flavin-adenine-dinucleotide and alcohol-oxidase

This review considers quinone-dependent alcohol dehydrogenases and FAD-dependent alcohol oxidases, enzymes that are present in numerous methylotrophic eu- and prokaryotes and significantly differ in their primary and quaternary structure. The cofactors of the enzymes are bound to the protein polypeptide chain through ionic and hydrophobic interactions. Microorganisms containing these enzymes are described. Methods for purification of the enzymes, their physicochemical properties, and spatial structures are considered. The supposed mechanism of action and practical application of these enzymes as well as their producers are discussed.

Topics: Alcohol Dehydrogenase; Alcohol Oxidoreductases; Animals; Benzoquinones; Flavin-Adenine Dinucleotide; Humans; Models, Molecular; Molecular Structure

Alcohol oxidase (AO) is the key enzyme of methanol metabolism in methylotrophic yeast species. It catalyses the first step of methanol catabolism, namely its oxidation to formaldehyde with concomitant production of hydrogen peroxide. In its mature active form, AO is a molecule of high molecular mass (600 kDa) that consists of eight identical subunits, each of which carry one non-covalently bound flavin adenine nucleotide (FAD) molecule as the prosthetic group. In vivo, the protein is compartmentalized into special cell organelles, termed peroxisomes. AO is an abundant protein and its synthesis is strictly regulated by repression/derepression and induction mechanisms that occur at the transcriptional level. Various aspects of its sorting and assembly/activation render AO a unique protein. Recent developments of AO synthesis, sorting and assembly/activation are highlighted in this paper.

Topics: Alcohol Oxidoreductases; Flavin-Adenine Dinucleotide; Flavoproteins; Peroxisomes; Yeasts

Alcohol oxidase (AO) catalyses the first step of methanol metabolism in yeasts. In vivo the enzyme is compartmentalized in special cell compartments, called peroxisomes. The enzyme along with the organelles are induced during growth of methylotrophic yeasts on methanol as the sole carbon source. Like all other peroxisomal matrix proteins, AO is encoded by a nuclear gene. Expression of the protein is regulated by a repression/derepression mechanism, but also by induction. Inactive monomeric precursor protein is synthesized in the cytosol and subsequently imported post-translationally into peroxisomes without further processing. Assembly into the active homo-octameric enzyme and binding of the prosthetic group flavin adenine dinucleotide occurs inside the organelle. When enhanced concentration of octameric alcohol oxidase are present in the organelles, the enzyme may form a crystalloid. Oligomerization is not dependent on translocation of AO precursors into their target organelle since octameric, active AO is detected in the cytosol and nucleus of peroxisome-deficient mutants of Hansenula polymorpha: at high expression rates large cytosolic AO crystalloids are formed, which occasionally are also encountered inside the nucleus of such mutants. This paper summarizes recent findings and views on the mechanisms involved in synthesis, import, assembly and crystallization of this important peroxisomal enzyme.

Topics: Alcohol Oxidoreductases; Amino Acid Sequence; Crystallization; Flavin-Adenine Dinucleotide; Gene Expression Regulation, Fungal; Genes, Fungal; Methanol; Microbodies; Molecular Sequence Data; Yeasts

Alcohol oxidase (AOX) is an important flavin adenine dinucleotide (FAD) dependent oxidoreductase, which is responsible for converting methanol into formaldehyde and hydrogen peroxide for the growth of methylotrophic yeast Candida boidinii. Although AOX plays a crucial role in methanol catabolism, the experimental structure of AOX from Candida boidinii has not been elucidated. This study reports the first complete in silico model of AOX from C. boidinii. This paper also reports the AOX structure modeled using the threading approach, followed by structure analysis and molecular dynamics simulation. The modeled structure was compared with the aryl alcohol oxidase structure (a glucose-methanol-choline family member, pdbID: 3fim). A docking study was performed to analyze the interaction between AOX and its cofactor FAD. The AOX modeled structure also exhibited high similarity with respect to the FAD binding sites, which are the substrate binding sites as seen with 3fim. It was observed that the adenosine part of FAD was deeply buried inside AOX while the isoalloxazine ring stuck to the surface. This paper reports the interaction of selective proton ionophores (CCCP and DNP) with AOX and also reports their binding sites. These proton ionophores showed competitive binding with FAD. The occupancy of the FAD binding sites by the proton ionophore may lead to blocking of the entry of FAD and thereby disruption of AOX import into peroxisomes.

