Regulation of alcohol oxidase synthesis in Hansenula polymorpha: Oversynthesis during growth on mixed substrates and induction by methanol

1980 ◽  
Vol 127 (2) ◽  
pp. 119-124 ◽  
Author(s):  
L. Eggeling ◽  
H. Sahm
Keyword(s):  
Hansenula Polymorpha ◽  
Alcohol Oxidase ◽  
Mixed Substrates

scholarly journals Pyruvate Carboxylase Is an Essential Protein in the Assembly of Yeast Peroxisomal Oligomeric Alcohol Oxidase

2003 ◽  
Vol 14 (2) ◽  
pp. 786-797 ◽  
Author(s):  
Paulina Ozimek ◽  
Ralf van Dijk ◽  
Kantcho Latchev ◽  
Carlos Gancedo ◽  
Dong Yuan Wang ◽  
...  
Keyword(s):  
Pyruvate Carboxylase ◽  
Tricarboxylic Acid Cycle ◽  
Hansenula Polymorpha ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Tricarboxylic Acid ◽  
Matrix Proteins ◽  
Growth Media ◽  
Fad Binding

Hansenula polymorpha ass3 mutants are characterized by the accumulation of inactive alcohol oxidase (AO) monomers in the cytosol, whereas other peroxisomal matrix proteins are normally activated and sorted to peroxisomes. These mutants also have a glutamate or aspartate requirement on minimal media. Cloning of the corresponding gene resulted in the isolation of the H. polymorpha PYC gene that encodes pyruvate carboxylase (HpPyc1p). HpPyc1p is a cytosolic, anapleurotic enzyme that replenishes the tricarboxylic acid cycle with oxaloacetate. The absence of this enzyme can be compensated by addition of aspartate or glutamate to the growth media. We show that HpPyc1p protein but not the enzyme activity is essential for import and assembly of AO. Similar results were obtained in the related yeast Pichia pastoris. In vitro studies revealed that HpPyc1p has affinity for FAD and is capable to physically interact with AO protein. These data suggest that in methylotrophic yeast pyruvate carboxylase plays a dual role in that, besides its well-characterized metabolic function as anapleurotic enzyme, the protein fulfils a specific role in the AO sorting and assembly process, possibly by mediating FAD-binding to AO monomers.

scholarly journals Flavin adenine dinucleotide binding is the crucial step in alcohol oxidase assembly in the yeast Hansenula polymorpha

Yeast ◽  
1996 ◽  
Vol 12 (10) ◽  
pp. 917-923 ◽  
Author(s):  
Melchior E. Evers ◽  
Vladimir Titorenko ◽  
Wim Harder ◽  
Ida van der Klei ◽  
Marten Veenhuis
Keyword(s):  
Flavin Adenine Dinucleotide ◽  
Hansenula Polymorpha ◽  
Alcohol Oxidase ◽  
Crucial Step

scholarly journals Routing of Hansenula polymorpha Alcohol Oxidase: An Alternative Peroxisomal Protein-sorting Machinery

2004 ◽  
Vol 15 (3) ◽  
pp. 1347-1355 ◽  
Author(s):  
Katja Gunkel ◽  
Ralf van Dijk ◽  
Marten Veenhuis ◽  
Ida J. van der Klei
Keyword(s):  
Amino Acids ◽  
Protein Translocation ◽  
Hansenula Polymorpha ◽  
Alcohol Oxidase ◽  
N Terminus ◽  
Dependent Protein ◽  
Peptides Synthesis ◽  
Translocation Pathway ◽  
Tpr Domain ◽  
Fad Binding

Import of Hansenula polymorpha alcohol oxidase (AO) into peroxisomes is dependent on the PTS1 receptor, HpPex5p. The PTS1 of AO (-LARF) is sufficient to direct reporter proteins to peroxisomes. To study AO sorting in more detail, strains producing mutant AO proteins were constructed. AO containing a mutation in the FAD binding fold was mislocalized to the cytosol. This indicates that the PTS1 of AO is not sufficient for import of AO. AO protein in which the PTS1 was destroyed (-LARA) was normally sorted to peroxisomes. Moreover, C-terminal deletions of up to 16 amino acids did not significantly affect AO import, indicating that the PTS1 was not necessary for targeting. Consistent with these observations we found that AO import occurred independent from the C-terminal TPR-domain of HpPex5p, known to bind PTS1 peptides. Synthesis of the N-terminal domain (amino acids 1-272) of HpPex5p in pex5 cells restored AO import, whereas other PTS1 proteins were mislocalized to the cytosol. These data indicate that AO is imported via a novel HpPex5p-dependent protein translocation pathway, which does not require the PTS1 of AO and the C-terminal TPR domains of HpPex5p, but involves FAD binding and the N-terminus of HpPex5p.

scholarly journals Substructure of crystalline peroxisomes in methanol-grown Hansenula polymorpha: evidence for an in vivo crystal of alcohol oxidase.

