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The physiological function of the plant enzyme alternative oxidase has long been a topic of debate. The cyanide-resistant alternative oxidase (AOX), along with the cytochrome c oxidase (COX), catalyzes the reduction of oxygen to water in the electron transport chain of mitochondrial respiration. Although respiration via the alternative pathway (AP) results in approximately one third of the ATP production as respiration via the cytochrome pathway (CP), the AP is utilized by all plants and some fungi and animals. This “energy wasteful” pathway has been proposed to reduce oxidative stress in plant cells under a variety of stressful conditions. Virtually all previous work on the AP has been performed on laboratory-grown plants in controlled environment conditions; thus, there is little knowledge of how the AP responds to unstable conditions and multiple environmental stresses in the field. This thesis presents new methodology for studying AP respiration and the AOX protein in field-grown plants, and investigates how the AP responds to natural changes in environmental conditions in the field in several plant species grown in diverse ecosystem types. The experimental work presented here also investigates how AP activity is related to changes in total rates of respiration, and questions whether abundance of the AOX protein determines electron partitioning to the AP. AP partitioning (or relative changes in AP partitioning) varied over seasonal timescales in each of the experimental studies. Chapter 3 reports on two species of Chionochloa, a native New Zealand tussock grass growing along an altitudinal gradient. In Chapter 4, seasonal variation was studied in two tree types: Populus x canadensis, a deciduous angiosperm, and Pinus radiata, an evergreen gymnosperm. Quercus rubra trees were studied along an urban-rural gradient originating in New York City in Chapter 5. In a highly exposed and variable environment, relative changes in AP partitioning in two species of Chionochloa were correlated with the previous day’s integrated light. In Quercus rubra, the AP was instead related to temperature changes: relative AP partitioning increased in response to seasonally low temperature in trees grown at colder, more rural field sites, while at the warmer, urban sites, it increased in response to high summer temperatures. Each of these environmental conditions that were related to increases in the AP (high light, low temperatures, and heat) are potentially stressful to plants. Thus, it is possible that the increases in AP respiration observed in these studies served to oxidize excess reducing equivalents generated through stressful conditions. In Chapter 4, although AP partitioning in Populus x canadensis and Pinus radiata varied seasonally, these changes were not directly related to environmental parameters. However, AP partitioning in Populus x canadensis was clearly shown to be dependent on measurement temperature. In each of the studies presented here, changes in the AP were not related to abundance of the AOX protein. AOX protein abundance showed consistent seasonal patterns in the two deciduous angiosperms, Populus sp. and Quercus sp, and was correlated with seasonal changes in temperature in Chionochloa spp. However, the lack of correlation between protein abundance and AP partitioning indicates that the AP is subject to post-translational control and likely varies more rapidly than protein levels. In each of Chapters 3 – 5, there was no clear impact of changes in AP partitioning on rates of total respiration. As the AP produces less ATP than the CP, I hypothesized that increases in AP activity would lead to higher respiration rates in order to meet a plant’s energy demands. However, in Populus x canadensis and Quercus rubra, respiration rates remained stable during sharp increases in AP partitioning, indicating that, at least under certain conditions, increases in AP activity are accompanied by a decrease in the CP. In some of the first research studying AP partitioning in field-grown plants, this thesis illuminates possible mechanisms, functions, and implications of the AP. Over a range of plant taxa and environmental settings, this work shows that the AP does respond to stressful conditions in the wild, but that this does not result in increased respiration. Lastly, the methods presented here to study AP activity and AOX proteins in the field enable future studies to further probe the specific responses of AOX to natural stresses.

In this study, the aim was to find an economical plant source for polyphenol oxidase (PPO) production as an alternative to mushroom and possible application areas by characterization and immobilization of the PPOs. For this purpose, tissues of various plants of no commercial value were screened for their PPO activities. Mulberry leaf tissues showed the highest PPO activity against 4-methyl catechol which was comparable to that of mushroom. Average Km and Vmax values of free mulberry leaf PPOs were found as 7 mM and 218 U/ml, respectively. Mulberry leaf PPOs were immobilized in a polypyrole matrix and the Km and Vmax values of immobilized PPOs were calculated as 35 mM and 3 U/ml, respectively. Mulberry leaf PPO was the most active at 45&deg / C and pH 7. By using electrophoretic analysis, laccase and catechol oxidase type activities of PPOs and in addition, peroxidase activity were detected. Molecular weights of laccase, peroxidase and catechol oxidase were found to be about 62, 64 and 62-64 kDa, with pI values of 8.0-8.5, 4.5 and 10, sequentially.