Topics: Alcohol Oxidoreductases; Algorithms; Binding Sites; Binding, Competitive; Candida; Flavin-Adenine Dinucleotide; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Molecular Docking Simulation; Molecular Dynamics Simulation; Principal Component Analysis; Protein Binding; Proton Ionophores

FAD-dependent alcohol oxidases (AOX) are key enzymes of methylotrophic organisms that can utilize lower primary alcohols as sole source of carbon and energy. Here we report the crystal structure analysis of the methanol oxidase AOX1 from Pichia pastoris. The crystallographic phase problem was solved by means of Molecular Replacement in combination with initial structure rebuilding using Rosetta model completion and relaxation against an averaged electron density map. The subunit arrangement of the homo-octameric AOX1 differs from that of octameric vanillyl alcohol oxidase and other dimeric or tetrameric alcohol oxidases, due to the insertion of two large protruding loop regions and an additional C-terminal extension in AOX1. In comparison to other alcohol oxidases, the active site cavity of AOX1 is significantly reduced in size, which could explain the observed preference for methanol as substrate. All AOX1 subunits of the structure reported here harbor a modified flavin adenine dinucleotide, which contains an arabityl chain instead of a ribityl chain attached to the isoalloxazine ring.

Topics: Alcohol Oxidoreductases; Crystallography, X-Ray; Flavin-Adenine Dinucleotide; Fungal Proteins; Pichia; Protein Structure, Quaternary; Protein Structure, Tertiary

The first step in methanol metabolism in methylotrophic yeasts, the oxidation of methanol and higher alcohols with molecular oxygen to formaldehyde and hydrogen peroxide, is catalysed by alcohol oxidase (AOX), a 600-kDa homo-octamer containing eight FAD cofactors. When these yeasts are grown with methanol as the carbon source, AOX forms large crystalline arrays in peroxisomes. We determined the structure of AOX by cryo-electron microscopy at a resolution of 3.4 Å. All residues of the 662-amino acid polypeptide as well as the FAD are well resolved. AOX shows high structural homology to other members of the GMC family of oxidoreductases, which share a conserved FAD binding domain, but have different substrate specificities. The preference of AOX for small alcohols is explained by the presence of conserved bulky aromatic residues near the active site. Compared to the other GMC enzymes, AOX contains a large number of amino acid inserts, the longest being 75 residues. These segments are found at the periphery of the monomer and make extensive inter-subunit contacts which are responsible for the very stable octamer. A short surface helix forms contacts between two octamers, explaining the tendency of AOX to form crystals in the peroxisomes.

Topics: Alcohol Oxidoreductases; Catalytic Domain; Cryoelectron Microscopy; Flavin-Adenine Dinucleotide; Models, Molecular; Pichia; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; Structure-Activity Relationship; Substrate Specificity

The alcohol oxidase (AOx) cDNA from Aspergillus terreus MTCC6324 with an open reading frame (ORF) of 2001 bp was constructed from n-hexadecane induced cells and expressed in Escherichia coli with a yield of ∼4.2 mg protein g-1 wet cell. The deduced amino acid sequences of recombinant rAOx showed maximum structural homology with the chain B of aryl AOx from Pleurotus eryngii. A functionally active AOx was achieved by incubating the apo-AOx with flavin adenine dinucleotide (FAD) for ∼80 h at 16°C and pH 9.0. The isoelectric point and mass of the apo-AOx were found to be 6.5±0.1 and ∼74 kDa, respectively. Circular dichroism data of the rAOx confirmed its ordered structure. Docking studies with an ab-initio protein model demonstrated the presence of a conserved FAD binding domain with an active substrate binding site. The rAOx was specific for aryl alcohols and the order of its substrate preference was 4-methoxybenzyl alcohol >3-methoxybenzyl alcohol>3, 4-dimethoxybenzyl alcohol > benzyl alcohol. A significantly high aggregation to ∼1000 nm (diameter) and catalytic efficiency (kcat/Km) of 7829.5 min-1 mM-1 for 4-methoxybenzyl alcohol was also demonstrated for rAOx. The results infer the novelty of the AOx and its potential biocatalytic application.

