Cytochrome c peroxidase from a methylotrophic yeast: physiological role and isolation (original) (raw)
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Yeast
The effect of various carbon compounds on the synthesis of alcohol oxidase in a medium with methanol was studied in the wild type strain of Pichia pinus as well as in gcrl and ecrl mutants defective in glucose and ethanol repression of methanol metabolic enzymes, respectively. Compounds repressing the synthesis of alcohol oxidase in the wild type strain were divided into four groups. Repression of alcohol oxidase by compounds of the first group (glucose, fructose, mannose, galactose, L-sorbose and xylose) was impaired only in the gcrl mutant and that by compounds of the second group (ethanol, acetate, 2-oxoglutarate and erythritol) only in the ecrl mutant. Repression by compounds of the third group (malate, dihydroxyacetone) was not impaired in both these regulatory mutants and that by compounds of the fourth group (succinate, fumarate, L-arabinose, sorbitol, salicin, xylitol and cellobiose) was partially reduced in both gcrl and ecrl strains.
Yeast, 1987
The effect of various carbon compounds on the synthesis of alcohol oxidase in a medium with methanol was studied in the wild type strain of Pichia pinus as well as in gcrl and ecrl mutants defective in glucose and ethanol repression of methanol metabolic enzymes, respectively. Compounds repressing the synthesis of alcohol oxidase in the wild type strain were divided into four groups. Repression of alcohol oxidase by compounds of the first group (glucose, fructose, mannose, galactose, L-sorbose and xylose) was impaired only in the gcrl mutant and that by compounds of the second group (ethanol, acetate, 2-oxoglutarate and erythritol) only in the ecrl mutant. Repression by compounds of the third group (malate, dihydroxyacetone) was not impaired in both these regulatory mutants and that by compounds of the fourth group (succinate, fumarate, L-arabinose, sorbitol, salicin, xylitol and cellobiose) was partially reduced in both gcrl and ecrl strains.
Biotechnology Progress, 1992
Single shifts in growth rate were imposed upon continuous cultures of the methylotrophic yeast Hansensula polymorpha growing under carbon limitation on 4 g/L methanol and 1 g/L glucose. Concentrations of system level parameters (glucose, methanol, and cell biomass) and intracellular components (methanol oxidase and hexokinase specific activity, methanol oxidase specific mRNA content, and the ratio of flavin adenine dinucleotide bound to methanol oxidase) suggest that the reduction in methanol oxidase specific activity upon a shift to a higher growth rate is limited, not a t the transcription level, but rather posttranslationally during peroxisome assembly. Subsequent [35Slmethionine pulse-chase techniques and immunoprecipitation of methanol oxidase monomers and octamers indicate, upon a dilution rate shift from 0.2 to 0.35 h-l, that monomeric methanol oxidase fails to polymerize, terminating peroxisome formation. Those peroxisomes formed at the lower growth rate slowly disintegrate at the higher growth rate, releasing bound flavin adenine dinucleotide.
Mutant Hansenula polymorpha Strain with Constitutive Alcohol Oxidase and Peroxisome Biosynthesis
Zeitschrift für Naturforschung C, 2002
A mutant of the methylotrophic yeast Hansenula polymorpha with constitutive alcohol oxidase (AOX) and peroxisome biosynthesis was obtained after UV treatment followed by cell plating on a medium containing methanol and 2-deoxy-D-glucose (DOG). DOG-resistant colonies of mutants were insensitive to catabolic repression by glucose and methanol. A selection procedure is described that allows the isolation of a mutant exhibiting a constitutive phenotype of AOX involved in methanol utilization. Furthermore, additional features of the constitutive presence of peroxisomes are demonstrated. 562 DOG-resistant colonies were tested, 24 of them demonstrating constitutive AOX formation. Based on quantitative analysis, one of the strains Ð DOG-13 was selected and its growth, biochemical and ultrastructural characteristics were examined. Its specific enzyme activity when cultivated on a yeast nitrogen base + 1% glucose (YNB + 1% Glucose) was found to reach 145 nmol.min Ð1 .mg Ð1 protein (compared to zero of the parent strain) after he 20 th hour of cultivation. This was confirmed by fine-structure analysis, showing typical peroxisomes, which number and size increased with the enzyme activity. This study demonstrates a constitutive AOX and peroxisome biosynthesis by the mutant strain H. polymorpha DOG-13 obtained.
Targeting signal of the peroxisomal catalase in the methylotrophic yeastHansenula polymorpha
FEBS Letters, 1992
The mcthylotrophic yeast, Hunscrtulu pol~ntorplta, harbours a unique catalase (EC 1.1 I, I .6), which is essential for growth on methanol as a carbon source and is located in peroxisomes. Its corresponding gene has been cloned and the nuclcotidc sequence determined. The deduced amino acid sequence displayed the tripeptide serinc-lysine-isaleucine at the extreme C-terminus, which is similar to sequences of other peroxisomal targeting signals, Exchange of the ultimate amino acid, isoleucinc, of catalase for serine rcvealcd a cytosolic enzyme activity and a concomitant loss of peroxisome function. We concluded that the tripeptide is csscntial for targeting of catalase in H. polymorph.
