Mechanistic comparison of the cobalt-substituted and wild-type copper amine oxidase from Hansenula polymorpha

A recent report by Mills and Klinman [Mills, S. A., and Klinman, J. P. (2000) J. Am. Chem. Soc. 122, 9897-9904] described the preparation and initial characterization of a cobalt-substituted form of the copper amine oxidase from Hansenula polymorpha (HPAO). This enzyme was found to be fully catalytically active at saturating substrate concentrations, but with a K(m) for O(2) approximately 70-fold higher than that of the copper-containing, wild-type enzyme. Herein, we report a detailed analysis of the mechanism of catalysis for the wild-type and the cobalt-substituted forms of HPAO. Both forms of enzyme are concluded to utilize the same mechanism for oxygen reduction, involving initial, rate-limiting electron transfer from the reduced cofactor of the enzyme to prebound dioxygen. Superoxide formed in this manner is stabilized by the active site metal, facilitating the transfer of a second electron and two protons to form the product hydrogen peroxide. The elevated K(m) for O(2) at the dioxygen binding site in Co-substituted HPAO, relative to that of wild-type HPAO, is proposed to be due to a change in the net charge at the adjacent metal site from +1 (cupric hydroxide) in wild-type enzyme to +2 (cobaltous H(2)O) in cobalt-substituted HPAO.     

Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha

We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.     

Role of a strictly conserved active site tyrosine in cofactor genesis in the copper amine oxidase from Hansenula polymorpha

The copper amine oxidases catalyze the O(2)-dependent, two-electron oxidation of amines to aldehydes at an active site that contains Cu(II) and topaquinone (TPQ) cofactor. TPQ arises from the autocatalytic, post-translational oxidation of a tyrosine side chain within the same active site. The contributions of individual active site amino acids to each of these chemical processes are being delineated. Previously, using the amine oxidase from the yeast Hansenula polymorpha (HPAO), mutations of a strictly conserved and structurally pivotal active site tyrosine (Y305) were studied and their effects on the catalytic cycle demonstrated [Hevel, J. M., Mills, S. A., and Klinman, J. P. (1999) Biochemistry 38, 3683-3693]. This study examines mutations at the same position for their effects on cofactor generation. While the Y305A mutation had moderate effects on the kinetics of catalysis (2.5- and 8-fold effects on k(cat) using ethylamine and benzylamine as substrates), the same mutation slows cofactor formation by approximately 45-fold relative to that of the wild-type (WT). Additionally, the Y305A mutant forms at least two species: primarily TPQ at lower pH and a species with a blue-shifted absorbance at high pH (lambda(max) = 400 nm). The 400 nm species does not react with phenylhydrazine or ethylamine and is stable toward pH buffer exchange, long-term storage (>3 weeks), incubation at high temperatures, or incubation with reductants and colorimetric peroxide quenching reagents. A similar species accumulates appreciably even at approximately neutral pH in the Y305F mutant, despite the fact that the rate of TPQ formation is reduced only 3-fold relative to that of WT HPAO. This small impact of Y305F on the rate of biogenesis contracts with a decrease in k(cat) (using ethylamine as the substrate) of 125-fold. The opposing effects of mutations at position 305 in biogenesis versus catalysis indicate that a single residue can be recruited for different roles during these processes.     

2,4,5-Trihydroxyphenylalanine quinone biogenesis in the copper amine oxidase from Hansenula polymorpha with the alternate metal nickel