Phenelzine (PLZ) is a monoamine oxidase (MAO) inhibitor that also inhibits the activity of GABA-transaminase (GABA-T), causing significant and long-lasting increases in brain GABA levels. Inhibition of MAO prior to PLZ administration has been shown to prevent the GABAergic effects of the drug, strongly suggesting that a metabolite of PLZ formed by the action of MAO is responsible for the GABAergic effects. While PLZ has been used clinically for decades for its antidepressant and antipanic effects, it has more recently been shown to be neuroprotective in an animal model of ischemia. The aim of the experiments described in this thesis was to identify the active metabolite of PLZ, and to determine the neurochemical mechanisms by which PLZ and this metabolite exert their neuroprotective effects (with a particular focus on degenerative mechanisms observed in cerebral ischemia and Alzheimers disease (AD)). The development of an analytical assay for -phenylethylidenehydrazine (PEH) was a major breakthrough in this project and permitted the positive identification of this compound as the active metabolite of PLZ. Further experiments demonstrated that PLZ and PEH could be neuroprotective in cerebral ischemia and AD not only by reducing excitotoxicity via increased GABAergic transmission, but also by (a) increasing brain ornithine, which could potentially lead to a decrease in glutamate synthesis and/or a decrease in polyamines (whose metabolism produces toxic aldehydes); (b) inhibiting the activity of human semicarbazide-sensitive amine oxidase (SSAO), an enzyme whose activity is increased in AD producing excessive amounts of the toxic aldehyde formaldehyde (FA); (c) by sequestering FA in vitro, forming a non-reactive hydrazone product. Since PEH appears to mediate or share the neurochemical effects of PLZ, two propargylated analogs of PEH were synthesized and tested for their potential as PEH prodrugs. Surprisingly these analogs were not particularly effective prodrugs in vivo, but they possessed an interesting neurochemical properties on their own (the ability to elevate brain levels of glycine), and warrant further investigation as potential antipsychotic agents. Together, these results suggest that PLZ and its active metabolite, PEH, should be further investigated for their neuroprotective potential in cerebral ischemia and in AD. / Neurochemistry

Selenium is an element that can modulate the dopaminergic neurotransmission. Studies show that diphenyl diselenide, an organic compound of selenium, has antioxidant activity improves depressive-like behavior and reduce the activity of the enzyme monoamine oxidase (MAO). However, there are few studies concerning about possible alterations of diphenyl diselenide in dopaminergic system. Thus, the purpose of the present study was to evaluate the effects of acute and sub-chronic treatment of diphenyl diselenide on amphetamine-induced behavioral and biochemical alterations in mice. In the acute treatment, the mice were treated with diphenyl diselenide (5 and 10 mg/kg, s.c.) or vehicle (10% Tween 80, s.c.) 30 min before administration of amphetamine (1.25 mg/kg, i.p.). After 25 min, locomotor activity was assessed with an open field and, also, the time of stereotypy and immobility was assessed in a glass cage. Sub-chronic treatment was conducted with seven administrations of diphenyl diselenide (5 and 10 mg/kg, s.c.), or its vehicle being one administration per day. On the eighth day, amphetamine (1.25 mg/kg, i.p.) was administered and the behavioral tests were conducted after 25 min. In both treatments ex vivo tests were performed: isoform activity MAO-A and MAO-B, and measurement of total protein and non-protein thiol levels, oxidation of diclorofluorescein. Amphetamine increased the number of crossing and rearing in the open field test and diphenyl diselenide prevented only the increase in the number of crossings when acutely administered to mice. Furthermore, amphetamine increased the time of immobility and stereotypy in mice. Diphenyl diselenide did not prevent these effects. By contrary, at 10 mg/kg, sub-chronic administration of diphenyl diselenide increased per se the time of immobility and stereotypy. It was also found a positive correlation between immobility and stereotypy in acute and sub-chronic treatment with diphenyl diselenide. It was also detected a decrease in brain MAO-B activity caused by sub-chronic treatment with diphenyl diselenide either alone or in combination of amphetamine. Any change was detected in oxidative stress parameters. In conclusion, sub-chronic administration of diphenyl