Topics: Alcohol Oxidoreductases; Alcohols; Amino Acid Sequence; Aspergillus; Base Sequence; Biophysical Phenomena; Circular Dichroism; DNA, Complementary; Electrophoresis, Gel, Two-Dimensional; Enzyme Stability; Flavin-Adenine Dinucleotide; Gene Amplification; Hydrogen-Ion Concentration; Kinetics; Light; Microsomes; Molecular Docking Simulation; Molecular Sequence Data; Peptide Mapping; Peptides; Proteomics; Recombinant Proteins; Scattering, Radiation; Spectrometry, Fluorescence; Substrate Specificity; Temperature

We report on the rerouting of peroxisomal alcohol oxidase (AO) to the secretory pathway of Hansenula polymorpha. Using the leader sequence of the Saccharomyces cerevisiae mating factor alpha (MFalpha) as sorting signal, AO was correctly sorted to the endoplasmic reticulum (ER), which strongly proliferated in these cells. The MFalpha presequence, but not the prosequence, was cleaved from the protein. AO protein was present in the ER as monomers that lacked FAD, and hence was enzymatically inactive. Furthermore, the recombinant AO protein was subject to gradual degradation, possibly because the protein did not fold properly. However, when the S. cerevisiae invertase signal sequence (ISS) was used, secretion of AO protein was observed in conjunction with bulk of the protein being localized to the ER. The amount of secreted AO protein increased with increasing copy numbers of the AO expression cassette integrated into the genome. The secreted AO protein was correctly processed and displayed enzyme activity.

Topics: Alcohol Oxidoreductases; Endoplasmic Reticulum; Flavin-Adenine Dinucleotide; Fungal Proteins; Microscopy, Immunoelectron; Peroxisomes; Pichia; Protein Precursors; Protein Sorting Signals; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

Peroxisome development is a dynamic process that is not yet completely understood. We use the methylotrophic yeast Hansenula polymorpha as model in our studies on peroxisome homeostasis. Cells of this species may contain different types of peroxisomes that differ in protein composition and capacity to incorporate matrix proteins. This protein import machinery is highly flexible and can accommodate unfolded and complex folded proteins.

Topics: Alcohol Oxidoreductases; Flavin-Adenine Dinucleotide; Peroxisomal Targeting Signal 2 Receptor; Peroxisome-Targeting Signal 1 Receptor; Peroxisomes; Pichia; Protein Transport; Receptors, Cytoplasmic and Nuclear

Using spectroscopic techniques we studied the effect of the nucleophilic reagents cyanide, cyanate and thiocyanate on three flavo-oxidases namely alcohol oxidase (AO), glucose oxidase (GOX) and D-amino acid oxidase (DAOX). All three ions, added at concentrations in the mM range, caused release of the flavin adenine dinucleotide (FAD) co-factors from the enzyme molecules. In the case of AO this was accompanied by significant conformational perturbations, which was not observed for GOX and DAOX. As suggested from fluorescence, absorption and circular dichroism spectral changes at least one phenolic hydroxyl group became ionized upon FAD release from AO and a new class of Trp residues, fluorescent only in apo-AO protein, was demasked.