Copper amine oxidase (CAO) is a dual-functioning enzyme that catalyzes the biosynthesis of a self-derived coenzyme and subsequent oxidative deamination of primary amines. The organic cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), is generated from the post-translational modification of an active site tyrosine (Y405) in a reaction shown to be dependent on both molecular oxygen and a mononuclear copper center. Previous investigations of Cu(II)-dependent cofactor formation in the Hansenula polymorpha amine oxidase (HPAO) provided evidence for the coordination of the precursor tyrosine in forming a ligand-to-metal charge transfer complex as a means of activating the tyrosyl ring for direct attack by triplet-state dioxygen. To further delineate the role of the metal in facilitating this complex series of reactions, apo-HPAO was reconstituted with alternate metals of varying reduction potentials and Lewis acidities [Ni(II), Co(II), Mn(II), Fe(II), and Fe(III)] and the consequence of each substitution on TPQ biogenesis examined. Ni(II) was found to support the transformation of the precursor tyrosine to the quinone cofactor to yield a mature enzyme competent for methylamine oxidation. Detailed kinetic analysis of the mechanism of TPQ biogenesis for the Ni(II)-substituted enzyme has led to the proposal of a direct electron transfer from the metal-coordinated tyrosinate to dioxygen as the dominant rate-limiting step.     

Crystal structure at 2.5 A resolution of zinc-substituted copper amine oxidase of Hansenula polymorpha expressed in Escherichia coli

Copper amine oxidases (CAOs) catalyze the two-electron oxidation of primary amines to aldehydes, utilizing molecular oxygen as a terminal electron acceptor. To accomplish this transformation, CAOs utilize two cofactors: a mononuclear copper, and a unique redox cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ or TOPA quinone). TPQ is derived via posttranslational modification of a specific tyrosine residue within the protein itself. In this study, the structure of an amine oxidase from Hansenula polymorpha has been solved to 2.5 A resolution, in which the precursor tyrosine is unprocessed to TPQ, and the copper site is occupied by zinc. Significantly, the precursor tyrosine directly ligands the metal, thus providing the closest analogue to date of an intermediate in TPQ production. Besides this result, the rearrangement of other active site residues (relative to the mature enzyme) proposed to be involved in the binding of molecular oxygen may shed light on how CAOs efficiently use their active site to carry out both cofactor formation and catalysis.     

An unexpected role for the active site base in cofactor orientation and flexibility in the copper amine oxidase from Hansenula polymorpha.

The role of the active site aspartate base in the aminotransferase mechanism of the copper amine oxidase from the yeast Hansenula polymorpha has been probed by site-directed mutagenesis. The D319E mutant catalyzes the oxidation of methylamine and phenethylamine, but not that of benzylamine. kcat/Km for methylamine is found to be 80-fold reduced compared to that of the wild type. Viscosogen and substrate and solvent deuteration have no effect on this parameter for D319E, which is suggestive of limitation of kcat/Km by a conformational change. This conformational change is proposed to be the movement of the cofactor into a productive orientation upon the binding of substrate. In the absence of substrate, a flipped cofactor orientation is likely, on the basis of resonance Raman evidence that the C5 carbonyl of the cofactor is less solvent accessible than the C3 hydrogen. kcat for D319E methylamine oxidase is reduced 200-fold compared to that of the wild type and is unaffected by substrate deuteration, but displays a substantial solvent isotope effect. A 428 nm absorbance is evident under conditions of saturating methylamine and oxygen with D319E. The D319N mutant is observed to produce a similar absorbance at 430 nm when treated with ammonia despite the fact that this mutant has no amine oxidase activity. Resonance Raman spectroscopy indicates the formation of a covalent ammonia adduct and identifies it as the deprotonated iminoquinone. In contrast, when the D319E mutant is reacted with ammonia, it gives predominantly a 340-350 nm species. This absorbance is ascribed to a localization of the cofactor oxyanion induced by binding of the cation at the active site and not to covalent adduct formation. Resonance Raman spectroscopic examination of the steady state species of D319E methylamine oxidation, in combination with the kinetic data, indicates that the 428 nm species is the deprotonated iminoquinone produced upon reoxidation of the reduced cofactor. A model is proposed in which a central role of the active site base is to position the free cofactor and several enzyme intermediates for optimal activity.     

