Impressive promiscuous biomimetic models of ascorbate, amine, and catechol oxidases.

Copper metalloenzymes ascorbate oxidase (AOase), amine oxidase (AmOase), and catechol oxidase (COase) possess copper(II) sites of coordination, which are trimeric, homodimeric, and dimeric, respectively. Two newly mononuclear copper(II) complexes, namely, [Cu(L)(bpy)](ClO4) (1) and [Cu(L)(phen)](ClO4) (2) where HL = Schiff base, have been synthesized. UV-visible, EPR and single-crystal X-ray diffraction examinations were used to validate the geometry in solution and solid state. For complex 1, the metal exhibits a coordination sphere between square pyramidal and trigonal bipyramidal geometry (τ, 0.49). A positive CuII/I redox potential indicates a stable switching between CuII and CuI redox states. Despite the monomeric origin, both homogeneous catalysts (1 or 2) in MeOH were found to favor three distinct chemical transformations, namely, ascorbic acid (H2A) to dehydroascorbic acid (DA), benzylamine (Ph-CH2-NH2) to benzaldehyde (Ph-CHO), and 3,5-di-tert-butylcatechol (3,5-DTBC) to 3,5-di-tert-butylquinone (3,5-DTBQ) [kcat: AOase, 9.6 (1) or 2.0 × 106 h-1(2); AmOase, 13.4 (1) or 9.4 × 106 h-1 (2); COase, 2.0 (1) or 1.9 × 103 h-1 (2)]. They exhibit higher levels of AOase activity as indicated by their kcat values compared to the AOase enzyme. The kcat values for COase activity in buffer solution [5.93 (1) or 2.95 × 105 h-1 (2)] are one order lower than those of the enzymes. This is because of the labile nature of the coordinated donor, the flexibility of the ligand, the simplicity of the catalyst-substrate interaction, and the positive CuII/I redox potential. Interestingly, more efficient catalysis is promoted by 1 and 2 concerning that of other mono- and dicopper(II) complexes.

Colorimetric detection of diamine using diamine oxidase and horseradish peroxidase co-incorporated hybrid microsphere as biomimetic cascade enzymes.

Dual-enzyme co-embedded materials have shown high potential for achieving efficient detection due to the convenience of two-enzyme cascade reactions. Herein, we developed a dual-enzyme hybrid microsphere (HM) based biosensor to detect diamines (histamine was included for ease of description) in aquatic products. The HM was made from diamine oxidase, horseradish peroxidase, and copper phosphate through the biomineralization method. Under optimal conditions, the system displayed linear color response to histamine of different concentrations ranging from 0 to 200 µg/mL. The detection limit of histamine was 0.15 µg/mL, showing higher sensitivity than the two-step free enzyme assay. Moreover, the detection system exhibited good specificity to diamines. The method was used to detect diamines in commercial samples, and the results were compared with those measured by the high-performance liquid chromatography method. Overall, the proposed assay exhibited high potential in diamine quantification and was readily extended to other cascade enzymatic reaction-based detection strategies.

Replacement of Tyrosines by Unnatural Amino Acid Aminophenylalanine Leads to Metal-Mediated Aniline Free Radical Formation in a Copper Amine Oxidase.

Copper amine oxidases (CAOs) catalyze the oxidative deamination of primary amines to aldehyde, ammonia, and hydrogen peroxide as products and are widely distributed in bacteria, plants, and eukaryotes. These enzymes initiate the single turnover, post-translational conversion of an active site tyrosine to the redox cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ), subsequently employing TPQ to catalyze steady-state amine oxidation. The mechanisms of TPQ biogenesis and steady-state amine oxidation have been studied extensively, with consensus mechanisms proposed for both reactions. One unresolved issue has been whether the Cu2+ center must undergo formal reduction to Cu1+ in the course of the reaction. Herein, we investigate the properties of the active site of a yeast (Hansenula polymorpha) amine oxidase (HPAO) that has undergone site-specific insertion of a para-aminophenylalanine (pAF) into the position of either the precursor tyrosine to TPQ (Y405) or the two strictly conserved neighboring tyrosines (Y305 and Y407). While our original intention was to interrogate cofactor biogenesis using a precursor unnatural amino acid (UAA) of altered redox potential and pKa, we instead observe an unanticipated reaction assigned to an intramolecular electron transfer from pAF to the active site copper ion. We establish the generality of the observed active site chemistry using exogenously added, aniline-containing substrates under conditions that prevent side chain amine oxidation. The results support previous proposals that the activation of the TPQ precursor occurs in the absence of a formal valence change at the active site copper site. The described reaction of pAFs with the active site redox Cu2+ center of HPAO provides a prototype for either the engineering of the enzymatic oxidation of exogenous anilines or the insertion of site-specific free radical probes within proteins.