diselenide can promote a behavioral sensitization that seems to be, at least in part, dependent of MAO-B inhibition. / O selênio é um elemento químico capaz importante para o funcionamento celular, envolvido também na modulação do sistema dopaminérgico. Estudos mostram que o disseleneto de difenila, um composto orgânico de selênio, possui atividade antioxidante, melhora o comportamento tipo-depressivo relacionado à inibição da atividade da enzima monoamino oxidase (MAO). No entanto, existem poucos estudos acerca dos efeitos do disseleneto de difenila sobre o sistema dopaminérgico. Desta forma, o objetivo do presente estudo foi avaliar os efeitos do tratamento agudo e sub-crônico do disseleneto de difenila sobre as alterações bioquímicas e comportamentais induzidas por anfetamina em camundongos. No tratamento agudo, os camundongos foram tratados com disseleneto de difenila (5 e 10 mg/kg, s.c.) ou veículo (10% de tween 80, s.c.) 30 minutos antes da administração de anfetamina (1,25 mg/kg, i.p.). Após 25 minutos, foram avaliados a atividade locomotora através do teste de campo aberto, além do tempo de estereotipia e imobilidade, avaliado em caixa espelhada. O tratamento subcrônico foi realizado com sete administrações de disseleneto de difenila ou veículo (5 e 10 mg/kg, s.c.) sendo uma administração por dia. No oitavo dia foi administrada a anfetamina (1,25 mg/kg, i.p.) e realizados os testes comportamentais 25 minutos após. Em ambos os tratamentos os testes ex-vivo realizados foram: atividade das isoformas MAO-A e MAO-B, níveis de tióis totais e não-protéico, oxidação da diclorofluoresceína. O tratamento com anfetamina aumentou o número de cruzamentos e de levantadas no teste do campo aberto e o disseleneto de difenila preveniu somente o número de cruzamentos quando administrado agudamente aos camundongos. Além disso, o tratamento com anfetamina aumentou o tempo de imobilidade e estereotipia em camundongos. O disseleneto de difenila não preveniu estes efeitos. Pelo contrário, na dose de 10 mg/kg, a administração subcrônica de disseleneto de difenila aumentou per se o tempo de imobilidade e de estereotipia. Uma correlação positiva entre o tempo de estereotipia e de imobilidade foi também encontrada tanto para o tratamento agudo como subcrônico com disseleneto de difenila. Também foi detectada uma diminuição na atividade cerebral da MAO-B causada pelo tratamento subcrônico com disseleneto de difenila tanto per se quanto em combinação com a anfetamina. Não foram encontradas alterações em parâmetros de estresse oxidativo. Em conclusão, o tratamento subcrônico com disseleneto de difenila pode promover uma sensibilização comportamental que parece ser, pelo menos em parte, dependente da inibição da MAO-B.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Evidências têm mostrado que as espécies reativas de oxigênio (ROS) geradas pela NAD(P)H oxidase são importantes moduladores de diversas funções celulares como migração, crescimento, proliferação e sobrevivência. Estudos recentes demonstraram o envolvimento da atividade da NAD(P)H oxidase no crescimento e sobrevivência de células de melanoma. Neste trabalho, investigamos o efeito da inibição da NAD(P)H oxidase por difenileneiodônio (DPI) sobre o crescimento das células de melanoma humano MV3 e observamos que este composto reduziu o crescimento destas células em aproximadamente 50%. A inibição da NAD(P)H oxidase induziu mudanças no formato celular, com arredondamento, diminuição do espraiamento e descolamento celular. Esta redução foi acompanhada por um rearranjo do citoesqueleto de actina, diminuição da fosforilação no resíduo Tyr397 da quinase de adesão focal (FAK) e redução na associação de FAK com actina e com a tirosina quinase c-Src. Isto indica que a inibição da geração de ROS está modulando negativamente vias de sinalização ativadas por integrinas, o que freqüentemente conduz a um tipo particular de morte celular conhecida por anoikis. Comprovando a ocorrência deste fenômeno, observamos que a inibição da atividade da NAD(P)H oxidase aumentou a apoptose das células de melanoma e induziu a ativação da caspase-3. Nossos resultados mostram ainda que a inibição da viabilidade celular por DPI foi revertida com o pré-tratamento das células MV3 com um inibidor de tirosina fosfatases (ortovanadato de sódio). Em resumo, este estudo mostra que a geração de ROS por NAD(P)H oxidase está envolvida nos mecanismos de sobrevivência em células de melanoma, uma vez que afetam as vias de sinalização dependentes de FAK-Src, através da inibição da atividade de proteína tirosina fosfatases. / NAD(P)H oxidase-derived reactive