Topics: Alcohol Oxidoreductases; Circular Dichroism; Cyanates; Cyanides; D-Amino-Acid Oxidase; Flavin-Adenine Dinucleotide; Flavoproteins; Glucose Oxidase; Indicators and Reagents; Oxidoreductases; Spectrometry, Fluorescence; Spectrophotometry; Thiocyanates

The digestive gland and other tissues of several species of terrestrial gastropod mollusc contain an aliphatic alcohol oxidase activity (EC1.1.3.13). The enzyme is FAD dependent, consumes oxygen and generates hydrogen peroxide and the corresponding aldehyde. Saturated primary alcohols are favoured as substrates with octanol preferred with an apparent Km of 3-4 microM. The activity is clearly distinguishable from previously reported molluscan aromatic alcohol oxidase (EC1.1.3.7) on the basis of FAD dependence, sensitivity to heat treatment and high salt concentration and with regard to substrate preferences. The aliphatic alcohol oxidase is membrane associated and most likely localised to the endoplasmic reticulum. Extraction of membranes with 1% Igipal solubilises the enzyme in active form. This enzyme is a further example of an oxidase apparently restricted to molluscs.

Topics: Alcohol Oxidoreductases; Alcohols; Animals; Digestive System; Flavin-Adenine Dinucleotide; Mollusca; Oxygen; Subcellular Fractions; Substrate Specificity

Aryl-alcohol oxidase (AAO), an FAD-dependent enzyme involved in lignin degradation, has been cloned from Pleurotus eryngii. The AAO protein is composed of 593 amino acids, 27 of which form a signal peptide. It shows 33% sequence identity with glucose oxidase from Aspergillus niger and lower homology with other oxidoreductases. The predicted secondary structures of both enzymes are very similar. For AAO, it is predicted to contain 13 putative alpha-helices and two major beta-sheets, each of the putative beta-sheets formed by six beta-strands. The ADP binding site and the signature-2 consensus sequence of the glucose-methanol-choline (GMC) oxidoreductases were also present. Moreover, residues potentially involved in catalysis and substrate binding were identified in the vicinity of the flavin ring. They include two histidines (H502 and H546) and several aromatic residues (Y78, Y92 and F501), as reported in other FAD oxidoreductases.

Topics: Alcohol Oxidoreductases; Amino Acid Sequence; Aspergillus niger; Binding Sites; Consensus Sequence; Flavin-Adenine Dinucleotide; Fungal Proteins; Glucose Oxidase; Lignin; Models, Molecular; Molecular Sequence Data; Pleurotus; Protein Structure, Secondary; Sequence Alignment; Sequence Homology, Amino Acid

Alcohol oxidase (AO) is a homo-octameric flavoenzyme which catalyzes methanol oxidation in methylotrophic yeasts. AO protein is synthesized in the cytosol and subsequently sorted to peroxisomes where the active enzyme is formed. To gain further insight in the molecular mechanisms involved in AO activation, we studied spectroscopically native AO from Hansenula polymorpha and Pichia pastoris and three putative assembly intermediates. Fluorescence studies revealed that both Trp and FAD are suitable intramolecular markers of the conformation and oligomeric state of AO. A direct relationship between dissociation of AO octamers and increase in Trp fluorescence quantum yield and average fluorescence lifetime was found. The time-resolved fluorescence of the FAD cofactor showed a rapid decay component which reflects dynamic quenching due to the presence of aromatic amino acids in the FAD-binding pocket. The analysis of FAD fluorescence lifetime profiles showed a remarkable resemblance of pattern for purified AO and AO present in intact yeast cells. Native AO contains a high content of ordered secondary structure which was reduced upon FAD-removal. Dissociation of octamers into monomers resulted in a conversion of beta-sheets into alpha-helices. Our results are explained in relation to a 3D model of AO, which was built based on the crystallographic data of the homologous enzyme glucose oxidase from Aspergillus niger. The implications of our results for the current model of the in vivo AO assembly pathway are discussed.

