Combinatorial synthesis of SSAO inhibitors using sonogashira coupling: SAR of aryl propargylic amines

The structure-activity relationships for semicarbazide-sensitive amine oxidase (SSAO) inhibitors based on arylpropynylamines was investigated using solution-phase combinatorial Sonogashira coupling. The results suggest that binding to the active site occurs by coordination of the amine to the proximal copper(II) and formation of a pi-complex between topaquinone and the electron-rich aryl group of the inhibitor.     

Irreversible inhibition of pig kidney copper-containing amine oxidase by sodium and lithium ions.

Copper amine oxidase was found to be inhibited in a complex way by small alkali metal ions. Classic enzyme kinetic studies showed that Li+ and Na+ were weak noncompetitive inhibitors, whereas the larger alkali metals K+, Rb+ and Cs+ were not inhibitors. However, freezing in the presence of Na+ or Li+ surprisingly resulted in complete and irreversible inactivation. In the case of Li+, it was possible to show that one ion per subunit was retained permanently in the inactivated enzyme, suggesting a structural rearrangement. The mechanism of inhibition was studied using a wide range of spectroscopic and analytic techniques. Only minor changes in the protein structure could be detected, except for a significant change in the geometry of the copper site. The unique topaquinone cofactor was apparently functional and able to proceed through the reductive half of the catalytic cycle, but the enzyme no longer reacted with oxygen. The effect of Na+ and Li+ was source-specific for pig kidney and bovine kidney amine oxidases, while the enzymes from bovine serum or plants were not inactivated, consistent with a mechanism dependent on small structural differences. A model for irreversible inactivation is proposed in which the cofactor is co-ordinated directly to copper, in analogy with the inactivation reported for Escherichia coli amine oxidase under crystal growth conditions.     

Cellular localization and metabolic function of n-butylamine-induced amine oxidases in the fungus Aspergillus niger AKU 3302

Using transmission electron microscopy, the amine oxidase activity in Aspergillus niger AKU 3302 was localized to the outer side of the cell wall but not inside the cell using the cerium perhydroxide deposition method. The presence of cerium in the deposit was confirmed by energy-dispersive microanalysis of X-rays. Interestingly, immunocytochemical localization using gold labeling with a specific antibody indicated the presence of amine oxidase protein inside the cell wall and not only on the outer surface. Besides labeling of the cell wall, a high level of labeling was also observed inside the cell in what seemed to be secretory vesicle structures. It is proposed that the highly active amine oxidase AO-I is located in the cell wall and serves primarily as a detoxifying agent, preventing amines from entering and damaging the cell. The amine oxidation exhibits an interesting spatial orientation involving a release of toxic hydrogen peroxide into the extracellular space. The inactive amine oxidase protein located inside the cell is most probably the amine oxidase AO-II, found in cell homogenates. It is also likely that the less active AO-II is an improperly folded precursor of AO-I, which acquired low-level activity after cell homogenization in the presence of Cu(II) and oxygen due to autooxidative formation of topaquinone.     

Purification and characterization of methylamine oxidase induced in Aspergillus niger AKU 3302

