Copper amine oxidases : structures, catalytic mechanisms, and role in pathophysiology

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As a result, only NH 3 was detected successfully Supplementary Fig. In addition, we found that H 2 O 2 was also produced through HarA-catalyzed harmaline degradation.

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Also, HarA showed no activity under strict anaerobic conditions. These findings indicated that O 2 acted as an electron acceptor and was converted into H 2 O 2 in HarA-catalyzed harmaline degradation. The stoichiometry of HarA-catalyzed harmaline degradation was examined. Post-translationally-synthesized cofactor topaquinone of HarA. Topaquinone TPQ within HarA was post-translationally synthesized from a specific tyrosine residue of HarA in the presence of copper ion and involved in HarA-catalyzed harmaline degradation.

UV-vis spectra of the purified enzymes are shown. The nonapeptide that was labeled with phenylhydrazine is denoted as a black star. We examined the substrate specificity of HarA using linear N -benzylidenemethylamine and N -benzylideneaniline or cyclic 2-methylenepiperidine [MP] imine substrates Supplementary Fig. Among them, MP was found to be a substrate for HarA. Linear imine substrates were inert for HarA.

Substrate specificity and kinetic properties of HarA. HarA catalyzed ring-opening reaction of cyclic imine within 2-methylenepiperidine MP. Michaelis—Menten analyses were performed using c harmaline or d MP as the substrate. Using various concentrations of harmaline and MP, we examined the kinetic properties of HarA.

Typical hyperbolic curves of product formation for each of the harmaline and MP concentration were obtained, which indicated that the reactions followed Michaelis—Menten kinetics Fig. The K m value 1. On the other hand, the V max value 0.

These findings indicate that the aromatic ring moiety was important for the interaction between an imine substrate and HarA. In addition, we also examined the substrate specificity of HarA toward amine substrates. HarA-catalyzed oxidations of aromatic substrates tryptamine and benzylamine were much faster than those of linear substrates methylamine, ethylamine, hexylamine, and octylamine Supplementary Fig. Through single-amino acid substitution of each of the above amino acid residues, we constructed a set of mutants i.

Even after overnight incubation, these mutants produced no reaction product. On the other hand, DE mutant catalyzed deamination of tryptamine, although the activity was less than that of wild-type enzyme Fig. Considering the previous results 10 , 11 , the other tested amino acid residues would contribute to the stability of TPQ and the substrate specificity of the enzyme. Identification of catalytic residues and reaction intermediates of HarA-catalyzed reaction.

To obtain insight into the reaction mechanism of HatA-catalyzed cyclic imine cleavage reaction, various biochemical analyses were performed. Dashed lines indicate the retention time in which each of the reaction products is detected.

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All spectra were obtained by subtracting the spectra of the YF mutant from those of native HarA. Arrows indicate the peaks that appeared upon the addition of each substrate. We spectroscopically analyzed reaction intermediates of harmaline degradation and oxidation of the amine moiety of an amine substrate. For Raman and UV-vis spectroscopic analyses, we were not able to use harmaline as a substrate for HarA for the following reasons: i the fluorescence of harmaline would have interfered with Raman spectroscopic analysis, and ii an absorbance band of harmaline in the UV-vis spectrum overlapped with that of TPQ.

These spectroscopic features were almost identical with those of a deprotonated imine intermediate of TPQ, which had been previously reported as the reaction intermediate of CAOs with amine substrates 9. These spectroscopic features strongly suggested the existence of a reaction intermediate other than an imine intermediate of TPQ, when the substrate was a cyclic imine. We investigated whether or not another CAO could catalyze harmaline degradation. An expression plasmid containing tynA was then constructed and introduced into E.

CAO from E. However, the harmaline-degrading activity of E. Harmaline-degrading activity of copper amine oxidase from E. Copper amine oxidase CAO from E. Lane M, marker proteins. One of the organisms is Banisteriopsis caapi Malpigbiaceae , from which Amazonian tribes make a botanical beverage, Ayahuasca, for sacred rituals.

Ayahuasca is famous for its strong hallucinogenic activity, and has the potential for medical use as a cure for drug addiction, depression, and anxiety disorders Notably, unique functions of harmaline, such as inhibition of monoamine oxidase A, are considered to contribute to these bioactivities. In addition, harmaline and harmine, which is a dehydrogenated derivative of harmaline, have been shown to exhibit potential therapeutic activity against diabetes On the other hand, the knowledge of their metabolism is limited.

In mammals, only O -demethylation, hydroxylation, and dehydrogenation are known as metabolic reactions for harmaline 17 , In nature, imines are generated through numerous metabolic pathways, such as biosynthetic pathways for secondary metabolites In addition, cyclic imines are present in the substructures of various bioactive secondary metabolites; e.

Synthetic cyclic imine sugars as potent inhibitors of various glycosidases are of medicinal importance Notably, in contrast to linear imines, cyclic imines are generally stable and hardly undergo hydrolysis. In fact, cyclic imine within harmaline was found to be stable with the respect to the spontaneous hydrolysis in water Supplementary Fig.