Topics: Alcohol Oxidoreductases; Circular Dichroism; Flavin-Adenine Dinucleotide; Fluorescence Polarization; Microbodies; Models, Molecular; Pichia; Protein Conformation; Protein Structure, Secondary; Sequence Homology, Amino Acid; Spectrometry, Fluorescence; Tryptophan

The content of modified FAD (mFAD) in isoforms of alcohol oxidase (AO) in the methylotrophic yeast Pichia methanolica MH4 is increased in the stationary growth phase of the culture or under lower dilution rates. The isoform with slower electrophoretic mobility has the higher mFAD content. The results of in vitro experiments and the occurrence of mFAD in AO-lacking mutants of Hansenula polymorpha DL-1 imply a biosynthetic origin of this cofactor. HPLC analysis of tryptic hydrolysates of the first and the ninth isoforms of AO from P. methanolica MH4 revealed two different types of subunits. Possible mechanism of mFAD formation and distribution between the AO isozymes is discussed.

Topics: Alcohol Oxidoreductases; Chromatography, High Pressure Liquid; Flavin-Adenine Dinucleotide; Fungal Proteins; Hydrolysis; Isoenzymes; Pichia

Alcohol oxidase from Candida methanosorbosa was purified about sixfold with a yield of 37.6% from the culture broth of Candida methanosorbosa M-2003. The enzyme preparation was homogeneous on slab gel electrophoresis. The purified enzyme had an optimal pH from 6.0 to 9.0 and was stable in the range 6.0-8.5. Its optimal temperature of reaction was 50 degrees C, and it was stable below 50 degrees C. In the presence of NaN3, the enzyme retained its initial activity at 30 degrees C for 35 days, indicating stability for a long term, so far. The isoelectric point was pH 4.3. Its molecular weight was 620, 000 by gel filtration chromatography and 80,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results indicate that the enzyme consists of 8 subunits.

Topics: Alcohol Oxidoreductases; Candida; Flavin-Adenine Dinucleotide; Hydrogen-Ion Concentration; Isoelectric Point; Molecular Weight; Substrate Specificity

We have studied the role of flavin adenine dinucleotide (FAD) in the in vivo assembly of peroxisomal alcohol oxidase (AO) in the yeast Hansenula polymorpha. In previous studies, using a riboflavin (Rf) autotrophic mutant, an unequivocal judgement could not be made, since Rf-limitation led to a partial block of AO import in this mutant. This resulted in the accumulation of AO precursors in the cytosol where they remained separated from the putative peroxisomal AO assembly factors. In order to circumvent the peroxisomal membrane barrier, we have now studied AO assembly in a peroxisome-deficient/Rf-autotrophic double mutant (delta per1.rif1) of H. polymorpha. By sucrose density centrifugation and native gel electrophoresis, three conformations of AO were detected in crude extracts of delta per1.rif1 cells grown under Rf-limitation, namely active octameric AO and two inactive, monomeric forms. One of the latter forms lacked FAD; this form was barely detectable in extracts wild-type and delta per1 cells, but had accumulated in the cytosol of rif1 cells. The second form of monomeric AO contained FAD; this form was also present in delta per1 cells but absent/very low in wild-type and rif1 cells. In vivo only these FAD-containing monomers associate into the active, octameric protein. We conclude that in H. polymorpha FAD binding to the AO monomer is mediated by a yet unknown peroxisomal factor and represents the crucial and essential step to enable AO oligomerization; the actual octamerization and the eventual crystallization in peroxisomes most probably occurs spontaneously.

Topics: Alcohol Oxidoreductases; Cytosol; Flavin-Adenine Dinucleotide; Microbodies; Mutation; Pichia; Protein Binding; Protein Conformation; Riboflavin

We have studied the in vitro inactivation/dissociation and subsequent reactivation/re-assembly of peroxisomal alcohol oxidases (AO) from the yeasts Hansenula polymorpha and Pichia pastoris. Both proteins are homo-oligomers consisting of eight identical subunits, each containing one FAD as the prosthetic group. They were both rapidly inactivated upon incubation in 80% glycerol, due to their dissociation into the constituting subunits, which however still contained FAD. Dilution of dissociated AO in neutral buffer lead to reactivation of the protein due to AO re-assembly, as was demonstrated by non-denaturing PAGE. After use of mixtures of purified AO from H. polymorpha and P. pastoris active hybrid AO oligomers were formed. When prior to dissociation FAD was chemically removed from AO, reactivation or re-assembly did not occur independent of externally added FAD.

