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Yasuyoshi Sakai

Publications and source records attributed to Yasuyoshi Sakai.

At least 37 records · Page 2Linked to original sources
PubMed2005

Anaerobic degradation of aromatic compounds by magnetospirillum strains: isolation and degradation genes.

Four Magnetospirillum strains degrading toluene, phenol, benzoate, and other aromatic compounds under anaerobic conditions were isolated from denitrifying enrichment cultures. One of the isolates, toluene-degrading strain TS-6, contained genes that are homologous to those encoding benzylsuccinate synthase (Bss) and benzoyl-CoA reductase (Bcr), two key enzymes of anaerobic toluene and benzoate degradation respectively in known denitrifying bacteria. Transcription of the genes was confirmed. It was controlled by growth substrates and oxygen conditions, but bcr genes were unexpectedly expressed in aerobic cells grown on benzoate. It was confirmed that the genus Magnetospirillum represents the third genus of denitrifying bacteria capable of degrading aromatic compounds under anaerobic conditions, besides the genera Thauera and Azoarcus.

Anaerobiosis↗
PubMed2005

Intracellular ATP correlates with mode of pexophagy in Pichia pastoris.

The methylotrophic yeast Pichia pastoris can degrade peroxisomes selectively though two distinct pexophagic pathways, viz., micropexophagy and macropexophagy. These micro- and macropexophagy pathways are induced by adaptation of methanol-grown cells to glucose-containing and ethanol-containing media respectively. However, our understanding of the molecular signal(s) that determine which pathway is activated or repressed in response to environmental changes is limited. In this study, the determinant for these pathways was sought using cells undergoing pexophagy under a variety of conditions. Micropexophagy and macropexophagy were distinguished in living cells by fluorescence microscopy. Our results indicate that glucose and ethanol were not specific inducers of micro- and macropexophagy respectively. Micropexophagy was found to be more sensitive to ATP-depletion than macropexophagy, suggesting that the micropexophagic process requires a higher level of ATP than the macropexophagic process. From these and other results, we postulate that intracellular ATP levels play an important role in determining which pexophagic pathway is activated.

Adenosine Triphosphate↗
PubMed2004

A sorting nexin PpAtg24 regulates vacuolar membrane dynamics during pexophagy via binding to phosphatidylinositol-3-phosphate.

Diverse cellular processes such as autophagic protein degradation require phosphoinositide signaling in eukaryotic cells. In the methylotrophic yeast Pichia pastoris, peroxisomes can be selectively degraded via two types of pexophagic pathways, macropexophagy and micropexophagy. Both involve membrane fusion events at the vacuolar surface that are characterized by internalization of the boundary domain of the fusion complex, indicating that fusion occurs at the vertex. Here, we show that PpAtg24, a molecule with a phosphatidylinositol 3-phosphate-binding module (PX domain) that is indispensable for pexophagy, functions in membrane fusion at the vacuolar surface. CFP-tagged PpAtg24 localized to the vertex and boundary region of the pexophagosome-vacuole fusion complex during macropexophagy. Depletion of PpAtg24 resulted in the blockage of macropexophagy after pexophagosome formation and before the fusion stage. These and other results suggest that PpAtg24 is involved in the spatiotemporal regulation of membrane fusion at the vacuolar surface during pexophagy via binding to phosphatidylinositol 3-phosphate, rather than the previously suggested function in formation of the pexophagosome.

Alcohol Oxidoreductases↗
PubMed2004

Molecular characterization of the glutathione-dependent formaldehyde dehydrogenase gene FLD1 from the methylotrophic yeast Pichia methanolica.

In this paper, we describe molecular characterization of the FLD1 gene, which encodes glutathione-dependent formaldehyde dehydrogenase (FLD), from the methylotrophic yeast Pichia methanolica. The P. methanolica FLD1 gene contains two exons corresponding to a gene product of 380 amino acid residues and a 225 bp intron, respectively, and its deduced amino acid sequence shows high similarity to those of Fld1ps from other methylotrophic yeasts (80-88%). In P. methanolica, FLD activity is mainly induced by methanol, and this induction is not completely repressed by glucose. Moreover, the expression of the PmFLD1 is strictly regulated, mainly at the mRNA level, its expression increasing with increasing methanol concentrations in the medium. These results suggest that FLD1 is involved in the detoxification of formaldehyde in methanol metabolism, and Fld1p coordinates the formaldehyde level in methanol-grown cells according to the methanol concentration on growth.

Aldehyde Oxidoreductases↗
PubMed2004

Alcohol dehydrogenases that catalyse methyl formate synthesis participate in formaldehyde detoxification in the methylotrophic yeast Candida boidinii.

