Stereochemistry of copper amine oxidase reactions

The stereochemical course of the oxidation of stereospecifically deuterated dopamine and tyramine, catalyzed by porcine plasma amine oxidase, has been investigated using 1H NMR spectroscopy. The oxidation proceeds with loss of the pro-R hydrogen at C-1. This stereochemistry is in contrast to that observed with the analogous copper containing oxidases isolated from pea seedlings (pro-S) and bovine plasma (nonstereospecific). There is no precedent for these three distinct stereochemical reaction courses to be followed by enzymes in the same class. Mechanistic differences among the three enzymes are evident from the profiles of solvent exchange into reaction products; however, these differences cannot account for the overall differential stereochemical courses observed.     

A theoretical study of the dioxygen activation by glucose oxidase and copper amine oxidase

Glucose oxidase (GO) and copper amine oxidase (CAO) catalyze the reduction of molecular oxygen to hydrogen peroxide. If a closed-shell cofactor (like FADH(2) in GO and topaquinone (TPQ) in CAO) is electron donor in dioxygen reduction, the formation of a closed-shell species (H(2)O(2)) is a spin forbidden process. Both in GO and CAO, formation of a superoxide ion that leads to the creation of a radical pair is experimentally suggested to be the rate-limiting step in the dioxygen reduction process. The present density functional theory (DFT) studies suggest that in GO, the creation of the radical pair induces a spin transition by spin orbit coupling (SOC) in O(2)(-)(rad), whereas in CAO, it is induced by exchange interaction with the paramagnetic metal ion (Cu(II)). In the rate-limiting step, this spin-transition is suggested to transform the O(2)(-)(rad)-FADH(2)(+)(rad) radical pair in GO and the Cu(II)-TPQ (triplet) species in CAO, from a triplet (T) to a singlet (S) state. For CAO, a mechanism for the O[bond]O cleavage step in the biogenesis of TPQ is also suggested.     

Partial conversion of Hansenula polymorpha amine oxidase into a "plant" amine oxidase: implications for copper chemistry and mechanism

The mechanism of the first electron transfer from reduced cofactor to O2 in the catalytic cycle of copper amine oxidases (CAOs) remains controversial. Two possibilities have been proposed. In the first mechanism, the reduced aminoquinol form of the TPQ cofactor transfers an electron to the copper, giving radical semiquinone and Cu(I), the latter of which reduces O2 (pathway 1). The second mechanism invokes direct transfer of the first electron from the reduced aminoquinol form of the TPQ cofactor to O2 (pathway 2). The debate over these mechanisms has arisen, in part, due to variable experimental observations with copper amine oxidases from plant versus other eukaryotic sources. One important difference is the position of the aminoquinol/Cu(II) to semiquinone/Cu(I) equilibrium on anaerobic reduction with amine substrate, which varies from almost 0% to 40% semiquinone/Cu(I). In this study we have shown how protein structure controls this equilibrium by making a single-point mutation at a second-sphere ligand to the copper, D630N in Hansenula polymorpha amine oxidase, which greatly increases the concentration of the cofactor semiquinone/Cu(I) following anaerobic reduction by substrate. The catalytic properties of this mutant, including 18O kinetic isotope effects, point to a conservation of pathway 2, despite the elevated production of the cofactor semiqunone/Cu(I). Changes in kcat/Km[O2] are attributed to an impact of D630N on an increased affinity of O2 for its hydrophobic pocket. The data in this study indicate that changes in cofactor semiquinone/Cu(I) levels are not sufficient to alter the mechanism of O2 reduction and illuminate how subtle features are able to control the reduction potential of active site metals in proteins.     

Structure and activity of Aspergillus nidulans copper amine oxidase

Aspergillus nidulans amine oxidase (ANAO) has the unusual ability among the family of copper and trihydroxyphenylalanine quinone-containing amine oxidases of being able to oxidize the amine side chains of lysine residues in large peptides and proteins. We show here that in common with the related enzyme from the yeast Pichia pastoris, ANAO can promote the cross-linking of tropoelastin and oxidize the lysine residues in α-casein proteins and tropoelastin. The crystal structure of ANAO, the first for a fungal enzyme in this family, has been determined to a resolution of 2.4 ?. The enzyme is a dimer with the archetypal fold of a copper-containing amine oxidase. The active site is the most open of any of those of the structurally characterized enzymes in the family and provides a ready explanation for its lysine oxidase-like activity.     