Recent advances in the application of microbial diamine oxidases and other histamine-oxidizing enzymes.

The consumption of foods fraught with histamine can lead to various allergy-like symptoms if the histamine is not sufficiently degraded in the human body. The degradation occurs primarily in the small intestine, naturally catalyzed by the human diamine oxidase (DAO). An inherent or acquired deficiency in human DAO function causes the accumulation of histamine and subsequent intrusion of histamine into the bloodstream. The histamine exerts its effects acting on different histamine receptors all over the body but also directly in the intestinal lumen. The inability to degrade sufficient amounts of dietary histamine is known as the 'histamine intolerance'. It would be preferable to solve this problem initially by the production of histamine-free or -reduced foods and by the oral supplementation of exogenous DAO supporting the human DAO in the small intestine. For the latter, DAOs from mammalian, herbal and microbial sources may be applicable. Microbial DAOs seem to be the most promising choice due to their possibility of an efficient biotechnological production in suitable microbial hosts. However, their biochemical properties, such as activity and stability under process conditions and substrate selectivity, play important roles for their successful application. This review deals with the advances and challenges of DAOs and other histamine-oxidizing enzymes for their potential application as processing aids for the production of histamine-reduced foods or as orally administered adjuvants to humans who have been eating food fraught with histamine.

Glycosylation site Asn168 is important for slow in vivo clearance of recombinant human diamine oxidase heparin-binding motif mutants.

Elevated plasma and tissues histamine concentrations can cause severe symptoms in mast cell activation syndrome, mastocytosis or anaphylaxis. Endogenous and recombinant human diamine oxidase (rhDAO) can rapidly and completely degrade histamine, and administration of rhDAO represents a promising new treatment approach for diseases with excess histamine release from activated mast cells. We recently generated heparin-binding motif mutants of rhDAO with considerably increased in vivo half-lives in rodents compared with the rapidly cleared wildtype protein. Herein, we characterize the role of an evolutionary recently added glycosylation site asparagine 168 in the in vivo clearance and the influence of an unusually solvent accessible free cysteine 123 on the oligomerization of diamine oxidase (DAO). Mutation of the unpaired cysteine 123 strongly reduced oligomerization without influence on enzymatic DAO activity and in vivo clearance. Recombinant hDAO produced in ExpiCHO-S™ cells showed a 15-fold reduction in the percentage of glycans with terminal sialic acid at Asn168 compared with Chinese hamster ovary (CHO)-K1 cells. Capping with sialic acid was also strongly reduced at the other glycosylation sites. The high abundance of terminal mannose and N-acetylglucosamine residues in the four glycans expressed in ExpiCHO-S™ cells compared with CHO-K1 cells resulted in rapid in vivo clearance. Mutation of Asn168 or sialidase treatment also significantly increased clearance. Intact N-glycans at Asn168 seem to protect DAO from rapid clearance in rodents. Full processing of all glycoforms is critical for preserving the improved in vivo half-life characteristics of the rhDAO heparin-binding motif mutants.

Biocatalytic Production of Aldehydes: Exploring the Potential of Lathyrus cicera Amine Oxidase.

Aldehydes are a class of carbonyl compounds widely used as intermediates in the pharmaceutical, cosmetic and food industries. To date, there are few fully enzymatic methods for synthesizing these highly reactive chemicals. In the present work, we explore the biocatalytic potential of an amino oxidase extracted from the etiolated shoots of Lathyrus cicera for the synthesis of value-added aldehydes, starting from the corresponding primary amines. In this frame, we have developed a completely chromatography-free purification protocol based on crossflow ultrafiltration, which makes the production of this enzyme easily scalable. Furthermore, we determined the kinetic parameters of the amine oxidase toward 20 differently substituted aliphatic and aromatic primary amines, and we developed a biocatalytic process for their conversion into the corresponding aldehydes. The reaction occurs in aqueous media at neutral pH in the presence of catalase, which removes the hydrogen peroxide produced during the reaction itself, contributing to the recycling of oxygen. A high conversion (>95%) was achieved within 3 h for all the tested compounds.

Microcrystal preparation for serial femtosecond X-ray crystallography of bacterial copper amine oxidase.