Crude extract of Aspergillus niger AKU 3302 mycelia incubated with methylamine showed a single amine oxidase activity band in a developed polyacrylamide gel that weakly cross-reacted with the antibody against a copper/topa quinone-containing amine oxidase (AO-II) from the same strain induced by n-butylamine. Since the organism cannot grow on methylamine and the already known quinoprotein amine oxidases of the organism cannot catalyze oxidation of methylamine, the organism was forced to produce another enzyme that could oxidize methylamine when the mycelia were incubated with methylamine. The enzyme was separated and purified from the already known two quinoprotein amine oxidases formed in the same mycelia. The purified enzyme showed a sharp symmetric sedimentation peak in analytical ultracentrifugation showing S20,w0 of 6.5s. The molecular mass of 133 kDa estimated by gel chromatography and 66.6 kDa found by SDS-PAGE confirmed the dimeric structure of the enzyme. The purified enzyme was pink in color with an absorption maximum at 494 nm. The enzyme readily oxidized methylamine, n-hexylamine, and n-butylamine, but not benzylamine, histamine, or tyramine, favorite substrates for the already known two quinoprotein amine oxidases. Inactivation by carbonyl reagents and copper chelators suggested the presence of a copper/topa quinone cofactor. Spectrophotometric titration by p-nitrophenylhydrazine showed one reactive carbonyl group per subunit and redox-cyclic quinone staining confirmed the presence of a quinone cofactor. pH-dependent shift of the absorption spectrum of the enzyme-p-nitrophenylhydrazone (469 nm at neutral to 577 nm at alkaline pH) supported the identity of the cofactor with topaquinone. Nothern blot analysis indicated that the methylamine oxidase encoding gene is largely different from the already known amine oxidase in the organism.     

The crystal structure of Pichia pastoris lysyl oxidase

Pichia pastoris lysyl oxidase (PPLO) is unique among the structurally characterized copper amine oxidases in being able to oxidize the side chain of lysine residues in polypeptides. Remarkably, the yeast PPLO is nearly as effective in oxidizing a mammalian tropoelastin substrate as is a true mammalian lysyl oxidase isolated from bovine aorta. Thus, PPLO is functionally related to the copper-containing lysyl oxidases despite the lack of any significant sequence similarity with these enzymes. The structure of PPLO has been determined at 1.65 A resolution. PPLO is a homodimer in which each subunit contains a Type II copper atom and a topaquinone cofactor (TPQ) formed by the posttranslational modification of a tyrosine residue. While PPLO has tertiary and quaternary topologies similar to those found in other quinone-containing copper amine oxidases, its active site is substantially more exposed and accessible. The structural elements that are responsible for the accessibility of the active site are identified and discussed.     

Structure and biogenesis of topaquinone and related cofactors

 The structure of a new biological redox cofactor – topaquinone (TPQ), the quinone of 2,4,5-trihydroxyphenylalanine – was elucidated in 1990. TPQ is the cofactor in most copper-containing amine oxidases. It is produced by post-translational modification of a strictly conserved active-site tyrosine residue. Recent work has established that TPQ biogenesis proceeds via a novel self-processing pathway requiring only the protein, copper, and molecular oxygen. The oxidation of tyrosine to TPQ by dioxygen is a six-electron process, which has intriguing mechanistic implications because copper is a one-electron redox agent, and dioxygen can function as either a two-electron or four-electron oxidant. This review adopts an historical perspective in discussing the structure and reactivity of TPQ in amine oxidases, and then assesses what is currently understood about the mechanism of the oxidation of tyrosine to produce TPQ. Aspects of the structures and chemistry of related cofactors, such as the Tyr-Cys radical in galactose oxidase and the lysine tyrosylquinone of lysyl oxidase, are also discussed. Received: 23 May 1998 / Accepted: 19 October 1998     

Medical implications from the crystal structure of a copper-containing amine oxidase complexed with the antidepressant drug tranylcypromine

The X-ray crystal structure of the copper-containing quinoprotein amine oxidase from E. coli has been determined in complex with the antidepressant drug tranylcypromine to 2.4 A resolution. The drug is a racemic mix of two enantiomers, but only one is seen bound to the enzyme. The other enantiomer is not acting as a substrate for the enzyme as no catalytic activity was detected when the enzyme was initially exposed to the drug. The inhibition of human copper amine oxidases could be a source of side-effects in its use as an antidepressant to inhibit the flavin-containing monoamine oxidases in the brain.     