Methyl formate synthesis during growth on methanol by methylotrophic yeasts has been considered to play a role in formaldehyde detoxification. An enzyme that catalyses methyl formate synthesis was purified from methylotrophic yeasts, and was suggested to belong to a family of alcohol dehydrogenases (ADHs). In this study we report the gene cloning and gene disruption analysis of three ADH-encoding genes in the methylotrophic yeast Candida boidinii (CbADH1, CbADH2 and CbADH3) in order to clarify the physiological role of methyl formate synthesis. From the primary structures of these three genes, CbAdh1 was shown to be cytosolic and CbAdh2 and CbAdh3 were mitochondrial enzymes. Gene products of CbADH1, CbADH2 and CbADH3 expressed in Escherichia coli showed both ADH- and methyl formate-synthesizing activities. The results of gene-disruption analyses suggested that methyl formate synthesis was mainly catalysed by a cytosolic ADH (CbAdh1), and this enzyme contributed to formaldehyde detoxification through glutathione-independent formaldehyde oxidation during growth on methanol by methylotrophic yeasts.

Alcohol Dehydrogenase↗
PubMed2004

Isolation and characterization of delta(6)-desaturase, an ELO-like enzyme and delta(5)-desaturase from the liverwort Marchantia polymorpha and production of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris.

The liverwort Marchantia polymorpha contains high proportions of arachidonic and eicosapentaenoic acids. In general, these C20 polyunsaturated fatty acids (PUFA) are synthesized from linoleic and alpha -linolenic acids, respectively, by a series of reactions catalyzed by Delta(6)-desaturase, an ELO-like enzyme involved in Delta(6) elongation and Delta(5)-desaturase. Here we report the isolation and characterization of the cDNAs, MpDES6, MpELO1 and MpDES5, coding for the respective enzymes from M. polymorpha. Co-expression of the MpDES6, MpELO1 and MpDES5 cDNAs resulted in the accumulation of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris. Interestingly, Delta(6) desaturation by the expression of the MpDES6 cDNA appears to occur both in glycerolipids and the acyl-CoA pool, although other lower-plant Delta(6)-desaturases are known to have a strong preference for glycerolipids.

Acetyl Coenzyme A↗
PubMed2004

Aerobic and anaerobic toluene degradation by a newly isolated denitrifying bacterium, Thauera sp. strain DNT-1.

A newly isolated denitrifying bacterium, Thauera sp. strain DNT-1, grew on toluene as the sole carbon and energy source under both aerobic and anaerobic conditions. When this strain was cultivated under oxygen-limiting conditions with nitrate, first toluene was degraded as oxygen was consumed, while later toluene was degraded as nitrate was reduced. Biochemical observations indicated that initial degradation of toluene occurred through a dioxygenase-mediated pathway and the benzylsuccinate pathway under aerobic and denitrifying conditions, respectively. Homologous genes for toluene dioxygenase (tod) and benzylsuccinate synthase (bss), which are the key enzymes in aerobic and anaerobic toluene degradation, respectively, were cloned from genomic DNA of strain DNT-1. The results of Northern blot analyses and real-time quantitative reverse transcriptase PCR suggested that transcription of both sets of genes was induced by toluene. In addition, the tod genes were induced under aerobic conditions, whereas the bss genes were induced under both aerobic and anaerobic conditions. On the basis of these results, it is concluded that strain DNT-1 modulates the expression of two different initial pathways of toluene degradation according to the availability of oxygen in the environment.

Aerobiosis↗
PubMed2004

Role of alpha-methylacyl coenzyme A racemase in the degradation of methyl-branched alkanes by Mycobacterium sp. strain P101.

A new isolate, Mycobacterium sp. strain P101, is capable of growth on methyl-branched alkanes (pristane, phytane, and squalane). Among ca. 10,000 Tn5-derived mutants, we characterized 2 mutants defective in growth on pristane or n-hexadecane. A single copy of Tn5 was found to be inserted into the coding region of mcr (alpha-methylacyl coenzyme A [alpha-methylacyl-CoA] racemase gene) in mutant P1 and into the coding region of mls (malate synthase gene) in mutant H1. Mutant P1 could not grow on methyl-branched alkanes. The recombinant Mcr produced in Escherichia coli was confirmed to catalyze racemization of (R)-2-methylpentadecanoyl-CoA, with a specific activity of 0.21 micromol . min(-1) . mg of protein(-1). Real-time quantitative reverse transcriptase PCR analyses indicated that mcr gene expression was enhanced by the methyl-branched alkanes pristane and squalane. Mutant P1 used (S)-2-methylbutyric acid for growth but did not use the racemic compound, and growth on n-hexadecane was not inhibited by pristane. These results suggested that the oxidation of the methyl-branched alkanoic acid is inhibited by the (R) isomer, although the (R) isomer was not toxic during growth on n-hexadecane. Based on these results, Mcr is suggested to play a critical role in beta-oxidation of methyl-branched alkanes in Mycobacterium. On the other hand, mutant H1 could not grow on n-hexadecane, but it partially retained the ability to grow on pristane. The reduced growth of mutant H1 on pristane suggests that propionyl-CoA is available for cell propagation through the 2-methyl citric acid cycle, since propionyl-CoA is produced through beta-oxidation of pristane.