A substrate-cofactor free radical intermediate in the reaction mechanism of copper amine oxidase.

Reduction of copper amine oxidase with substrate led to the appearance of a free radical which can be detected in anaerobiosis by ESR and optical spectroscopy. The origin of this radical was examined through studies of the semiquinones of 6-hydroxydopamine, an analogue of the recently identified cofactor 6-hydroxydopa. The ESR spectrum of the 6-hydroxydopamine radical was too narrow to account for the enzyme radical signal; however, after spontaneous reaction with primary amines the hyperfine splittings and spectral width obtained by modulation broadening became very similar to those observed for the oxidase radical species. This effect was ascribed to covalent binding of a nitrogen atom directly to the aromatic ring structure, suggesting that the amine oxidase radical is an amino-6-hydroxydopa semiquinone. Identical ESR spectra were obtained using the amines putrescine, cadaverine, p-[(dimethylamino)methyl]benzylamine, and ethylenediamine; these oxidase substrates gave identical enzyme radical spectra as well. The interaction between cofactor and substrate was proved unambiguously by the technique of isotopic labeling: addition of [15N2]ethylenediamine instead of the normal 14N-labeled compound changed the ESR spectra of both the enzyme radical and its 6-hydroxydopamine counterpart. The results were confirmed by optical spectroscopy measurements; 6-hydroxydopamine and oxidized 6-hydroxydopamine gave spectra identical to those of reduced and oxidized amine oxidase, respectively. The 6-hydroxydopamine radical showed a sharp peak at 440 nm; upon addition of amines the maximum shifted to 460 nm, as found for the enzyme. It is proposed that copper amine oxidase represents the first example of a mixed substrate-cofactor radical within the family of tyrosine radical enzymes.     

Roles of the two copper ions in bovine serum amine oxidase

With a view to obtaining information on the roles of the two copper ions in bovine serum amine oxidase (BSAO), spectroscopic and magnetic studies on several BSAO derivatives have been carried out. Cu-depleted BSAO (Cu-depBSAO) exhibits no enzyme activity and only a low absorption intensity at ca. 475 nm, which is the characteristic absorption maximum of the chromophore in BSAO. The binding of 1 mol of Cu to 1 mol of Cu-depBSAO slightly but definitely increases the enzyme activity and the absorptivity, although they are much lower than those of native BSAO. The incorporation of 2 mol of Cu into Cu-depBSAO gives rise to a similar high activity and absorptivity as those of the native enzyme. Electron paramagnetic resonance (EPR) spectra of the BSAO derivatives reveal that two copper ions in the enzyme molecule are environmentally identical. Titrations of BSAO, Cu-depBSAO, and Cu-half-depleted BSAO (Cu-half-depBSAO), containing 1 mol of copper per mole of protein, with phenylhydrazine (an inhibitor of BSAO) indicate that only 1 mol of phenylhydrazine reacts with 1 mol of the enzyme. In other words the enzyme possesses only one chromophore or one active site, though the molecule is composed of two electrophoretically identical subunits. The binding constants between phenylhydrazine and BSAO, Cu-depBSAO, or Cu-half-depBSAO were estimated to be 5 X 10(6), 5 X 10(4), and 1 X 10(5) M-1, respectively. The binding of phenylhydrazine to the chromophore is assisted by the presence of two copper ions by a factor of 100.(ABSTRACT TRUNCATED AT 250 WORDS)     

A flash-photolysis study of the reactions of acaa 3-ttype cytochrome oxidase with dioxygen and carbon monoxide

The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase fromBacillus PS3 in the presence of O₂ has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochromec subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 s, 30 s, 100 s, and 1 ms at 445 nm. The initial, 10-s absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving hemea and Cu_A are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochromec, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O₂ were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from hemea ₃ toa in PS3 oxidase compared to the bovine enzyme.     