Properties of copper-free pig kidney amine oxidase: role of topa quinone

Copper removal from pig kidney amine oxidase containing Cu/topaquinone (TPQ) has been obtained using CN(-) in the presence of the poor substrate p-(dimethylamino)benzylamine. Upon removal of copper, the enzyme loses its activity while the TPQ cofactor remains in its oxidized form. The addition of copper to the apo-form fully restores the active enzyme. The CN(-) treatment in the presence of sodium dithionite or good substrates (cadaverine or benzylamine) also removes copper but the TPQ cofactor is irreversibly reduced and the addition of copper does not regenerate the active enzyme. Ni(II) and Zn(II) do not bind the apo-protein in contrast to Co(II) which is incorporated to the same extent as Cu(II). However, Co-reconstituted enzyme only shows a very low activity. These results demonstrate that copper is essential for the catalytic mechanism because it maintains the correct active site geometry.     

Modulation of bovine serum amine oxidase activity by hydrogen peroxide

Bovine serum amine oxidase (BSAO), reduced by excess amine under limited turnover conditions, was over 80% inactivated by H(2)O(2) upon oxygen exhaustion. The UV-Vis spectrum and the reduced reactivity with carbonyl reagents showed that the cofactor topaquinone (TPQ) was stabilized in reduced form. The protein large M(r) (170 kDa) prevented the identification of modified residues by amino acid analyses. Minor changes of the Cu(2+) EPR signal and the formation of a radical at g = 2.001, with intensity a few percent of that of the Cu(2+) signal, unaffected by a temperature increase, suggest that Cu(2+)-bound histidines were not oxidized and the radical was not the Cu(+)-semiquinolamine in equilibrium with Cu(2+)-aminoquinol. It may derive from the modification of a conserved residue in proximity of the active site, possibly the tyrosine at hydrogen-bonding distance of TPQ C-4 ionized hydroxyl. The inactivation reaction appears to be a general feature of copper-containing amine oxidases. It may be part of an autoregulatory process in vivo, possibly relevant to cell adhesion and redox signaling.     

Escherichia coli mechanisms of copper homeostasis in a changing environment

Escherichia coli is equipped with multiple systems to ensure safe copper handling under varying environmental conditions. The Cu(I)-translocating P-type ATPase CopA, the central component in copper homeostasis, is responsible for removing excess Cu(I) from the cytoplasm. The multi-copper oxidase CueO and the multi-component copper transport system CusCFBA appear to safeguard the periplasmic space from copper-induced toxicity. Some strains of E. coli can survive in copper-rich environments that would normally overwhelm the chromosomally encoded copper homeostatic systems. Such strains possess additional plasmid-encoded genes that confer copper resistance. The pco determinant encodes genes that detoxify copper in the periplasm, although the mechanism is still unknown. Genes involved in copper homeostasis are regulated by MerR-like activators responsive to cytoplasmic Cu(I) or two-component systems sensing periplasmic Cu(I). Pathways of copper uptake and intracellular copper handling are still not identified in E. coli.     

Identification of topaquinone and its consensus sequence in copper amine oxidases.

The nature of the active site cofactor and the amino acid sequence flanking this structure have been determined in a range of copper amine oxidases. For enzymes from porcine plasma, porcine kidney, and pea seedlings, proteolytic digestion was performed on phenylhydrazone or p-nitrophenylhydrazone derivatives. Thermolysin treatment leads to relatively small active site peptides, which have been characterized by Edman degradation and by resonance Raman spectroscopy. Resonance Raman spectra of peptides show identical peak positions and intensities relative to each other and to a model p-nitrophenylhydrazone derivative of topaquinone hydantoin, establishing topaquinone as the cofactor in each instance. Edman degradation of peptides provides active site sequences for comparison to previous determinations with bovine serum and yeast amine oxidases. The available data establish a consensus sequence of Asn, Topa, Asp/Glu. Trypsin leads to significantly longer peptides, which reveal a high degree of sequence identity between plasma proteins from bovine and porcine sources (89%), with significantly decreased identity between the porcine serum and intracellular amine oxidases (56%). A lower degree of identity (45%) is observed between the pea seedling and mammalian enzymes. As an alternative to the isolation of active site peptides for topaquinone identification, visible spectra of intact proteins have been investigated. It is shown that p-nitrophenylhydrazone derivatives of native enzymes, active site-derived peptides, and a topaquinone model exhibit identical behavior, absorbing at 457-463 nm at neutral pH (pH 7.2) and at 575-587 nm in basic solution (1-2 M KOH). These spectral properties, which appear unique to topaquinone, provide a rapid and simple test for the presence of this cofactor in intact enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigation of spectroscopic intermediates during copper-binding and TPQ formation in wild-type and active-site mutants of a copper-containing amine oxidase from yeast