Alkanes↗
PubMed2004

The pro-peptide of Streptomyces mobaraensis transglutaminase functions in cis and in trans to mediate efficient secretion of active enzyme from methylotrophic yeasts.

Transglutaminase (TGase) from the actinomycete Streptomyces mobaraensis is a useful enzyme in the food industry, and development of an efficient production system for it would be desirable. Herein we report secretion of TGase in an enzymatically active form by methylotrophic yeasts as expression hosts. Secretory production of active TGase required a pro-peptide from TGase. When an artificial Kex2-endopeptidase recognition site was placed between the pro-peptide and mature TGase, secretion and in vitro maturation of TGase depended on Kex2-dependent cleavage. Unexpectedly, coexpression of unlinked pro-peptide with mature TGase yielded efficient secretion of the active enzyme. These results indicate that the pro-peptide from TGase functions not only in an intramolecular but also in an intermolecular manner. Site-directed mutagenesis of putative N-glycosylation sites increased the productivity of the active TGase further. A recombinant Candida boidinii strain was found to secrete active TGase up to 1.83 U/ml (about 90 mg/l) after 119 h of cultivation.

Candida↗
PubMed2003

Modification of a ubiquitin-like protein Paz2 conducted micropexophagy through formation of a novel membrane structure.

Microautophagy is a versatile process in which vacuolar or lysosomal membranes directly sequester cytosolic targets for degradation. Recent genetic evidence suggested that microautophagy uses molecular machineries essential for macroautophagy, but the details of this process are still unknown. In this study, a ubiquitin-like protein Paz2 essential for micropexophagy in the yeast Pichia pastoris has been shown to receive modification through the function of Paz8 and Gsa7, yielding a modified form Paz2-I, similar to the ubiquitin-like lipidation of Aut7 that is essential for macroautophagy in Saccharomyces cerevisiae. We identified a novel membrane structure formed after the onset of micropexophagy, which we suggest is necessary for the sequestration of peroxisomes by the vacuole. Assembly of this newly formed membrane structure, which is followed by localization of Paz2 to it, was found to require a properly functioning Paz2-modification system. We herein show that Paz2 and its modification system conduct micropexophagy through formation of the membrane structure, which explains the convergence between micropexophagy and macroautophagy with regard to de novo membrane formation.

Amino Acid Sequence↗
PubMed2003

Peroxisome degradation requires catalytically active sterol glucosyltransferase with a GRAM domain.

Fungal sterol glucosyltransferases, which synthesize sterol glucoside (SG), contain a GRAM domain as well as a pleckstrin homology and a catalytic domain. The GRAM domain is suggested to play a role in membrane traffic and pathogenesis, but its significance in any biological processes has never been experimentally demonstrated. We describe herein that sterol glucosyltransferase (Ugt51/Paz4) is essential for pexophagy (peroxisome degradation), but not for macroautophagy in the methylotrophic yeast Pichia pastoris. By expressing truncated forms of this protein, we determined the individual contributions of each of these domains to pexophagy. During micropexophagy, the glucosyltransferase was associated with a recently identified membrane structure: the micropexophagic apparatus. A single amino acid substitution within the GRAM domain abolished this association as well as micropexophagy. This result shows that GRAM is essential for proper protein association with its target membrane. In contrast, deletion of the catalytic domain did not impair protein localization, but abolished pexophagy, suggesting that SG synthesis is required for this process.

Autophagy↗
PubMed2003

Existence of catalase-less peroxisomes in Sf21 insect cells.

Catalase activity, a peroxisomal marker enzyme, was not detectable in any of the subcellular fractions of Spodoptera frugiperda (Sf) 21 insect cells, although marker enzymes in other organelles were distributed in the fractions in a manner similar to that seen in mammalian cells. When a green fluorescent protein fused with peroxisome targeting signal 1 at the C-terminal (GFP-SKL) was expressed in Sf21 cells, punctate fluorescent dots were observed in the cytoplasm. The fraction where GFP-SKL was concentrated exhibited long-chain and very-long-chain fatty acid beta-oxidation activities in the presence of KCN and the density of this fraction was slightly higher than that of mitochondria. Immunoelectron microscopy studies with anti-SKL antibody demonstrated that Sf21 cells have immunoreactive peroxisome-like organelles which are structurally distinct from mitochondria, endoplasmic reticulum, and lysosomes. In contrast to peroxisomal matrix proteins, adrenoleukodystrophy protein, a peroxisomal membrane protein, was not located to peroxisomes. This suggests that the targeting signal for PMP in insect cells is distinct from that in mammalian cells. These results demonstrate that Sf21 insect cells have unique catalase-less peroxisomes capable of beta-oxidation of fatty acids.