Molecular cloning and characterization of copper amine oxidase from Huperzia serrata

A cDNA encoding a novel copper amine oxidase (CAO) was cloned and sequenced from the Chinese club moss Huperzia serrata (Huperziaceae), which produces the Lycopodium alkaloid huperzine A. A 2043-bp open reading frame encoded an Mr 76,854 protein with 681 amino acids. The deduced amino acid sequence shared 44-56% identity with the known CAOs of plant origin, and contained the active site consensus sequence of Asn-Tyr-Asp/Glu. The phylogenetic tree analysis revealed that HsCAO from the primitive vascular plant H. serrata is closely related to Physcomitrella patens subsp CAO. The recombinant enzyme, heterologously expressed in Escherichia coli, catalyzed the oxidative deamination of aliphatic and aromatic amines. Among them, the enzyme accepted cadaverine as the best substrate to catalyze the oxidative deamination to Δ(1)-piperideine, which is the precursor of the Lycopodium alkaloids. Furthermore, a homology modeling and site-directed mutagenesis studies predicted the active site architecture, which suggested the crucial active site residues for the observed substrate preference. This is the first report of the cloning and characterization of a CAO enzyme from the primitive Lycopodium plant.     

Molecular cloning and heterologous expression of pea seedling copper amine oxidase

The cDNA coding for copper amine oxidase has been cloned from etiolated pea seedlings (Pisum sativum). The deduced amino acid sequence, consisting of 674 residues including the signal peptide, agreed well with those reported for the enzymes from a different cultivar of P. sativum and other plant sources, except for several evolutionary replacements located mostly on the molecular surface. A heterologous expression system for the cloned pea enzyme was constructed with the yeast Pichia pastoris, using the AOX1 promoter and the yeast alpha-factor secretion signal. Adding copper to the culture medium increased the secretion of an active, quinone-containing enzyme. Furthermore, the inactive enzyme produced in a copper-deficient medium was activated considerably by subsequent incubation with excess cupric ions. These results strongly suggest that the Tyr-derived redox cofactor, 2,4,5-trihydroxyphenylalanylquinone (topa quinone, TPQ), is produced in the plant enzyme by post-translational modification that proceeds through the copper-dependent, self-processing mechanism, as in the enzymes from bacteria and yeast.     

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.     

The metal function in the reactions of bovine serum amine oxidase with substrates and hydrazine inhibitors

 Bovine serum amine oxidase (BSAO) reacts with 2-hydrazinopyridine, which binds the organic cofactor 2,4,5-trihydroxyphenylalanine quinone, forming a band at 435 nm. The band shifts to 526 nm around 60  °C, to 415 nm upon denaturation, but only shifts to 429 nm upon Cu²⁺ depletion. Its wavelength and intensity suggest that the adduct has the azo conformation, whilst the same adduct of crystallineEscherichia coli amine oxidase (ECAO) shows the hydrazone conformation in the X-ray structure. The steady state kinetics of aminomethyl- and aminoethylpyridines confirm that the formation of the product Schiff base, analogous to the azo form of the 2-hydrazinopyridine adduct, is not hindered in solution. The structural stability of the adduct in the absence of Cu²⁺ is taken to imply hydrogen bonding of the pyridyl nitrogen to a conserved aspartate, as in the ECAO adduct. Thus the ECAO adduct provides a good model for a transient intermediate leading to formation of the BSAO azo adduct. On the basis of this model and of the catalytic competence of Co²⁺-substituted BSAO, confirmed by the present data, a catalytic reaction scheme is proposed. Received: 2 December 1998 / Accepted: 22 March 1999     

Ectopic expression of maize polyamine oxidase and pea copper amine oxidase in the cell wall of tobacco plants

