Abstract Mammalian peroxisomes contain two parallel multifunctional enzymes (MFE), MFE type 1 and MFE type 2 (MFE-2), which are responsible for the degradation of fatty acids. They both catalyze the second and third reactions of the β-oxidation pathway, but through reciprocal stereochemical courses. MFE-2 possesses (2E)-enoyl-CoA hydratase-2 and (3R)-hydroxyacyl-CoA dehydrogenase activities. In addition, the carboxy-terminal part is similar to the sterol carrier protein type 2 (SCP-2). The purpose of this work was to study the structure-function relationship of functional domains of mammalian MFE-2 by recombinant DNA technology, enzyme kinetics and X-ray crystallography. The work started with the identification of conserved regions in MFE-2. This information was utilized when dehydrogenase, hydratase-2 and/or SCP-2-like domain were produced as separate recombinant proteins. Subsequently, both dehydrogenase and SCP-2-like domains were crystallized and their crystal structures were solved. The structure of the dehydrogenase region of rat MFE-2 contains the basic α/β short-chain alcohol dehydrogenase/reductase (SDR) fold and the four-helix bundle at the dimer interface, which is typical of dimeric SDR enzymes. However, the structure has a novel carboxy-terminal domain not seen among the known structures. This domain lines the active site cavity of the neighbouring monomer, reflecting cooperative behaviour within a homodimer. The monomeric SCP-2-like domain of human MFE-2 has the same fold as rabbit SCP-2. The structure includes a hydrophobic tunnel occupied by an ordered Triton X-100 molecule, demonstrating the ligand-binding site. Compared to the unliganded rabbit SCP-2 structure, the position of the carboxy-terminal helix is different. The movement of this helix in the liganded human SCP-2-like domain resulted in the exposure of a peroxisomal targeting signal, suggesting ligand-assisted protein import into peroxisomes. The roles of conserved protic residues in the hydratase-2 region of human MFE-2 were studied by mutating them to alanine. In the first step, the ability of mutated variants to utilize oleic acid in vivo was tested with Saccharomyces cerevisiae fox-2 cells (devoid of endogenous MFE-2). Subsequently, in vitro characterization of the mutant enzymes revealed two amino acid residues, Glu366 and Asp510, vital for hydratase-2 activity. The results indicate that the acid-base catalysis is valid for hydratase-2.

Student Number : 0009756Y - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / A versatile nitrile-degrading bacterium was isolated through enrichment culturing of soil samples from Johannesburg, South Africa. It was identified as Rhodococcus rhodochrous and submitted to the ATCC culture collection as strain BAA-870. This organism was determined to be a potential biocatalyst in that it contains a two enzyme system with strong nitrile-converting activity comprising nitrile hydratase and amidase. The development of a suitable assay for measuring the activity of the enzymes of interest was explored. A pHsensitive indicator-based assay was found to be suitable only for colorimetrically identifying highly concentrated enzymes with acid-forming activity. An ophthaldialdehyde- based fluorimetric assay was found to be applicable to conversions of select compounds, but the assay could not be used to measure the activity of Rhodoccocus rhodochrous ATCC BAA-870. High performance liquid chromatography was the most suitable method for reliable and quantitative measurement of nitrile hydrolysis, and is applicable to monitoring activities of whole-cell and cell-free extracts. Initial analysis of six compounds, benzonitrile, benzamide, benzoic acid, hydrocinnamonitrile, 3-hydroxy-3- phenylpropionitrile and 3-hydroxy-3-phenylpropionic acid, was performed by HPLC to measure linearly the average retention area, amount and absorbance of the compounds up to 10 mM concentrations. The conversion of the substrates benzonitrile, benzamide and 3- hydroxy-3-phenylpropionitrile were further analysed with respect to time and enzyme concentration. Conversion of benzonitrile to benzamide by the nitrile hydratase was rapid and could be measured in 10 minutes. Conversion of benzamide to benzoic acid by the amidase was considered the rate-limiting step and could be followed for 90 minutes of the reaction at the concentrations tested. Conversion of 3-hydroxy-3-phenylpropionitrile was linearly measured over 20 minutes. Mass spectral analysis was used to confirm, at a structural level, relatively less volatile reactant compounds with a higher thermal stability, including benzamide, 3-hydroxy-3-phenylpropionitrile and 3-hydroxy-3-phenylpropionic acid. Protein concentration studies indicated that activity