Investigation of Cu(I)-dependent 2,4,5-trihydroxyphenylalanine quinone biogenesis in Hansenula polymorpha amine oxidase

Copper, a mediator of redox chemistries in biology, is often found in enzymes that bind and reduce dioxygen. Among these, the copper amine oxidases catalyze the oxidative deamination of primary amines utilizing a type(II) copper center and 2,4,5-trihydroxyphenylalanine quinone (TPQ), a covalent cofactor derived from the post-translational modification of an active site tyrosine. Previous studies established the dependence of TPQ biogenesis on Cu(II); however, the dependence of cofactor formation on the biologically relevant Cu(I) ion has remained untested. In this study, we demonstrate that the apoform of the Hansenula polymorpha amine oxidase readily binds Cu(I) under anaerobic conditions and produces the quinone cofactor at a rate of 0.28 h(-1) upon subsequent aeration to yield a mature enzyme with kinetic properties identical to the protein product of the Cu(II)-dependent reaction. Because of the change in magnetic properties associated with the oxidation of copper, electron paramagnetic resonance spectroscopy was employed to investigate the nature of the rate-limiting step of Cu(I)-dependent cofactor biogenesis. Upon aeration of the unprocessed enzyme prebound with Cu(I), an axial Cu(II) electron paramagnetic resonance signal was found to appear at a rate equivalent to that for the cofactor. These data provide strong evidence for a rate-limiting release of superoxide from a Cu(II)(O(2)(.)) complex as a prerequisite for the activation of the precursor tyrosine and its transformation for TPQ. As copper is trafficked to intracellular protein targets in the reduced, Cu(I) state, these studies offer possible clues as to the physiological significance of the acquisition of Cu(I) by nascent H. polymorpha amine oxidase.     

The nature of O2 reactivity leading to topa quinone in the copper amine oxidase from Hansenula polymorpha and its relationship to catalytic turnover

The copper amine oxidases (CAOs) catalyze the O(2)-dependent, two-electron oxidation of amines to aldehydes at an active site that contains Cu(II) and topaquinone (TPQ) cofactor. TPQ arises from the autocatalytic, post-translational oxidation of a tyrosine side chain in the active site. Monooxygenation within the ring of tyrosine at a single Cu(II) site is unique in biology and occurs as an early step in the formation of TPQ. The mechanism of this reaction has been further examined in the CAO from Hansenula polymorpha (HPAO). When a Clark electrode fitted to a custom-made, gastight apparatus over a range of initial concentrations of O(2) was used, rates of O(2) consumption at levels greater than air are seen to be reduced relative to earlier results, yielding K(D)(apparent) = 216 microM for O(2). This is consistent with a mechanism in which O(2) binds reversibly to the active site, triggering a conformational change that promotes ligation of tyrosinate to Cu(II). The activated Cu(II)-tyrosinate species has been proposed to react with O(2) in a rate-limiting step, although it was also possible that breakdown of a putative peroxy-intermediate controlled TPQ formation. To test the latter hypothesis, Cu(II)-free HPAO was prepared with 3,5-ring-[(2)H(2)]-tyrosine incorporated throughout the primary sequence. The absence of an isotope effect on the rate of TPQ formation eliminates cleavage of this C-H bond in a proposed Cu(II)-aryl-peroxide intermediate as a rate limiting step. The role of methionine 634, previously found to moderate O(2) binding during the catalytic cycle, is shown here to serve a similar function in TPQ formation. As with catalysis, the rate of TPQ formation correlates with the volume of the hydrophobic side chain at position 634, implicating similar binding sites for O(2) during catalysis and cofactor biogenesis.     