oxygen species (ROS) have emerged as critical mediators of several cell functions as diverse as migration, growth, proliferation and survival. Recent evidence has show that NAD(P)H oxidase activity is essential to melanoma proliferation and survival. We reported that NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI) inhibited melanoma growth. NAD(P)H oxidase inhibition induced changes in cell shape with cell spreading decrease, rounding up and detachment. These phenomena were accompanied by rearrangement of actin network and a decrease in both focal adhesion (FAK) phosphorylation in Tyr397 residue and in FAK association to actin and c-Src, indicating that inhibition of ROS generation would down- modulate integrin-mediated signaling, what often results in a particular type of apoptotic cell death, known as anoikis. We observed that NAD(P)H oxidase inhibitor induced apoptosis in melanoma cells with activation of caspase-3. We results show that the effects promoted by NAD(P)H oxidase inhibition on melanoma growth were completely abolished by the pre-treatment of MV3 cells with the protein tyrosine phosphatases inhibitor sodium orthovanadate. In conclusion, our results strongly suggest that ROS generated by NAD(P)H oxidase complex transmit cell survival signals in melanoma cells through the FAK-Src pathway, probably inhibiting protein tyrosine phosphatases.

La biomasse végétale est une alternative durable et écologique pour les ressources fossiles. L'exploitation et la valorisation de cette biomasse sont rendues possibles grâce à la capacité naturelle des enzymes fongiques à dégrader et modifier cette biomasse. Parmi ces enzymes, les glyoxal oxydases génératrices de H2O2 (GLOX) restent un groupe peu étudié avec un seul exemple de protéine caractérisée dans la littérature à partir d’un champignon dégradant le bois.Dans cette thèse, trois GLOX, précédemment identifiées dans le génome du champignon dégradant le bois Pycnoporus cinnabarinus (PciGLOX), ont été sélectionnées, produites par voie hétérologue et caractérisées. La caractérisation a révélé des différences entre les trois PciGLOX dans la stabilité des protéines, la spécificité du substrat et l’efficacité catalytique. Les protéines PciGLOX sont produites sous leur forme inactive et leur mécanisme d'activation a été étudié. La capacité des GLOX à catalyser la réaction d'oxydation du 5-hydroxyméthylfurfural (HMF), d’intérêt industriel, a été étudiée pour la première fois dans ce travail. Le HMF a été oxydé par PciGLOX en acide 5-hydroxyméthyl-2-furancarboxylique (HMFCA) comme produit principal. Le HMFCA est difficile à produire par catalyse chimique et est utilisé dans la production de polyesters et de produits pharmaceutiques. PciGLOX ont également été capables de produire l’acide furandicarboxylique (FDCA), qui est un précurseur dans les procédés de production du bioplastique. Ce travail ouvre de nouvelles perspectives pour étudier plus en détail le rôle de GLOX dans la dégradation de la lignocellulose, et dans les applications biotechnologiques. / Plant biomass is a sustainable and eco-friendly alternative for fossil fuels. The exploitation and valorisation of plant biomass is possible through biotechnological processes that rely on the natural ability of fungal enzymes to degrade and modify this biomass. Among these enzymes are H2O2-generating glyoxal oxidases (GLOX), which haven’t been extensively studied with only one example in the literature on GLOX from wood-degrading fungi. In this thesis three GLOX, previously identified in the genome of the wood-degrading fungus Pycnoporus cinnabarinus (PciGLOX), were heterologously produced and characterisation. The three PciGLOX showed differences in their stability, substrate preferences and catalytic properties. The ability of GLOX to catalyse the biotechnologically important oxidation reaction of 5-hydroxymethylfurfural (HMF) was investigated for the first time in this work. PciGLOX oxidized HMF to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), which is difficult to produce via chemical catalysis and is used in polyesters and pharmaceutical products production. PciGLOX were also able to oxidize HMF derivatives leading to the formation of the final product furandicarboxylic acid (FDCA), which is a bioplastic precursor. PciGlOX proteins are produced in their inactive form and their activation mechanism was investigated in this thesis. This work opens new prospects to investigate more the role of GLOX in plant biomass degradation and biotechnology, and the possible optimization techniques of the catalytic properties of this enzyme.