Topics: Alcohol Oxidoreductases; Enzyme Activation; Flavin-Adenine Dinucleotide; Microbodies; Pichia; Protein Conformation; Protein Denaturation

The spatial localization of the coenzyme FAD in the quaternary structure of the alcohol oxidase from the yeast Pichia pinus was studied by tritium planigraphy and ESR methods. In the present paper we measured the specific radioactivity of FAD labelled as a part of the alcohol oxidase complex. The specific-radioactivity ratio for two FAD portions (FMN and AMP) was calculated. ESR experiments show 4 A (0.4 nm) to be the depth of immersion of paramagnetic isoalloxazines into alcohol oxidase octamer molecules. It is suggested that FAD molecules are bound to the surface of the octamer, rather than to the subunit interfaces. The orientation of the prosthetic group FAD in the alcohol oxidase protein is discussed.

Topics: Adenosine Monophosphate; Alcohol Oxidoreductases; Algorithms; Electron Spin Resonance Spectroscopy; Ferricyanides; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Kinetics; Pichia; Protein Binding; Tritium

The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was replaced by a stereochemical analog, which is spontaneously formed from natural FAD in alcohol oxidases from methylotrophic yeasts. Reconstitution of p-hydroxybenzoate hydroxylase from apoprotein and modified FAD is a rapid process complete within seconds. Crystals of the enzyme-substrate complex of modified FAD-containing p-hydroxybenzoate hydroxylase diffract to 2.1 A resolution. The crystal structure provides direct evidence for the presence of an arabityl sugar chain in the modified form of FAD. The isoalloxazine ring of the arabinoflavin adenine dinucleotide (a-FAD) is located in a cleft outside the active site as recently observed in several other p-hydroxybenzoate hydroxylase complexes. Like the native enzyme, a-FAD-containing p-hydroxybenzoate hydroxylase preferentially binds the phenolate form of the substrate (pKo = 7.2). The substrate acts as an effector highly stimulating the rate of enzyme reduction by NADPH (kred > 500 s-1). The oxidative part of the catalytic cycle of a-FAD-containing p-hydroxybenzoate hydroxylase differs from native enzyme. Partial uncoupling of hydroxylation results in the formation of about 0.3 mol of 3,4-dihydroxybenzoate and 0.7 mol of hydrogen peroxide per mol NADPH oxidized. It is proposed that flavin motion in p-hydroxybenzoate hydroxylase is important for efficient reduction and that the flavin "out" conformation is associated with the oxidase activity.

Topics: 4-Hydroxybenzoate-3-Monooxygenase; Alcohol Oxidoreductases; Bacterial Proteins; Crystallography, X-Ray; Flavin-Adenine Dinucleotide; Fungal Proteins; Models, Molecular; Pichia; Pseudomonas fluorescens; Spectrophotometry; Stereoisomerism

The peroxisomal flavoprotein alcohol oxidase (AO) is an octamer (600 kDa) consisting of eight identical subunits, each of which contains one flavin adenine dinucleotide molecule as a cofactor. Studies on a riboflavin (Rf) auxotrophic mutant of the yeast Hansenula polymorpha revealed that limitation of the cofactor led to drastic effects on AO import and assembly as well as peroxisome proliferation. Compared to wild-type control cells Rf-limitation led to 1) reduced levels of AO protein, 2) reduced levels of correctly assembled and activated AO inside peroxisomes, 3) a partial inhibition of peroxisomal protein import, leading to the accumulation of precursors of matrix proteins in the cytosol, and 4) a significant increase in peroxisome number. We argue that the inhibition of import may result from the saturation of a peroxisomal molecular chaperone under conditions that normal assembly of a major matrix protein inside the target organelle is prevented.