Further insight into the mechanism of stereoselective proton abstraction by bacterial copper amine oxidase

During the catalytic reaction of copper amine oxidase, one of the two prochiral hydrogen atoms at the C1 position of substrate amine is stereoselectively abstracted by a conserved Asp residue serving as a general base. Using stereospecifically deuterium-labeled enantiomers of 2-phenylethylamine, we previously showed that the pro-S alpha-proton is abstracted by the enzyme from Arthrobacter globiformis (AGAO) [Uchida, M., et al. (2003) Biosci. Biotechnol. Biochem. 67, 2664-2667]. More recently, we have also demonstrated that the pro-S selectivity of alpha-proton abstraction is fully retained even in the reaction of a mutant AGAO lacking the catalytic base [Chiu, Y.-C., et al. (2006) Biochemistry 45, 4105-4120]. On the basis of these findings, we have proposed that the stereoselectivity of alpha-proton abstraction is primarily determined by the conformation of the Schiff base intermediate formed between the substrate and the topa quinone cofactor (TPQ), stabilized by the binding of the distal part of the substrate to a hydrophobic pocket of the enzyme. In this conformation, the pro-S hydrogen atom to be abstracted is nearly perpendicular to the plane of the Schiff base-TPQ conjugate system, achieving the maximum overlap of sigma- and pi-orbitals. To further elucidate the stereochemical details, we have synthesized stereospecifically deuterium-labeled enantiomers of ethylamine, a very poor substrate for AGAO, in addition to those structurally related to the preferred substrate, 2-phenylethylamine. In marked contrast to the nearly complete pro-S selectivity of alpha-proton abstraction for most substrates that have been examined, the stereoselectivity for ethylamine decreased significantly to as little as 88%. The crystal structure of AGAO soaked with ethylamine showed very poor electron densities for the substrate Schiff base intermediate, showing that its conformation is not defined uniquely. Thus, the stereoselectivity of alpha-proton abstraction during the copper amine oxidase reaction is closely associated with the conformational flexibility of the substrate Schiff base intermediate.     

Peroxisomal remnant structures in Hansenula polymorpha Pex5 cells can develop into normal peroxisomes upon induction of the PTS2 protein amine oxidase

We have analyzed the properties of peroxisomal remnants in Hansenula polymorpha pex5 cells. In such cells PTS1 matrix protein import is fully impaired. In H. polymorpha pex5 cells, grown on ethanol/ammonium sulfate, conditions that repressed the PTS2 protein amine oxidase (AMO), peroxisomal structures were below the limit of detection. In methanol/ammonium sulfate-grown cells, normal peroxisomes are absent, but a few small membranous structures were observed that apparently represented peroxisomal ghosts since they contained Pex14p. These structures were the target of a Pex10p.myc fusion protein that was produced in pex5 cells under the control of the homologous alcohol oxidase promoter (strain pex5::P(AOX).PEX10.MYC). Glycerol/methanol/ammonium sulfate-grown cells of this transformant were placed in fresh glucose/methylamine media, conditions that fully repress the synthesis of the Pex10p.myc fusion protein but induce the synthesis of AMO. Two hours after the shift Pex10p.myc-containing structures were detectable that had accumulated newly synthesized AMO protein and which during further cultivation developed in normal peroxisomes. Concurrently, the remaining portion of these structures was rapidly degraded. These findings indicate that peroxisomal remnants in pex5 cells can develop into peroxisomes. Also, as for normal peroxisomes in H. polymorpha, apparently a minor portion of these structures actually take part in the development of these organelles.     

Binding of dioxygen to non-metal sites in proteins: exploration of the importance of binding site size versus hydrophobicity in the copper amine oxidase from Hansenula polymorpha