As biocélulas a combustível enzimáticas (BFCs) são dispositivos eletroquímicos que convertem energia química em energia elétrica, utilizando enzimas como biocatalisadores. Quando miniaturizada, uma BFC pode ser implantada em animais vertebrados e invertebrados, vislumbrando-se sua utilização na produção de energia elétrica para alimentar microdispositivos biomédicos e microssensores em pequenos insetos. No entanto, ainda é um desafio obter BFCs implantáveis e miniaturizadas, com uma potência suficiente (dezenas de microwatts) para alimentar microcircuitos eletrônicos de maneira estável e em longo prazo. Diante do exposto, esta tese de doutorado apresenta um estudo das propriedades eletroquímicas de eletrodos enzimáticos, visando a aplicação em BFCs de glicose/O2 miniaturizadas e implantáveis. Para isso, utilizaram-se fibras flexíveis de carbono (FCFs) modificadas com as enzimas bilirrubina oxidase (BOx) no cátodo e glicose desidrogenase (GDh) NAD-dependente no ânodo, a fim de se obter a redução de O2 e a oxidação de glicose, respectivamente. Os resultados obtidos mostram que FCFs previamente submetidas a um tratamento químico de oxidação com permanganato de potássio e com posterior eletrodepolimerização do mediador vermelho neutro produzem bioânodos estáveis e robustos. Estes eletrodos, combinados com biocátodos compostos por FCFs na ausência de mediadores redox, foram utilizados em BFCs miniaturizadas, que foram implantadas em formigas da espécie Atta sexdens rubrupilosa. A potência máxima da BFC operando in vivo foi 13,5 ± 3,8 µW cm-2 em 190 ± 58,9 mV, com corrente máxima de 143 ± 40,2 µA cm-2 e a voltagem de circuito aberto de 260 ± 99,6 mV. Acredita-se que estes valores ainda possam ser otimizados e este trabalho contribui para mostrar que a flexibilidade das FFC, a presença de um mediador de elétrons polimérico no ânodo, o uso do tratamento químico de oxidação com permanganato de potássio das fibras e a miniaturização dos eletrodos são elementos importantes, e que podem ser considerados no desenvolvimento de biocélulas a combustível implantáveis. / Enzymatic biofuel cells (BFCs) are electrochemical devices that convert chemical energy into electrical energy using enzymes as biocatalysts. When miniaturized, BFCs can be implanted in vertebrate and invertebrate animals and, their use to produce electrical energy to feed biomedical microdevices and micro-sensors in small insects can be observed. However, it is still challenging to obtain implantable and miniaturized BFCs, with sufficient power (tens of microwatts) to power electronic microcircuits in a stable and long-term manner. In view of the above, this PhD thesis presents a study of the electrochemical properties of enzymatic electrodes, aiming to use them in miniaturized and implantable glucose/O2 BFCs. In order to obtain a reduction in O2 and oxidation of glucose, flexible carbon fibers (FCFs) modified with bilirubin oxidase (BOx) enzymes in the cathode and glucose dehydrogenase (GDh) at the anode, respectively, were used. The results show that FCFs previously submitted to a chemical treatment of oxidation with potassium permanganate and, subsequently, electropolymerization of the neutral red mediator produce stable and robust bioanodes. These electrodes, combined with biocathodes consisting of FCFs in the absence of redox mediators, were used in miniaturized BFCs, which were implanted in Atta sexdens rubrupilosa ant species. The BFC maximum power source, operating in vivo, was 13.5 ± 3.8 μW cm-2 at 190 ± 58.9 mV, with a maximum current of 143 ± 40.2 μA cm-2 and the open circuit voltage was 260 ± 99.6 mV. Although these values can be optimized, this research shows that the flexibility of the FCF, the presence of a polymer electron mediator on the anode, using the chemical treatment of oxidation with potassium permanganate of the fibers and electrode miniaturization are important elements, which can be considered in the development of implantable biofuels.