To test the feasibility of altering polyamine levels by influencing their catabolic pathway, we obtained transgenic tobacco (Nicotiana tabacum) plants constitutively expressing either maize (Zea mays) polyamine oxidase (MPAO) or pea (Pisum sativum) copper amine oxidase (PCuAO), two extracellular and H(2)O(2)-producing enzymes. Despite the high expression levels of the transgenes in the extracellular space, the amount of free polyamines in the homozygous transgenic plants was similar to that in the wild-type ones, suggesting either a tight regulation of polyamine levels or a different compartmentalization of the two recombinant proteins and the bulk amount of endogenous polyamines. Furthermore, no change in lignification levels and plant morphology was observed in the transgenic plants compared to untransformed plants, while a small but significant change in reactive oxygen species-scavenging capacity was verified. Both the MPAO and the PCuAO tobacco transgenic plants produced high amounts of H(2)O(2) only in the presence of exogenously added enzyme substrates. These observations provided evidence for the limiting amount of freely available polyamines in the extracellular space in tobacco plants under physiological conditions, which was further confirmed for untransformed maize and pea plants. The amount of H(2)O(2) produced by exogenously added polyamines in cell suspensions from the MPAO transgenic plants was sufficient to induce programmed cell death, which was sensitive to catalase treatment and required gene expression and caspase-like activity. The MPAO and PCuAO transgenic plants represent excellent tools to study polyamine secretion and conjugation in the extracellular space, as well as to determine when and how polyamine catabolism actually intervenes both in cell wall development and in response to stress.     

Towards the development of selective amine oxidase inhibitors. Mechanism-based inhibition of six copper containing amine oxidases.

Four substrate analogs, 4-(2-naphthyloxy)-2-butyn-1-amine (1), 1,4-diamino-2-chloro-2-butene (2), 1,6-diamino-2,4-hexadiyne (3), and 2-chloro-5-phthalimidopentylamine (4) have been tested as inhibitors against mammalian, plant, bacterial, and fungal copper-containing amine oxidases: bovine plasma amine oxidase (BPAO), equine plasma amine oxidase (EPAO), pea seedling amine oxidase (PSAO), Arthrobacter globiformis amine oxidase (AGAO), Escherichia coli amine oxidase (ECAO), and Pichia pastoris lysyl oxidase (PPLO). Reactions of 1,4-diamino-2-butyne with selected amine oxidases were also examined. Each substrate analog contains a functional group that chemical precedent suggests could produce mechanism-based inactivation. Striking differences in selectivity and rates of inactivation were observed. For example, between two closely related plasma enzymes, BPAO is more sensitive than EPAO to 1 and 3, while the reverse is true for 2 and 4. In general, inactivation appears to arise in some cases from TPQ cofactor modification and in other cases from alkylation of protein residues in a manner that blocks access of substrate to the active site. Notably, 1 completely inhibits AGAO at stoichiometric concentrations and is not a substrate, but is an excellent substrate of PSAO and inhibition is observed only at very high concentrations. Structural models of 1 in Schiff base linkage to the TPQ cofactor in AGAO and PSAO (for which crystal structures are available) reveal substantial differences in the degree of interaction of bound 1 with side-chain residues, consistent with the widely divergent activities. Collectively, these results suggest that the development of highly selective amine oxidase inhibitors is feasible.     

Structure and nucleotide sequence of Euphorbia characias copper/TPQ-containing amine oxidase gene

A cDNA encoding for a copper containing amine oxidase has been isolated and sequenced from young leaves of Euphorbia characias, a perennial mediterranean shrub. A single long open reading frame of 2068 pb encodes a protein composed of 653 amino acids with a molecular mass of about 74 kDa. A putative 24-aminoacid signal peptide precedes the sequence of the mature protein, with characteristics of a secretion signal peptide. Alignments of Euphorbia amine oxidase cDNA nucleotide sequence with that of amine oxidase from the seedlings of the pulses lentil, pea, and chickpea reveal several conserved regions, especially in the C-terminus, with a homology 90%–97%. The near 5 region shows several insertions, deletions, and different nucleotide sequence with ca. 60% homology. The enzyme contains 1%–2% carbohydrate deduced by deglycosylation experiments. Five cysteine residues are present in the deduced aminoacid sequence with a single disulfide bridge as judged by titration with cysteine reagents.