Copper amine oxidases (CAOs) contain 2,4,5-trihydroxyphenylalanyl quinone (TPQ) and a copper ion in their active sites, catalyzing amine oxidation to aldehyde and ammonia concomitant with the reduction of molecular oxygen to hydrogen peroxide. Kinetic studies on the CAO from bovine serum (BSAO) [Su and Klinman (1999) Biochemistry 37, 12513-12525] and the recent reports on the cobalt substituted form of the enzyme from Hansenula polymorpha (HPAO) [Mills and Klinman (2000) J. Am. Chem. Soc. 122, 9897-9904, and Mills et al. (2002) Biochemistry, 41, 10577-10584] support pre-binding of molecular oxygen prior to a rate-limiting electron transfer from the reduced form of TPQ (p-aminohydroquinone form) to dioxygen. Although there is significant sequence homology between BSAO and HPAO, k(cat)/K(m)(O2) for BSAO under the optimal condition is one order of magnitude lower than that for HPAO. From a comparison of amino acid sequences for BSAO and HPAO, together with the X-ray crystal structure of HPAO, a plausible dioxygen pre-binding site has been identified that involves Y407, L425, and M634 in HPAO; the latter two residues are altered in BSAO to A490 and T695. To determine which of these residues plays a greater role in dioxygen chemistry, k(cat)/K(m)(O2) was determined in HPAO for the M634 --> T and L425 --> A mutants. The L425 --> A mutation does not alter k(cat)/K(m)(O2) to a large extent, whereas the M634 --> T decreased k(cat)/K(m)(O2) by one order of a magnitude, creating a catalyst that is similar to BSAO. A series of mutants at M634 (to F, L, and Q) were, therefore, prepared in HPAO and characterized with regard to k(cat)/K(m)(O2) as a function of pH. Structure reactivity correlations show a linear relationship of rate with side chain volume, rather than hydrophobicity, indicating that dioxygen reactivity increases with the bulk of the residue at position 634. This site also shows specificity for O2, in relation to the co-gas N2, since substitution of the inert gas N2 by either Ar or He has no effect on measured rates. In particular, He gas is expected to have little affinity for protein at 1 atmospheric pressure, implying little or no binding by N2 as well.     

Routing of Hansenula polymorpha alcohol oxidase: an alternative peroxisomal protein-sorting machinery

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.     

Thermodynamic behaviour and conformational changes of alcohol oxidase of yeasts Hansenula polymorpha

We studied influence of heating, ethanol and sodium azide on the structural and conformational changes of the alcohol oxidase from yeast Hansenula polymorpha. The increase of fluorescence of alcohol oxidase -cofactor, flavin adenine dinucleotide, was revealed when heated to 60 degrees C while the enzymatic activity of alcohol oxidase remained unchanged. Differential scanning microcalorimetry revealed that ethanol stabilized the protein structure and increased the temperature of melting, Based on the data of circular dichroism, we concluded that the percentage of helicities in the secondary structure of alcohol oxidase was not influenced by both ethanol and sodium azide, however ethanol significantly modified the circular dichroism spectrum associated with the tertiary structure of alcohol oxidase.     

Relationship of stopped flow to steady state parameters in the dimeric copper amine oxidase from Hansenula polymorpha and the role of zinc in inhibiting activity at alternate copper-containing subunits

The expression of a copper amine oxidase (CAO) from Hansenula polymorpha in Saccharomyces cerevisiae under differing culture conditions leads to the incorporation of varied levels of CAO-bound zinc. The presence of substantial amount of zinc results in two distinctive enzyme species, designated as the fast and slow enzymes. Both forms are rapidly reduced by substrate methylamine with a rate constant of 199 s(-1) but behave remarkably differently in their oxidation rates; the fast enzyme is oxidized by dioxygen at a rate of 22.1 s(-1), whereas the slow enzyme reacts at a rate of 1.8 x 10(-4) s(-1). The apparent kcat of the enzyme preparation is linearly proportional to the fraction of the fast enzyme, with an extrapolated value of 6.17 s(-1) when the enzyme is 100% in its "fast" form. A comparison of rate constants for cofactor reduction and reoxidation steps, measured in stopped flow experiments, to the extrapolated kcat implicates additional steps in the steady state reaction. Measurement of the proportion of oxidized (ETPQ(ox)) and reduced cofactor (ETPQ(red)) under steady state conditions indicates approximately 50% of each cofactor form at 0.8 or 2 mM methylamine. Kinetic isotope effect measurements using deuterated amine substrate lead to the following steady state values: (D)(k(red)) = 8.5 (0.5), (D)(kcat) = 1.7 (0.1), and (D)(kcat/K(m)) = 4.3 (0.2). The collective data allow the calculation of partially rate-determining constants during the reductive half-reaction (ca. 200 s(-1) for binding of substrate to ETPQ(ox) and 27.9 s(-1) for release of aldehyde product or a protein isomerization from ETPQ(red)); an additional step with a rate constant of 13.2 s(-1) is assigned to the oxidative half-reaction, most likely for the release of product hydrogen peroxide. These results, together with the sole detection of oxidized and reduced cofactor during rapid scanning stopped flow experiments, indicate that four steps contribute to kcat, with the first electron transfer from cofactor to O2 contributing ca. 29%. An investigation of the relationship between the copper content and the extent of the fast enzyme shows that only the copper-containing homodimer is capable of rapid reoxidation and that zinc-copper heterodimers are incapable of rapid turnover at either subunit. This implies communication between the metal sites of the two subunits per dimer that impacts O2 binding and/or electron transfer from reduced cofactor to bound O2.     