Copper amine oxidases possess the unusual ability to generate autocatalytically their organic cofactor, which is subsequently utilized in turnover. This cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), is formed within the active site of these enzymes by the oxidation of a single tyrosine residue. In vitro, copper(II) and oxygen are both necessary and sufficient for the conversion of tyrosine to TPQ. In this study, the biogenesis of TPQ has been characterized in an amine oxidase from Hansenula polymorpha expressed as the apo-enzyme in Escherichia coli. With the WT enzyme, optical absorbances which are copper or oxygen dependent are observed and characterized. Active-site mutants are used to investigate further the nature of these spectral species. Evidence is presented which suggests that tyrosine is activated for reaction with oxygen by liganding to Cu(II). In the following paper in this issue [Schwartz, B., Dove, J. E., and Klinman, J. P. (2000) Biochemistry 39, 3699-3707], the initial reaction of precursor protein with oxygen is characterized kinetically. Taken together, the available data suggest a mechanism for the oxidation of tyrosine to TPQ where the role of the copper is to activate substrate.     

Linkage between catecholate siderophores and the multicopper oxidase CueO in Escherichia coli

The multicopper oxidase CueO had previously been demonstrated to exhibit phenoloxidase activity and was implicated in intrinsic copper resistance in Escherichia coli. Catecholates can potentially reduce Cu(II) to the prooxidant Cu(I). In this report we provide evidence that CueO protects E. coli cells by oxidizing enterobactin, the catechol iron siderophore of E. coli, in the presence of copper. In vitro, a mixture of enterobactin and copper was toxic for E. coli cells, but the addition of purified CueO led to their survival. Deletion of fur resulted in copper hypersensitivity that was alleviated by additional deletion of entC, preventing synthesis of enterobactin. In addition, copper added together with 2,3-dihydroxybenzoic acid or enterobactin was able to induce a Phi(cueO-lacZ) operon fusion more efficiently than copper alone. The reaction product of the 2,3-dihydroxybenzoic acid oxidation by CueO that can complex Cu(II) ions was determined by gas chromatography-mass spectroscopy and identified as 2-carboxymuconate.     

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.     

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 a copper-containing amine oxidase from Mycobacterium sp. strain JC1 DSM 3803 grown on benzylamine

A bacterial semicarbazide-sensitive amine oxidase (SSAO) was purified and characterized from Mycobacterium sp. strain JC1 DSM 3803 grown on benzylamine. During the purification procedures, the enzyme was tending to aggregate and exhibited heterogeneity in native PAGE. The heterogeneous forms having amine oxidase (AO) activity could be separated by their native molecular weights using gel-filtration chromatography. Most of the AOs behaved as dimers (M(r) 150,000) composed of a 75-kDa subunit, but some aggregated to form tetramers (M(r) 300,000). Besides their native molecular weight, subunit composition and V(max) value, both forms (dimer and tetramer) have almost identical biochemical properties (e.g. subunit size, optimum pH and temperature, activation energy, K(m) value on benzylamine, substrate and inhibitor specificities). When AO activity was observed by activity staining, the best-oxidized substrate was benzylamine, although the AO also oxidized tyramine and histamine. The AO was strongly inhibited by semicarbazide and isoniazid, but KCN did not affect its activity. The purified enzyme was shown to contain 2.39 mol of copper per mole of subunit, but there were no evidences of topaquinone co-factor involvement, when tested by absorption spectrum analysis and redox-cycling staining for quinoprotein detection.     