Recent advances in serial femtosecond X-ray crystallography (SFX) using X-ray free-electron lasers have paved the way for determining radiation-damage-free protein structures under nonfreezing conditions. However, the large-scale preparation of high-quality microcrystals of uniform size is a prerequisite for SFX, and this has been a barrier to its widespread application. Here, a convenient method for preparing high-quality microcrystals of a bacterial quinoprotein enzyme, copper amine oxidase from Arthrobacter globiformis, is reported. The method consists of the mechanical crushing of large crystals (5-15 mm3), seeding the crushed crystals into the enzyme solution and standing for 1 h at an ambient temperature of ∼26°C, leading to the rapid formation of microcrystals with a uniform size of 3-5 µm. The microcrystals diffracted X-rays to a resolution beyond 2.0 Šin SFX measurements at the SPring-8 Angstrom Compact Free Electron Laser facility. The damage-free structure determined at 2.2 Šresolution was essentially identical to that determined previously by cryogenic crystallography using synchrotron X-ray radiation.

Heparin-binding motif mutations of human diamine oxidase allow the development of a first-in-class histamine-degrading biopharmaceutical.

Background: Excessive plasma histamine concentrations cause symptoms in mast cell activation syndrome, mastocytosis, or anaphylaxis. Anti-histamines are often insufficiently efficacious. Human diamine oxidase (hDAO) can rapidly degrade histamine and therefore represents a promising new treatment strategy for conditions with pathological histamine concentrations. Methods: Positively charged amino acids of the heparin-binding motif of hDAO were replaced with polar serine or threonine residues. Binding to heparin and heparan sulfate, cellular internalization and clearance in rodents were examined. Results: Recombinant hDAO is rapidly cleared from the circulation in rats and mice. After mutation of the heparin-binding motif, binding to heparin and heparan sulfate was strongly reduced. The double mutant rhDAO-R568S/R571T showed minimal cellular uptake. The short α-distribution half-life of the wildtype protein was eliminated, and the clearance was significantly reduced in rodents. Conclusions: The successful decrease in plasma clearance of rhDAO by mutations of the heparin-binding motif with unchanged histamine-degrading activity represents the first step towards the development of rhDAO as a first-in-class biopharmaceutical to effectively treat diseases characterized by excessive histamine concentrations in plasma and tissues. Funding: Austrian Science Fund (FWF) Hertha Firnberg program grant T1135 (EG); Sigrid Juselius Foundation, Medicinska Understödsförening Liv och Hälsa rft (TAS and SeV).

Three-in-one via syringe needle-based device: sampling, microextraction and peroxidase-like catalysis for colorimetric detection of the change of biogenic amines levels with time in meat.

In this work, a syringe needle-based integrated method was designed for the detection of biogenic amines (BAs) in raw meat samples. Based on a sequential process, the needle-based sampling, micro liquid-phase extraction and peroxidase-like catalysis were adopted for the sample collection, target analytes extraction and colorimetric analysis, respectively. The proposed method exhibited high selectivity towards BAs (the total amount of histamine, putrescine and cadaverine was utilized to present the level of BAs), where the linear range is 5-50 µM and 50-1000 µM, and the limit of detection is 1.52 µM. Specifically, the whole process could be completed in a single syringe needle. In addition, due to the minimized sampling, the change of BAs levels with time in different area of real samples (fish) can be conveniently investigated. This method has the advantages of simplicity, low cost, high sensitivity and selectivity, endowing it a promising candidate for food analysis.

Measuring Histamine and Cytokine Release from Basophils and Mast Cells.

Basophils and mast cells are known for their capability to release both preformed and newly synthesized inflammatory mediators. In this chapter, we describe how to stimulate and detect histamine released from basophils in whole blood, purified basophils, in vitro cultured mast cells, and in situ skin mast cells (the latter by microdialysis), using either a solid phase assay or flow cytometry. We also give an example of an activation protocol for basophil and mast cell cytokine release and discuss approaches for cytokine detection.

Neutron crystallography of copper amine oxidase reveals keto/enolate interconversion of the quinone cofactor and unusual proton sharing.

Recent advances in neutron crystallographic studies have provided structural bases for quantum behaviors of protons observed in enzymatic reactions. Thus, we resolved the neutron crystal structure of a bacterial copper (Cu) amine oxidase (CAO), which contains a prosthetic Cu ion and a protein-derived redox cofactor, topa quinone (TPQ). We solved hitherto unknown structures of the active site, including a keto/enolate equilibrium of the cofactor with a nonplanar quinone ring, unusual proton sharing between the cofactor and the catalytic base, and metal-induced deprotonation of a histidine residue that coordinates to the Cu. Our findings show a refined active-site structure that gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions.