Copper amine oxidase: cunning cofactor and controversial copper

Copper amine oxidases have a complex reaction cycle that converts a primary amine and molecular oxygen into the aldehyde, ammonia and hydrogen peroxide. Coupling structural studies of freeze-trapped reaction intermediates in crystals with kinetic and spectroscopic experiments in solution has generated a detailed molecular picture of catalysis. Although dioxygen has been directly observed bound to the copper at a late stage in the reaction cycle, whether copper is the initial binding site remains controversial.     

Cyanide as a copper-directed inhibitor of amine oxidases: implications for the mechanism of amine oxidation

 The interactions of five copper-containing amine oxidases with substrates and substrate analogues in the presence of the copper ligands cyanide, azide, chloride, and 1,10-phenanthroline have been investigated. While cyanide inhibits, to varying degrees, the reaction of phenylhydrazine with porcine kidney amine oxidase (PKAO), porcine plasma amine oxidase (PPAO), bovine plasma amine oxidase (BPAO), and pea seedling amine oxidase (PSAO), it enhances the reaction of Arthrobacter P1 amine oxidase (APAO) with this substrate analogue. This indicates that cyanide exerts an indirect effect on topa quinone (TPQ) reactivity via coordination to Cu(II) rather than through cyanohydrin formation at the TPQ organic cofactor. Moreover, cyanide binding to the mechanistically relevant TPQ^• semiquinone form of substrate-reduced APAO and PSAO was not observable by EPR or resonance Raman spectroscopy. Hence, cyanide most likely inhibits enzyme reoxidation by binding to Cu(I) and trapping the Cu(I)-TPQ^• form of amine oxidases, and thus preventing the reaction of O₂ with Cu(I). In contrast, ligands such as azide, chloride, and 1,10-phenanthroline, which preferentially bind to Cu(II), inhibit by stabilizing the aminoquinol Cu(II)-TPQ_red redox state, which is in equilibrium with Cu(I)-TPQ^•. Received: 12 December 1996 / Accepted: 20 March 1997     

Purification and characterization of alcohol oxidase from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha

Alcohol oxidase (AOX) has been purified 8-fold from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha C-105 (gcr1 catX) with impaired glucose-induced catabolite repression and completely devoid of catalase. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis and HPLC. Some physicochemical and biochemical properties of AOX were studied in detail: molecular weight (approximately 620 kD), isoelectric point (pI 6.1), and UV-VIS, circular dichroism (CD), and fluorescence spectra. The content of different secondary structure motifs of the enzyme has been calculated from the CD spectra using a computer program. It was found that the native protein contains about 50% alpha-helix, 25% beta-sheet, and about 20% random structures. The kinetic parameters for different substrates, such as methanol, ethanol, and formaldehyde, were measured using a Clark oxygen electrode. The rate of enzymatic oxidation of formaldehyde by alcohol oxidase from H. polymorpha is only twice lower compared to the best substrate of the enzyme, methanol.     

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

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.