Study of Catalase Enzyme in Methylotrophic Yeasts
Biotechnology & Biotechnological Equipment, 2008
Methylotrophic yeasts possess a respiratory type of metabolism and during growth an accumulation of potentially cytotoxic species (O 2-) and hydrogen peroxide (H 2 O 2) takes place (13, 27). The catalase [E.C.1.11.1.6] and superoxide dismutase [E.C.1.15.1.1] enzymes play a key role in cellular defense against the reactive species (9). The intracellular localization of catalase enzyme has been under debate for many years. It is a topic wildly investigated and the initial localization in peroxisomes has been determined by a series of cytochemical and biochemical studies which also describe its position in other cellular compartments. This approach has been used by number of authors (8, 18, 20, 32). Michailova et al. (20) have isolated a pure heavy mitochondrial fraction from Candida boidinii and have provided evidences for catalase activity. At present it is well documented with Saccharomyces cerevisiae yeast cells that mitochondria possess catalase A enzyme (24). It has been shown that catalase A, although primary considered as a peroxisomal protein, could also be independently target to mitochondria (25). Given this data, it is important to perform broader investigations on catalase enzyme in methylotrophic yeasts. In the present work, we have studied the mitochondrial localization of catalase using three different strains methylotrophic yeasts: Pichia pastoris, Pichia pini and Hansenula polymorpopha and demonstrated that similarly to S. cerevisiae yeast, methylotrophic ones possess constitutive transport of catalase into both organelles. Materials and Methods Microorganisms and growth conditions The yeasts used in this investigation were, as follows: Pichia pastoris X-33 (Invitrogen), Hansenula polymorpha CBS 4732 and Pichia pini NBIMCC 8360. The strains were cultivated in liquid YP medium (1% Yeast Extract, 1% Bacto-Peptone) supplemented either with 2 % glucose (YPD), 1 % methanol (YPM) or 1 % glycerol (YPG) at 30 o C on a reciprocal shaker (204 rpm). Cell-free extract preparation Cells from 6, 12, 20, 30, 48 and 72 h of cultivation were harvested by centrifugation at 800 x g for 10 minutes and washed twice with distilled H 2 O. Cell wall disruption was carried out by spheroplasting according to the procedure of Defontaine et al. (3). The cell debris was removed by centrifugation at 1000 g and 4 • C for 10 min and the cell free extracts were triply frozen and thawed to break open organelles, and centrifuged at 15 000 g and 4 • C for 10 min. The supernatants obtained were used for enzymatic analyses. Subcellular fractionation and Nycodenz gradients For cell fractionation, yeast cells were grown 20 h to reach end of logarithmic phase. Spheroplasts were generated by the procedure of Defontaine et al. (3) and unlysed cells, nuclei and cell debris were removed by centrifugation at 1000 g and 4 • C for 10 min. The supernatant containing the crude yeast cell organelles was again centrifuged at 25 000 g and 4 • C for 20 min, and the crude organelle fraction was resuspended in a total
Biotechnology for Biofuels
Background: Ogataea (Hansenula) polymorpha is one of the most thermotolerant xylose-fermenting yeast species reported to date. Several metabolic engineering approaches have been successfully demonstrated to improve hightemperature alcoholic fermentation by O. polymorpha. Further improvement of ethanol production from xylose in O. polymorpha depends on the identification of bottlenecks in the xylose conversion pathway to ethanol. Results: Involvement of peroxisomal enzymes in xylose metabolism has not been described to date. Here, we found that peroxisomal transketolase (known also as dihydroxyacetone synthase) and peroxisomal transaldolase (enzyme with unknown function) in the thermotolerant methylotrophic yeast, Ogataea (Hansenula) polymorpha, are required for xylose alcoholic fermentation, but not for growth on this pentose sugar. Mutants with knockout of DAS1 and TAL2 coding for peroxisomal transketolase and peroxisomal transaldolase, respectively, normally grow on xylose. However, these mutants were found to be unable to support ethanol production. The O. polymorpha mutant with the TAL1 knockout (coding for cytosolic transaldolase) normally grew on glucose and did not grow on xylose; this defect was rescued by overexpression of TAL2. The conditional mutant, pYNR1-TKL1, that expresses the cytosolic transketolase gene under control of the ammonium repressible nitrate reductase promoter did not grow on xylose and grew poorly on glucose media supplemented with ammonium. Overexpression of DAS1 only partially restored the defects displayed by the pYNR1-TKL1 mutant. The mutants defective in peroxisome biogenesis, pex3Δ and pex6Δ, showed normal growth on xylose, but were unable to ferment this sugar. Moreover, the pex3Δ mutant of the non-methylotrophic yeast, Scheffersomyces (Pichia) stipitis, normally grows on and ferments xylose. Separate overexpression or co-overexpression of DAS1 and TAL2 in the wild-type strain increased ethanol synthesis from xylose 2 to 4 times with no effect on the alcoholic fermentation of glucose. Overexpression of TKL1 and TAL1 also elevated ethanol production from xylose. Finally, co-overexpression of DAS1 and TAL2 in the best previously isolated O. polymorpha xylose to ethanol producer led to increase in ethanol accumulation up to 16.5 g/L at 45 °C; or 30-40 times more ethanol than is produced by the wild-type strain.
Molecular and cellular biology, 1989
The crystalloid core in peroxisomes of the methylotrophic yeast Hansenula polymorpha is composed of the octameric flavoprotein methanol oxidase (MOX). We transformed yeast cells with a high-copy-number vector harboring the cloned MOX gene in order to study the effects on regulation, protein import, and peroxisome biosynthesis. In transformed wild-type cells, no increase in expression of MOX was detectable. Mutants defective in MOX activity were isolated by a specific selection procedure. Two structural MOX mutants are described that allow overproduction of a fully active enzyme upon transformation at quantities of about two-thirds of the total cellular protein. The overproduced protein was imported into peroxisomes, altering their morphology (in thin sections) and stability in cell lysates; the organelles showed a tendency to form rectangular bodies, and their lumina were completely filled with the crystalloid structure. The overall size of the peroxisomes was increased severalfold ...