Comparison of Inhibitor and Substrate Selectivity between Rodent and Human Vascular Adhesion Protein-1.

Vascular adhesion protein-1 (VAP-1) is an ectoenzyme that functions as a copper-containing amine oxidase and is involved in leukocyte adhesion at sites of inflammation. Inhibition of VAP-1 oxidative deamination has become an attractive target for anti-inflammatory therapy with demonstrated efficacy in rodent models of inflammation. A previous comparison of purified recombinant VAP-1 from mouse, rat, monkey, and human gene sequences predicted that rodent VAP-1 would have higher affinity for smaller hydrophilic substrates/inhibitors because of its narrower and more hydrophilic active site channel. An optimized in vitro oxidative deamination fluorescence assay with benzylamine (BA) was used to compare inhibition of five known inhibitors in recombinant mouse, rat, and human VAP-1. Human VAP-1 was more sensitive compared to rat or mouse VAP-1 (lowest IC50 concentration) to semicarbazide but was least sensitive to hydralazine and LJP-1207. Hydralazine had a lower IC50 in rats compared to humans, although not significant. However, the IC50 of hydralazine was significantly higher in the rat compared to mouse VAP-1. The larger hydrophobic compounds from Astellas (compound 35c) and Boehringer Ingelheim (PXS-4728A) were hypothesized to have higher binding affinity for human VAP-1 compared to rodent VAP-1 since the channel in human VAP-1 is larger and more hydrophobic than that in rodent VAP-1. Although the sensitivity of these two inhibitors was the lowest in the mouse enzyme, we found no significant differences between mouse, rat, and human VAP-1. Michaelis-Menten kinetics of the small primary amines phenylethylamine and tyramine were also compared to the common marker substrate BA demonstrating that BA had the highest affinity among the substrates. Rat VAP-1 had the highest affinity for all three substrates and mouse VAP-1 had intermediate affinity for BA and phenylethylamine, but tyramine was not a substrate for mouse VAP-1 under these assay conditions. These results suggest that comparing oxidative deamination in mouse and rat VAP-1 may be important if using these species for preclinical efficacy models.

Autophagy induction by exogenous polyamines is an artifact of bovine serum amine oxidase activity in culture serum.

Polyamines are small polycationic alkylamines involved in many fundamental cellular processes, including proliferation, nucleic acid synthesis, apoptosis, and protection from oxidative damage. It has been proposed that in addition to these functions, elevated levels of polyamines promote longevity in various biological systems, including yeast, Drosophila, and murine models. A series of in vitro mechanistic studies by multiple investigators has led to the conclusion that addition of exogenous spermidine promotes longevity through autophagy induction; however, these experiments were confounded by the use of mammalian cell culture systems supplemented with fetal bovine serum. Using cell viability assays, LC3B immunoblots, and live-cell fluorescence microscopy, we report here that in the presence of ruminant serum, exogenously added polyamines are quickly oxidized by the copper-containing bovine serum amine oxidase. This polyamine oxidation resulted in the production of harmful byproducts including hydrogen peroxide, ammonia, and reactive aldehydes. Our data demonstrate that it is critically important to prevent confounding bovine serum amine oxidase-induced cytotoxicity in mechanistic studies of the roles of polyamines in autophagy.

Zinc-α2-glycoprotein as an inhibitor of amine oxidase copper-containing 3.

Zinc-α2-glycoprotein (ZAG) is a major plasma protein whose levels increase in chronic energy-demanding diseases and thus serves as an important clinical biomarker in the diagnosis and prognosis of the development of cachexia. Current knowledge suggests that ZAG mediates progressive weight loss through ß-adrenergic signalling in adipocytes, resulting in the activation of lipolysis and fat mobilization. Here, through cross-linking experiments, amine oxidase copper-containing 3 (AOC3) is identified as a novel ZAG binding partner. AOC3-also known as vascular adhesion protein 1 (VAP-1) and semicarbazide sensitive amine oxidase (SSAO)-deaminates primary amines, thereby generating the corresponding aldehyde, H2O2 and NH3. It is an ectoenzyme largely expressed by adipocytes and induced in endothelial cells during inflammation. Extravasation of immune cells depends on amine oxidase activity and AOC3-derived H2O2 has an insulinogenic effect. The observations described here suggest that ZAG acts as an allosteric inhibitor of AOC3 and interferes with the associated pro-inflammatory and anti-lipolytic functions. Thus, inhibition of the deamination of lipolytic hormone octopamine by AOC3 represents a novel mechanism by which ZAG might stimulate lipolysis. Furthermore, experiments involving overexpression of recombinant ZAG reveal that its glycosylation is co-regulated by oxygen availability and that the pattern of glycosylation affects its inhibitory potential. The newly identified protein interaction between AOC3 and ZAG highlights a previously unknown functional relationship, which may be relevant to inflammation, energy metabolism and the development of cachexia.

