufficient cis elements for AOD1 regulation and found to be an efficient promoter for heterologous gene expression.     

Improved purification of Candida boidinii formate dehydrogenase

Formate dehydrogenase (FDH, EC 1.2.1.2) from Candida boidinii was purified to homogeneity. The two step procedure comprised anion exchange chromatography (2.9-fold purification, 85% step yield, elution with 35 mM KCl), followed by dye-ligand affinity chromatography on immobilized Cibacron Blue 3GA (1.4-fold purification, 75% step yield, elution with 0.15 mM NAD⁺/2 mM Na₂SO₃). The procedure afforded FDH at 63.8% overall yield and a specific activity of 7.2 units/mg. The purity of the final FDH preparation was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), high performance gel filtration liquid chromatography (gfHPLC) and N-terminal amino acid sequencing. The analytical techniques showed the presence of a single polypeptide chain that corresponds to the molecular weight of 41 kDa (as determined by SDS-PAGE) and 81 kDa (as determined by gfHPLC).     

Age structure of methanol-assimilating Candida boidinii cells

A population of Candida boidinii was grown under the chemostat conditions at a dilution rate of 0.14-0.20 h-1 in a mineral medium with methanol as a sole source of carbon and energy. Its age structure was analysed taking account of the reproductive age (the number of cycles) and the cyclic age of the cells (the phase of a cell cycle). Four morphological phases of the cycle, namely, one phase of preparation for budding and three successive budding phases, were compared with the phases G1, S, G2 and M. In terms of their reproductive age, the cells can be arranged as follows: (1) cells that formed no buds at all, 43 +/- 5%; (2) cells after one cycle of budding, 28 +/- 4%; (3) cells from which two and more daughter cells have separated, 29 +/- 4%. The age structure of an yeast population must be analysed when it is necessary to estimate its physiological heterogeneity or to elaborate the cultivation of microorganisms involving the control over the age structure of the population.     

Changes in proteinase activities and subcellular distribution during inactivation of alcohol oxidase in Candida boidinii.

Adaptation of methanol-grown Candida boidinii to ethanol utilization was accompanied by an increase in proteolytic activities, which behaved like known vacuolar enzymes. Degradation of alcohol oxidase protein was partially prevented by the serine proteinase inhibitor phenylmethanesulphonyl fluoride, but not by the carboxyl proteinase inhibitor pepstatin. Fractionation of cell-free extracts, by high-speed zonal centrifugation, of methanol-grown C. boidinii showed non-sedimentable and sedimentable proteolytic activities. Naturally occurring inhibitors of vacuolar proteinases were non-sedimentable. Fractionation of extracts prepared from methanol-grown cells which had been adapted to ethanol utilization for 5 h revealed significant changes in the sedimentability and distribution of proteolytic and acid phosphatase activities. These results suggest the possible involvement of a vacuolar process during alcohol oxidase degradation.     

Degradative inactivation of the peroxisomal enzyme, alcohol oxidase, during adaptation of methanol-grown Candida boidinii to ethanol.

Adaptation of methanol-grown C. boidinii to ethanol-utilization in non-growing cells resulted in decreased activity of the peroxisomal enzyme alcohol oxidase. Re-appearance of alcohol oxidase activity was dependent on protein synthesis de novo. Degradation of alcohol oxidase protein was shown to parallel the decrease in activity. Adaptation of methanol-grown cells to ethanol-utilization resulted in increased absorbance due to cytochromes and decreased absorbance due to flavoprotein. Decrease in alcohol oxidase activity was associated with loss of the flavin coenzyme, FAD, from the organisms and the appearance of flavins (FAD, FMN, riboflavin) in the surrounding medium. Electron microscopic observations showed that general degradation of whole peroxisomes rather than specific loss of crystalline cores (alcohol oxidase protein) occurred during the adaptation.     

High-resolution structures of formate dehydrogenase from Candida boidinii

The understanding of the mechanism of enzymatic recovery of NADH is of biological and of considerable biotechnological interest, since the essential, but expensive, cofactor NADH is exhausted in asymmetric hydrogenation processes, but can be recovered by NAD(+)-dependent formate dehydrogenase (FDH). Most accepted for this purpose is the FDH from the yeast Candida boidinii (CbFDH), which, having relatively low thermostability and specific activity, has been targeted by enzyme engineering for several years. Optimization by mutagenesis studies was performed based on physiological studies and structure modeling. However, X-ray structural information has been required in order to clarify the enzymatic mechanism and to enhance the effectiveness and operational stability of enzymatic cofactor regenerators in biocatalytic enantiomer synthesis as well as to explain the observed biochemical differences between yeast and bacterial FDH. We designed two single-point mutants in CbFDH using an adapted surface engineering approach, and this allowed crystals suitable for high-resolution X-ray structural studies to be obtained. The mutations improved the crystallizability of the protein and also the catalytic properties and the stability of the enzyme. With these crystal structures, we explain the observed differences from both sources, and form the basis for further rational mutagenesis studies.     

Sorting of peroxisomal membrane protein PMP47 from Candida boidinii into peroxisomal membranes of Saccharomyces cerevisiae

A gene encoding PMP47, a peroxisomal integral membrane protein of the methylotrophic yeast Candida boidinii, was isolated from a genomic library. DNA sequencing of PMP47 revealed an open reading frame of 1269 base pairs capable of encoding a protein of 46,873 Da. At least two membrane-spanning regions in the protein are predicted from the sequence. Since the 3 amino acids at the carboxyl terminus are -AKE, PMP47 lacks a typical peroxisomal sorting signal. No significant similarities in primary structure between PMP47 and known proteins were observed, including PMP70, a rat peroxisomal membrane protein whose sequence has recently been reported (Kamijo, K., Taketani, S., Yokota, S., Osumi, T., and Hashimoto, T. (1990). J. Biol. Chem. 265, 4534-4540). In order to study the import and assembly of PMP47 into peroxisomes by genetic approaches, the gene was expressed in the yeast Saccharomyces cerevisiae. When PMP47 was expressed in cells grown on oleic acid to induce peroxisomes, the protein was observed exclusively in peroxisomes as determined by marker enzyme analysis of organelle fractions. Most of the PMP47 co-purified with the endogenous peroxisomal membrane proteins on isopycnic sucrose gradients. Either in the native host or when expressed in S. cerevisiae, PMP47 was not extractable from peroxisomal membranes by sodium carbonate at pH 11, indicating an integral membrane association. These results indicate that PMP47 is competent for sorting to and assembling into peroxisomal membranes in S. cerevisiae.     

Theoretical insights into the AIE mechanism of assembles

Fluorophores in aggregated state are commonly used in optoelectronic devices, and the molecular packing are complex and diverse, including crystal, amorphous aggregate in solution, thin film, ordered supramolecular assemblies, and highly ordered cell membrane. In addition, the luminous behavior of the aggregated state can be precisely regulated by external stimuli such as hydrostatic pressure. In this review, we summarize the representative progress on the application of multiscale modeling protocol to illustrate the underlying mechanism of fluorescent emission of organic dyes in different assembles. The aim is to obtain the molecular packing in different forms of assembles and then to understand their underlying mechanism of stimuli-responsive fluorescent behavior at the molecular level. This is essential for the rational design, synthesis, and efficient application of fluorescent dyes.