1981 ◽  
Vol 1 (10) ◽  
pp. 949-957 ◽  
Author(s):  
M Veenhuis ◽  
W Harder ◽  
J P van Dijken ◽  
F Mayer
Keyword(s):  
Osmotic Shock ◽  
Hansenula Polymorpha ◽  
Three Dimensional ◽  
Alcohol Oxidase ◽  
Molecular Architecture ◽  
Three Dimensional Model ◽  
Structural Element ◽  
Free Mobility ◽  
Crystalline Matrix

The substructural organization of completely crystalline peroxisomes present in Hansenula polymorpha cells grown under methanol limitation in a chemostat was investigated by different cytochemical and ultrastructural techniques. Time-dependent cytochemical staining experiments indicated that activities of the two main constituents of these organelles, namely, alcohol oxidase and catalase, were present throughout the crystalline matrix. Catalase was completely removed from isolated peroxisomes by osmotic shock treatment. After such treatment, the ultrastructure of the crystalline matrix of the organelles remained virtually intact. Because alcohol oxidase activity was still present in this matrix, it was concluded that alcohol oxidase protein is the only structural element of the peroxisomal crystalloids. The molecular architecture of the crystalloids was investigated in ultrathin cryosections which permitted recognition of individual molecules in the crystalline matrix. Depending on the plane of sectioning, different crystalline patterns were observed. Tilting experiments indicated that these images were caused by superposition of octameric alcohol oxidase molecules arranged in a tetragonal lattice. A three-dimensional model of the crystalloid is presented. The repeating unit of this structure is composed of four alcohol oxidase molecules. The crystalloid represents an open structure, which may explain the observed free mobility of catalase molecules.

In vivo inactivation of peroxisomal alcohol oxidase in Hansenula polymorpha by KCN is an irreversible process

1988 ◽  
Vol 151 (1) ◽  
pp. 26-33 ◽  
Author(s):  
Ida J. van der Klei ◽  
Marten Veenhuis ◽  
Klaas Nicolay ◽  
Wim Harder
Keyword(s):  
Irreversible Process ◽  
Hansenula Polymorpha ◽  
Alcohol Oxidase

Thermal and operational stabilities of Hansenula polymorpha alcohol oxidase

2004 ◽  
Vol 27 (1) ◽  
pp. 37-45 ◽  
Author(s):  
A.M. Azevedo ◽  
J.M.S. Cabral ◽  
D.M.F. Prazeres ◽  
T.D. Gibson ◽  
L.P. Fonseca
Keyword(s):  
Hansenula Polymorpha ◽  
Alcohol Oxidase

Electron Microscopy of Alcohol Oxidase Crystals from Pichia Pastoris

1990 ◽  
Vol 48 (1) ◽  
pp. 108-109
Author(s):  
Janet Vonck ◽  
Ernst F.J. van Bruggen
Keyword(s):  
Pichia Pastoris ◽  
Hansenula Polymorpha ◽  
Alcohol Oxidase ◽  
Unit Cell ◽  
Electron Microscopic ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cell Dimensions ◽  
Microscopic Studies ◽  
Unit Cell Dimensions

Several yeast species are able to grow on methanol. When they are grown in a methanol-restricted culture, their peroxisomes contain large crystalline inclusions, consisting of alcohol oxidase (AOX). A monomer of AOX has a molecular weight of ca. 74,000. Inside the peroxisome, AOX occurs as octamers.Electron microscopic studies of AOX from Hansenula polymorpha have revealed that the eight subunits are slightly elongated and form two layers of four, which are twisted relative to each other. The molecule measures ca. 12 nm in all directions.Recently, crystals suitable for x-ray diffraction have been formed of AOX from Pichia pastoris . The space group is P21, with unit cell dimensions a=157.3Å, b=171.45Å, c=231.6Å, β=94°. These dimensions indicate that the unit cell contains four octamers, too much to solve by x-ray crystallography alone. Therefore, we have started an EM study of the crystals, to get information about the organization of the molecules in the crystal lattice.

Production of catalase-free alcohol oxidase by Hansenula polymorpha

1988 ◽  
Vol 28 (1) ◽  
pp. 14-19 ◽  
Author(s):  
Marco L. F. Giuseppin ◽  
Hendrikus M. J. van Eijk ◽  
Cornelis Verduyn ◽  
Iddo Bante ◽  
Johannes P. van Dijken
Keyword(s):  
Hansenula Polymorpha ◽  
Alcohol Oxidase ◽  
Free Alcohol

scholarly journals Alcohol Oxidase Is a Novel Pathogenicity Factor for Cladosporium fulvum, but Aldehyde Dehydrogenase Is Dispensable

2001 ◽  
Vol 14 (3) ◽  
pp. 367-377 ◽  
Author(s):  
G. Segers ◽  
N. Bradshaw ◽  
D. Archer ◽  
K. Blissett ◽  
R. P. Oliver
Keyword(s):  
Hansenula Polymorpha ◽  
Infected Plant ◽  
Alcohol Oxidase ◽  
Carbon Starvation ◽  
Cladosporium Fulvum ◽  
In Planta ◽  
Western Blots ◽  
Pathogenicity Factor ◽  
Fungal Genes

Cladosporium fulvum is a mitosporic ascomycete pathogen of tomato. A study of fungal genes expressed during carbon starvation in vitro identified several genes that were up regulated during growth in planta. These included genes predicted to encode acetaldehyde dehydrogenase (Aldh1) and alcohol oxidase (Aox1). An Aldh1 deletion mutant was constructed. This mutant lacked all detectable ALDH activity, had lost the ability to grow with ethanol as a carbon source, but was unaffected in pathogenicity. Aox1 expression was induced by carbon starvation and during the later stages of infection. The alcohol oxidase enzyme activity has broadly similar properties (Km values, substrate specificity, pH, and heat stability) to yeast enzymes. Antibodies raised to Hansenula polymorpha alcohol oxi-dase (AOX) detected antigens in Western blots of starved C. fulvum mycelium and infected plant material. Antigen reacting with the antibodies was localized to organelles resembling peroxisomes in starved mycelium and infected plants. Disruption mutants of Aox1 lacked detectable AOX activity and had markedly reduced pathogenicity as assayed by two different measures of fungal growth. These results identify alcohol oxidase as a novel pathogenicity factor and are discussed in relation to peroxisomal metabolism of fungal pathogens during growth in planta.

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