Copper­containing amine oxidase purified from Lathyrus sativus as a modulator of human neutrophil functions.

Over the last few decades, copper­containing amine oxidase (Cu­AO) from vegetal sources, and belonging to the class of diamine oxidase, has been documented to exhibit beneficial effects in both in vivo and ex vivo animal models of inflammatory or allergic conditions, including asthma­like reaction and myocardial or intestinal ischemia­reperfusion injuries. The aim of the present study was to assess the potential of vegetal Cu­AO as an anti­inflammatory and an antiallergic agent and to clarify its antioxidant properties. In cell­free systems, the reactive oxygen species and reactive nitrogen species scavenging properties of Cu­AO that is purified from Lathyrus sativus were investigated. Its effect on the formyl­methionyl­leucyl­phenylalanine peptide (fMLP)­activated cellular functions of human neutrophils were subsequently analyzed. The obtained results demonstrated that Cu­AO is not a scavenger of superoxide or nitric oxide, and does not decompose hydrogen peroxide. However, it inhibits the fMLP­dependent superoxide generation, elastase release and cell migration, and interferes with the process of calcium flux, supporting the idea that plant Cu­AO can interact with human neutrophils to modulate their inflammatory function. Therefore, the importance of these properties on the possible use of vegetal Cu­AO to control inflammatory conditions, particularly intestinal inflammation, is discussed in the current study.

Novel approach using activated cellulose film for efficient immobilization of purified diamine oxidase to enhance enzyme performance and stability.

The presence of various contaminants in foodstuffs has led to serious public health concerns. Diamine oxidase (DAO) has attracted tremendous attention for guarding food safety as well as clinical and environmental industries. In this study, DAO from Pisum sativum (Pea) seedlings was extracted and purified by dialysis and gel filtration. Purified DAO was covalently immobilized onto the surface of nitrocellulose membrane using glutaraldehyde. The obtained bioaffinity support has efficiently shown high yield immobilization of DAO from pea seedlings. The optimal conditions of free and immobilized DAO activity were evaluated against the substrate, Putrescine dihydrochloride. The influence of pH, temperature, storage stability, and reusability of immobilized enzyme with comparison to the free enzyme was studied and the results showed that the stabilities were significantly enhanced compared with free counterpart. Residual activity of the immobilized enzyme was 59% of the initial activity after being recycled 10 times. We approve that this novel low cost immobilized DAO carrier presents a new approach in large scale applications.

Human Copper-Containing Amine Oxidases in Drug Design and Development.

Two members of the copper-containing amine oxidase family are physiologically important proteins: (1) Diamine oxidase (hDAO; AOC1) with a preference for diamines is involved in degradation of histamine and (2) Vascular adhesion protein-1 (hVAP-1; AOC3) with a preference for monoamines is a multifunctional cell-surface receptor and an enzyme. hVAP-1-targeted inhibitors are designed to treat inflammatory diseases and cancer, whereas the off-target binding of the designed inhibitors to hDAO might result in adverse drug reactions. The X-ray structures for both human enzymes are solved and provide the basis for computer-aided inhibitor design, which has been reported by several research groups. Although the putative off-target effect of hDAO is less studied, computational methods could be easily utilized to avoid the binding of VAP-1-targeted inhibitors to hDAO. The choice of the model organism for preclinical testing of hVAP-1 inhibitors is not either trivial due to species-specific binding properties of designed inhibitors and different repertoire of copper-containing amine oxidase family members in mammalian species. Thus, the facts that should be considered in hVAP-1-targeted inhibitor design are discussed in light of the applied structural bioinformatics and structural biology approaches.

Amperometric biogenic amine biosensors based on Prussian blue, indium tin oxide nanoparticles and diamine oxidase- or monoamine oxidase-modified electrodes.