ATP Binding Cassette Transporter, Subfamily D, Mem↗
PubMed2003

Wax ester production by bacteria.

The enzymological and genetic aspects of microbial metabolism of hydrocarbons have been extensively revealed. Such molecular information is useful for understanding the bioremediation of oil spill environments and production of hydrocarbon-specific fine chemicals.

Bacteria↗
PubMed2003

Physiological role of S-formylglutathione hydrolase in C(1) metabolism of the methylotrophic yeast Candida boidinii.

The methylotrophic yeast Candida boidinii exhibits S-formylglutathione hydrolase activity (FGH, EC 3.1.2.12), which is involved in the glutathione-dependent formaldehyde oxidation pathway during growth on methanol as the sole carbon source. The structural gene, FGH1, was cloned from C. boidinii, and its predicted amino acid sequence showed more than 60 % similarity to those of FGHs from Paracoccus denitrificans and Saccharomyces cerevisiae, and human esterase D. FGH from C. boidinii contained a C-terminal tripeptide, SKL, which is a type I peroxisome-targeting signal, and a bimodal distribution of FGH between peroxisomes and the cytosol was demonstrated. The FGH1 gene was disrupted in the C. boidinii genome by one-step gene disruption. The fgh1Delta strain was still able to grow on methanol as a carbon source under methanol-limited chemostat conditions with low dilution rates (D<0.05 h(-1)), conditions under which a strain with disruption of the gene for formaldehyde dehydrogenase (another enzyme involved in the formaldehyde oxidation pathway) could not survive. These results suggested that FGH is not essential but necessary for optimal growth on methanol. This is believed to be the first report of detailed analyses of the FGH1 gene in a methylotrophic yeast strain.

Base Sequence↗
PubMed2003

Formaldehyde fixation contributes to detoxification for growth of a nonmethylotroph, Burkholderia cepacia TM1, on vanillic acid.

During bacterial degradation of methoxylated lignin monomers, such as vanillin and vanillic acid, formaldehyde is released through the reaction catalyzed by vanillic acid demethylase. When Burkholderia cepacia TM1 was grown on vanillin or vanillic acid as the sole carbon source, the enzymes 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) were induced. These enzymes were also expressed during growth on Luria-Bertani medium containing formaldehyde. To understand the roles of these enzymes, the hps and phi genes from a methylotrophic bacterium, Methylomonas aminofaciens 77a, were introduced into B. cepacia TM1. The transformant strain constitutively expressed the genes for HPS and PHI, and these activities were two- or threefold higher than the activities in the wild strain. Incorporation of [14C]formaldehyde into the cell constituents was increased by overexpression of the genes. Furthermore, the degradation of vanillic acid and the growth yield were significantly improved at a high concentration of vanillic acid (60 mM) in the transformant strain. These results suggest that HPS and PHI play significant roles in the detoxification and assimilation of formaldehyde. This is the first report that enhancement of the HPS/PHI pathway could improve the degradation of vanillic acid in nonmethylotrophic bacteria.

Aldehyde-Lyases↗
PubMed2003

Propane monooxygenase and NAD+-dependent secondary alcohol dehydrogenase in propane metabolism by Gordonia sp. strain TY-5.

A new isolate, Gordonia sp. strain TY-5, is capable of growth on propane and n-alkanes with C(13) to C(22) carbon chains as the sole source of carbon. In whole-cell reactions, significant propane oxidation to 2-propanol was detected. A gene cluster designated prmABCD, which encodes the components of a putative dinuclear-iron-containing multicomponent monooxygenase, including the large and small subunits of the hydroxylase, an NADH-dependent acceptor oxidoreductase, and a coupling protein, was cloned and sequenced. A mutant with prmB disrupted (prmB::Kan(r)) lost the ability to grow on propane, and Northern blot analysis revealed that polycistronic transcription of the prm genes was induced during its growth on propane. These results indicate that the prmABCD gene products play an essential role in propane oxidation by the bacterium. Downstream of the prm genes, an open reading frame (adh1) encoding an NAD(+)-dependent secondary alcohol dehydrogenase was identified, and the protein was purified and characterized. The Northern blot analysis results and growth properties of a disrupted mutant (adh1::Kan(r)) indicate that Adh1 plays a major role in propane metabolism. Two additional NAD(+)-dependent secondary alcohol dehydrogenases (Adh2 and Adh3) were also found to be involved in 2-propanol oxidation. On the basis of these results, we conclude that Gordonia sp. strain TY-5 oxidizes propane by monooxygenase-mediated subterminal oxidation via 2-propanol.

Alcohol Dehydrogenase↗