As far as we know, cyclic imine-opening enzymes have never been previously found; cleaving activity of CAO toward cyclic imines including harmaline has never been reported. Although flavin-dependent monoamine oxidases MAOs catalyze similar oxidative deamination to that of CAOs, notably, harmaline and its derivatives have been known to be a strong competitive inhibitor of MAOs rather than their substrate Therefore, harmaline-degrading activity would be a unique feature of CAOs.

Similar to TPQ, some cofactors, such as lysine tyrosylquinone LTQ and tryptophan tryptophylquinone TTQ , are also known to be post-translationally generated from specific amino acid residues This finding indicates that these CAOs are also able to catalyze imine-cleaving reactions if imine substrates bind to the active sites of the enzymes. Indeed, CAO from E. In addition, human CAOs, such as AOC2 retina-specific copper amine oxidase and AOC3 vascular adhesion protein, VAP-1 , are known to have substrate specificities that are similar to those of HarA and ECAO; these enzymes are more specific for aromatic substrates, such as benzylamine or tryptamine, than methylamine or ethylamine 30 , Moreover, notably, harmaline or its derivatives have been detected in human tissues such as the eyes of cataract patients, where AOC2 is specifically expressed 7 , Many studies have been performed to elucidate the mechanism of CAO-catalyzed amine oxidation.

In the proposed mechanism 34 , 35 , 36 , TPQ forms substrate Schiff base with the substrate, an amine.

Copper Amine Oxidases. Structures, Catalytic Mechanisms and Role in Pathophysiology.

Substrate Schiff base is converted into product Schiff base and then hydrolyzed by the aspartate residue in the active site of CAO, releasing an aldehyde product. Next, the copper ion activates O 2 to accept electrons from the resulting semiquinone. The results of single-amino acid mutagenesis showed that Asp of HarA is crucial for enzyme activity toward both harmaline and amine substrates. As the aspartate residue and copper ion have been shown to also be crucial for CAO-catalyzed amine oxidation 34 , 35 , 36 , the above results indicate that amine oxidation and harmaline-degrading reaction share these catalytic components aspartate residue and copper ion.

A semiquinone intermediate has been identified as one of the reaction intermediates which are derived from TPQ of CAO-catalyzed amine oxidation 9. As these features are similar to those of a reaction intermediate of HarA with MP, it is indicated that this intermediate was also observed in our experiment.

The existence of semiquinone in both HarA-catalyzed MP degradation and deamination of amine substrates suggests that these reactions proceed via the formation of so far known intermediates i. Schematic diagram of a possible reaction mechanism of HarA-catalyzed reactions. A possible reaction mechanism of two-step degradation of harmaline by HarA. The reaction found in this study is indicated by a red arrow. Each substrate forms substrate Schiff base with TPQ. Substrate Schiff base is converted to product Schiff base not shown in the figure through deprotonation by deprotonated Asp Hydrolysis of product Schiff base is facilitated by protonation of the intermediate by protonated Asp The previous study 9 shows that some amine substrates form an inactive deprotonated imine intermediate with TPQ.

Although substrate Schiff base and product Schiff base are unstable, the above inactive deprotonated imine intermediate is detectable on Raman spectroscopy. In our resonance Raman spectroscopic analyses, we observed a benzylamine adduct, which would be the inactive deprotonated imine intermediate, in HarA Fig. In the proposed reaction mechanism of CAOs 9 , nucleophilic attack on product Schiff base by H 2 O is facilitated by protonation of the intermediate. As reported previously for the methylamine adducts of phenylethylamine oxidase and bovine serum amine oxidase 9 , the deprotonated and inactive state of the benzylamine adduct of HarA may be favored by flipping of TPQ and interaction with copper or amino acid residues under anaerobic conditions Supplementary Fig.

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One of these oxygen atoms would be incorporated into 2-AIMA through hydrolysis of product Schiff base. On the other hand, the other H 2 O-derived oxygen atom would be added to harmaline through nucleophilic attack by activated H 2 O on an electrophilic carbon before the formation of substrate Schiff base. The above result of oxygen incorporation suggests that H 2 O is activated in the initial step of the reaction by any of the active site components, which are polar amino acid residues, TPQ and copper. On the other hand, DE mutant catalyzed the deamination of tryptamine, although this enzyme completely lacked harmaline-degrading activity Fig.

These results indicate that HarA lost the ability for the hydrolysis of harmaline through DE mutation, while Glu could act as an active site base instead of Asp for oxidative deamination of an amine substrate, as previously reported for CAOs of E. Therefore, Asp may play an essential role for the hydrolysis of cyclic imine, and length of the side chain of the amino acid residue would be crucial for the activity.

Therefore, CAOs would be unique dual functional enzymes catalyzing cyclic imine hydrolysis besides oxidative deamination of amine substrates.