Biogenic amine biosensors, based on screen-printed carbon electrodes (SPCE) modified with Prussian blue (PB) and indium tin oxide nanoparticles (ITONP), are reported. PB/ITONP-modified SPCE was further modified with diamine oxidase (DAO) or monoamine oxidase (MAO) enzymes to construct the biosensors. The morphology of the modified electrodes was studied by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX) and atomic force microscopy (AFM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to enlighten the electrochemical properties of the modified electrodes at each step of biosensor fabrication. Electrode surface composition and experimental conditions were optimized and analytical performance characteristics of the biosensors were studied. Several biogenic amines were tested and both biosensors responded to histamine, putrescine and cadaverine. DAO/ITONP/PB/SPCE biosensor exhibited the highest response to histamine 6.0 × 10-6-6.9 × 10-4 M with a sensitivity of 1.84 µA mM-1. On the other hand, the highest sensitivity was obtained for cadaverine with the MAO/ITONP/PB/SPCE biosensor. The analytical utility of the presented biosensors were illustrated by the determination of cadaverine and histamine in cheese sample.

Evaluation of porcine diamine oxidase for the conversion of histamine in food-relevant amounts.

Histamine exists in a multitude of foods and displays an emerging role within food intolerances. Our aim was to identify the activity of porcine diamine oxidase (DAO) required for the in vitro degradation of histamine amounts that are found in typical meals containing histamine (75 mg, equaled 150 mg/L). Furthermore, we investigated an actual dietary supplement that is commercially available for histamine intolerant individuals for its histamine reduction capability. Kinetic investigations of porcine DAO showed a substrate inhibition by histamine concentrations greater than 56 mg/L (0.5 mM). The stability of free porcine DAO was tested in a fed state simulated intestinal fluid and exhibited a half-life period of around 19 min. A total of 50 nanokatal (nkat) free porcine DAO, which equaled the amount of enzyme isolated from around 100 g pig kidney, were necessary for the in vitro reduction of around 90% of the histamine. The dietary supplement that contains a pig kidney extract did not show DAO activity. Instead, the used histamine (0.75 mg) was apparently reduced due to the adsorption of histamine onto a capsule component by 18.9 ± 2.3% within 5 hr. Although the capsule preparation retained its overall structure and shape for at least 90 min in simulated gastric fluid, the apparent histamine reduction was significantly reduced to 12.1 ± 2.3% (P ≤ 0.05). In conclusion, an alternative to the pig kidney DAO or an improved capsule preparation is needed to ensure an adequate supplementation for histamine-intolerant humans. PRACTICAL APPLICATION: Histamine intolerance is an emerging issue in our society and the intolerance-related physiological symptoms are currently not reliably treatable due to a lack of scientific investigation. A commercially available dietary supplement for histamine intolerance does not fulfil the requirements for a satisfactory histamine reduction in intolerant humans. The activity of the histamine degrading enzyme diamine oxidase, required for a satisfactory histamine degradation, is by far higher than the theoretical amount apparently given in the dietary supplement. With this knowledge, it is obvious that improved food supplements must be developed to help histamine intolerant humans.

Evolution and functional classification of mammalian copper amine oxidases.

Mammalian copper-containing amine oxidases (CAOs), encoded by four genes (AOC1-4) and catalyzing the oxidation of primary amines to aldehydes, regulate many biological processes and are linked to various diseases including inflammatory conditions and histamine intolerance. Despite the known differences in their substrate preferences, CAOs are currently classified based on their preference for either primary monoamines (EC 1.4.3.21) or diamines (EC 1.4.3.22). Here, we present the first extensive phylogenetic study of CAOs that, combined with structural analyses of the CAO active sites, provides in-depth knowledge of their relationships and guidelines for classification of mammalian CAOs into AOC1-4 sub-families. The phylogenetic results show that CAOs can be classified based on two residues, X1 and X2, from the active site motif: T/S-X1-X2-N-Y-D. Residue X2 discriminates among the AOC1 (Tyr), AOC2 (Gly), and AOC3/AOC4 (Leu) proteins, while residue X1 further classifies the AOC3 (Leu) and AOC4 (Met) proteins that so far have been poorly identified and annotated. Residues X1 and X2 conserved within each sub-family and located in the catalytic site seem to be the key determinants for the unique substrate preference of each CAO sub-family. Furthermore, one residue located at 10 Šdistance from the catalytic site is different between the sub-families but highly conserved within each sub-family (Asp in AOC1, His in AOC2, Thr in AOC3 and Asn in AOC4) and likely contributes to substrate selectivity. Altogether, our results will benefit the design of new sub-family specific inhibitors and the design of in vitro tests to detect individual CAO levels for diagnostic purposes.