A biomassa vegetal é a maior fonte de carbono renovável disponível, e é composta de celulose, hemicelulose e lignina. A lignina, um heteropolímero polifenólico complexo, é uma barreira a utilização mais eficiente da biomassa. Fungos desenvolveram formas de superar o obstáculo imposto pela lignina, através da atividade de enzimas como peroxidases, e da ação de pequenas moléculas reativas. As duas formas dependem da presença de peróxido de hidrogênio, que é produzido pela atividade de enzimas como aril-álcool oxidases (AAOs). AAOs são enzimas pertencentes a superfamília GMC de oxidoredutases e, portanto, possuem FAD como grupo prostético. Realizam a conversão de álcoois aromáticos à aldeídos, com concomitante produção de peróxido de hidrogênio. Devido à capacidade de oxidação de substratos variados, e dependente somente de oxigênio molecular, AAOs têm potencial aplicação na indústria biotecnológica, tanto como produtoras de peróxido de hidrogênio quanto na produção de químicos-plataforma a partir da biomassa. No entanto, AAOs ainda têm poucos exemplares caracterizados, e a maior parte dos estudos publicados é referente a enzimas do gênero de fungos basidiomicetos Pleurotus sp.. A enzima MtAAOx do fungo ascomiceto termofílico M. thermophila foi produzida de forma heteróloga em A. nidulans, e algumas de suas propriedades bioquímicas e estruturais foram determinadas. MtAAOx é uma enzima extracelular monomérica glicosilada, com grupo FAD dissociável, e capaz de oxidar álcoois aromáticos fenólicos e não fenólicos, incluindo um monômero da lignina, coniferil álcool, substrato para o qual a enzima teve mais afinidade. MtAAOx, além de oxidar álcoois aromáticos, também foi capaz de oxidar um substrato heterocíclico, 5-HMF, e de utilizar outro aceptor de elétrons além do oxigênio. Esses resultados, aliados a baixa similaridade entre sequencias de aminoácidos indicam que MtAAOx tem sítio catalítico e canal de acesso do substrato distintos das AAOs até o momento caracterizadas. A enzima MtAAOx deglicosilada teve estabilidade térmica reduzida em comparação com a enzima nativa. A atividade enzimática foi afetada positivamente com a presença de alguns cátions, entre eles Ca2+. Além de afetar a atividade, Ca2+proporcionou ganho de estabilidade térmica, com aumento da temperatura de melting em 5 ºC. Esta enzima é uma nova aril-álcool oxidase caracterizada, a primeira de um fungo ascomiceto, e acredita-se que tenha papel na degradação da lignina da biomassa vegetal. / Plant biomass is the largest source of available renewable carbon, and is composed of cellulose, hemicellulose and lignin. Lignin, a complex polyphenolic heteropolymer, is a barrier to a more efficient use of biomass. Fungi have developed ways to overcome the obstacle imposed by lignin through the activity of enzymes such as peroxidases and the action of small reactive molecules. The two forms depend on the presence of hydrogen peroxide, which is produced by the activity of enzymes such as aryl-alcohol oxidases (AAOs). AAOs are enzymes belonging to the GMC superfamily of oxidoreductases and therefore have FAD as the prosthetic group. They perform the conversion of aromatic alcohols to aldehydes, with simultaneous production of hydrogen peroxide. Because of their capacity of oxidizing various substrates, and dependence only on molecular oxygen, AAOs have potential applications in the biotech industry, both as hydrogen peroxide producers and in the production of platform chemicals derived from biomass. However, characterized AAOs are few, most of the published studies being on enzymes of basidiomycete fungi from genus Pleurotus sp.. The MtAAOx enzyme of the thermophilic ascomycete M. thermophila was produced heterologously in A. nidulans, and some of its biochemical and structural properties were determined. MtAAOx is a glycosylated, monomeric, extracellular enzyme with a dissociable FAD group, capable of oxidizing phenolic and non-phenolic aromatic alcohols, including a lignin monomer, coniferyl alcohol, the substrate for which the enzyme has the highest affinity. MtAAOx, besides oxidizing aromatic alcohols, was also able to oxidize a heterocyclic substrate, 5-HMF, and to use another electron acceptor in addition to oxygen. These results, combined with the low similarity between amino acid sequences, indicate that MtAAOx has a catalytic site and substrate access channel distinct from characterized AAOs\'. The deglycosylated MtAAOx enzyme had reduced thermal stability compared to the native enzyme. The enzymatic activity was positively affected by the presence of some cations, including Ca2+. In addition to affecting the activity, Ca2+ improved thermal stability, with 5 °C increase of the melting temperature. This enzyme is a novel aryl-alcohol oxidase characterized, the first of an ascomycete fungus, and is believed to play a role in the lignin degradation of plant biomass.