X-ray snapshots of quinone cofactor biogenesis in bacterial copper amine oxidase

The quinone cofactor TPQ in copper amine oxidase is generated by posttranslational modification of an active site tyrosine residue. Using X-ray crystallography, we have probed the copper-dependent autooxidation process of TPQ in the enzyme from Arthrobacter globiformis. Apo enzyme crystals were anaerobically soaked with copper; the structure determined from this crystal provides a view of the initial state: the unmodified tyrosine coordinated to the bound copper. Exposure of the copper-bound crystals to oxygen led to the formation of freeze-trapped intermediates; structural analyses indicate that these intermediates contain dihydroxyphenylalanine quinone and trihydroxyphenylalanine. These are the first visualized intermediates during TPQ biogenesis in copper amine oxidase.     

The role of copper in bovine serum amine oxidase

Summary The role of copper in bovine serum amine oxidase was investigated by studying the effect of copper-binding inhibitors on the reactions of the pyrroloquinoline quinone carbonyl and on the reaction with oxygen. Hydrazines and hydrazides were used as carbonyl reagents and one of the hydrazines, benzylhydrazine, which was found to behave as a pseudo-substrate, was used to probe the reaction with oxygen. The presence ofN,N-diethyldithiocarbamate, a chelator that binds copper irreversibly, did not prevent the reactions at the carbonyl, but slowed down their rate and modified the conformation of the adducts. The same happened to the reaction with oxygen, which was slowed down but not abolished. Copper, which was never seen in the reduced state, thus appears to control all reactions without being directly involved in the binding of either hydrazines or oxygen. The enzyme functionality was in fact preserved upon substitution of copper with cobalt. The specific activity of the cobalt-substituted enzyme was only reduced to about 40% the native amine oxidase value. This is the first case so far in which the role of copper can be performed by a different metal ion.Abbreviations BSAO bovine serum amine oxidase - DDC N,N-diethyldithiocarbamate - PQQ pyrroloquinoline quinone     

Confirmation of the presence of a Cu(II)/topa quinone active site in the amine oxidase from fenugreek seedlings

Amine oxidase from etiolated seedlings of fenugreek (Trigonellafoenum—graecum) has been isolated by a purification procedureinvolving three chromato—graphic steps. The homogeneousenzyme is of pink colour with a visible absorption maximum at500 nm. The dimeric enzyme (2 75 kDa) is a slightly acidicprotein (pl 6.8) containing 8% neutral sugars. N—ter—minalamino acid sequence of the enzyme shows a high degree of similarityto other plant and microbial copper—containing amine oxidases.The best substrates of the enzyme are aliphatic diamines andsome polyamines, whereas inhibitors are substrate analogues,copper complexing agents, some alkaloids and several other compounds.Spectrophotometric titra—tions with phenylhydrazines demonstratedone reactive carbonyl group per subunit of the enzyme and redox—cyclicquinone staining after native electrophor—esis indicatedthe presence of a quinone cofactor. Differential pulse polarographyshowed the existence of a copper/quinone—containing activesite. The resonance Raman spectroscopy and the pH—dependentshift of the absorption spectrum of the enzyme p—nitrophenylhydrazoneconfirm unambiguously the identity of the cofactor with topaquinone. EPR spectra of the enzyme are in accordance with thoseof tetragonal cupric complexes as known for other copper—containingamine oxidases. Besides the copper, Mn(II)ions were detectedthat partially occupy another metal site in the enzyme, buttheir catalytical importance is unlikely. Key words: Fenugreek, Trigonella foenum—graceum, amine oxidase, topa quinone