Topics: Alcohol Oxidoreductases; Biological Transport, Active; Enzyme Activation; Flavin-Adenine Dinucleotide; Immunohistochemistry; Microbodies; Microscopy, Electron; Molecular Chaperones; Mutation; Pichia; Protein Conformation; Riboflavin

Alcohol oxidase, a major peroxisomal protein of methanol-utilizing yeasts, may possess two different forms of flavin adenine dinucleotide, classical FAD and so-called modified FAD (mFAD). Conversion of FAD into mFAD was observed both in purified preparations of the enzyme and in cells grown in batch and continuous culture. The relative amount of mFAD in the enzyme varied from 5 to 95%, depending on the growth or storage conditions. The presence of mFAD led to a slight decrease in Vmax and a significant (about one order) decrease in the Km of alcohol oxidase with respect to methanol. The kinetics of modification measured in purified preparations of the enzyme obeyed first-order kinetics (k = 0.78 h-1). The modification process was strongly inhibited by methanol, formaldehyde or hydroxylamine. Modification observed in continuous culture under steady state conditions depended on the dilution rate and could also be described as a spontaneous first-order reaction (kapp = 0.27 h-1). FAD modification could only be detected in alcohol oxidase and not in other yeast peroxisomal flavoenzymes, such as D-amino acid oxidase from Candida boidinii.

Topics: Alcohol Oxidoreductases; Candida; Flavin-Adenine Dinucleotide; Formaldehyde; Fungal Proteins; Hydroxylamine; Hydroxylamines; Kinetics; Methanol; Pichia

Alcohol oxidase of methylotrophic yeast is an FAD-containing enzyme. When in its active form, the enzyme is an octamer and located in the peroxisomes. To study the importance of FAD-binding on the activity, octamerization and intracellular localization of the enzyme, alcohol oxidase of Hansenula polymorpha was mutated in its presumed nucleotide-binding domain, which is formed by the N-terminal sequence. Whereas mutations of a glutamic acid residue (E42) reduced the stability of the octamer, it hardly affected enzyme activity and expression. However, replacements of three conserved glycines (G13, G15 and G18) and a conserved glutamic acid (E39) within the fold had severe effects. The mutations not only resulted in loss of enzyme activity but in reduced protein levels as well, probably due to decreased stability of the mutant alcohol oxidase. However, octamerization of the protein still occurred. The existence of inactive octameric proteins provides information about the formation pathway of this octameric flavoprotein.

Topics: Alcohol Oxidoreductases; Amino Acid Sequence; Binding Sites; Enzyme Stability; Flavin-Adenine Dinucleotide; Gene Expression Regulation, Fungal; Microscopy, Immunoelectron; Molecular Sequence Data; Mutagenesis, Site-Directed; Pichia; Protein Conformation; Recombination, Genetic; Sequence Homology, Nucleic Acid; Structure-Activity Relationship

The fate of alcohol oxidase (AO) in chemostat-grown cells of Hansenula polymorpha, after its inactivation by KCN, was studied during subsequent cultivation of the cyanide-treated cells in fresh methanol media. Biochemical experiments showed that the cyanide-induced inactivation of AO was due to the release of flavin adenine dinucleotide (FAD) from the holo enzyme. However, dissociation of octameric AO into subunits was not observed. Subsequent growth of intact cyanide-treated cells in fresh methanol media was paralleled by proteolytic degradation of part of the peroxisomes present in the cells. The recovery of AO activity, concurrently observed in these cultures, was accounted for by synthesis of new enzyme protein. Reactivation of previously inactivated AO was not observed, even in the presence of FAD in such cultures. Newly synthesized AO protein was incorporated in only few of the peroxisomes present in the cells. 31P nuclear magnetic resonance (NMR) studies showed that cyanide-treatment of the cells led to a dissipation of the pH gradient across the peroxisomes membrane. However, restoration of this pH gradient was fast when cells were incubated in fresh methanol medium after removal of the cyanide.

Topics: Alcohol Oxidoreductases; Cyanides; Flavin-Adenine Dinucleotide; Fungal Proteins; Hydrogen-Ion Concentration; Microbodies; Microscopy, Electron; Pichia; Potassium Cyanide; Saccharomycetales