against benzonitrile was probably due to a nitrile hydratase with potent activity rather than a concentrated enzyme, since formation of benzamide from benzonitrile showed first order reaction kinetics at protein concentrations less than 0.2 mg/ml. Formation of benzoic acid from benzamide was linear up to 1.3 mg total protein and product formation from 3-hydroxy-3-phenylpropionitrile was linear up to 1.4 mg total protein. Overlapping activities against benzonitrile and 3- hydroxy-3-phenylpropionitrile indicate that the nitrile hydratase has differing substrate specificity for the two compounds, with higher activity toward the small aromatic mononitrile, benzonitrile, than the arylaliphatic b-hydroxy nitrile, 3-hydroxy-3- phenylpropionitrile. The nitrile-converting activity of Rhodococcus rhodochrous ATCC BAA-870 would be suitable for biocatalysis as the conversions take place under a wide pH range, require low concentrations of enzyme and reactions are fast. Separation of nitrileconverting activities in Rhodococcus rhodochrous ATCC BAA-870 was undertaken using various chromatography methods to establish a simple, one-step protocol for biocatalytic enzyme preparations. HPLC was not suited to assaying nitrile-converting activity in chromatofocusing fractions, and chromatofocusing Ampholyte buffers were found to interfere with activity measurements. Gel exclusion chromatography of the soluble protein extract from Rhodococcus rhodochrous ATCC BAA-870 indicated the enzyme/s responsible for nitrile hydratase activity are high molecular weight proteins ranging from 40 to 700 kDa in size, while the amidase native enzyme is proposed to be roughly 17 to 25 kDa. SDS-PAGE analysis of gel exclusion and ion exchange chromatography fractions indicated nitrile converting activity in Rhodococcus rhodochrous ATCC BAA-870 is likely due to multimer-forming enzymes made up of 84, 56, 48 and 21 kDa subunits. It is postulated that nitrile hydratase is made up of ab and a2b2 tetramers that may form larger enzyme aggregates. Ion exchange chromatography was used to separate nitrile hydratase with high activity against benzonitrile and 3-hydroxy-3-phenylpropionitrile from amidase activity, and showed that an additional, substrate specific nitrile hydratase may exist in the organism.

Abstract The hydratase/isomerase superfamily consists of enzymes having a common evolutionary origin but acting in a wide variety of metabolic pathways. Many of the superfamily members take part in β-oxidation, one of the processes of fatty acid degradation. One of these β-oxidation enzymes is the Δ3-Δ 2-enoyl-CoA isomerase, which is required for the metabolism of unsaturated fatty acids. It catalyzes the shift of a double bond from the position C3 of the substrate to the C2 position. In this study, the Δ 3-Δ 2-enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae was identified, overexpressed as a recombinant protein and characterized. Subsequently, its structure and function were studied by X-ray crystallography. The yeast Δ 3-Δ 2-enoyl-CoA isomerase polypeptide contains 280 amino acid residues, which corresponds to a subunit size of 32 kDa. Six enoyl-CoA isomerase subunits assemble to form a homohexamer. According to structural studies, the hexameric assembly can be described as a dimer of trimers. The yeast Δ 3-Δ 2-enoyl-CoA isomerase is located in peroxisomes, the site of fungal β-oxidation, and is a necessary prerequisite for the β-oxidation of unsaturated fatty acids; the enoyl-CoA isomerase knock-out was unable to grow on such carbon sources. In the crystal structure of the yeast Δ 3-Δ 2-enoyl-CoA isomerase, two domains can be recognized, the N-terminal spiral core domain for catalysis and the C-terminal α-helical trimerization domain. This overall fold resembles the other known structures in the hydratase/isomerase superfamily. Site-directed mutagenesis suggested that Glu158 could be involved in the enzymatic reaction. Structural studies confirmed this, as Glu158 is optimally positioned at the active site for interaction with the substrate molecule. The oxyanion hole stabilizing the transition state of the enzymatic reaction is formed by the main chain NH groups of Ala70 and Leu126. The yeast Δ 3-Δ 2-enoyl-CoA isomerase hexamer forms by dimerization of two trimers, as in the other superfamily members. An extensive comparison of the five known structures of this family showed that the mode of assembly into hexamers is not a conserved feature of this superfamily, since the distance between the trimers and the orientation of the trimers with respect to each other varied. Marked differences were also detected between the two yeast enoyl-CoA isomerase crystal forms used in this study, one being crystallized at low pH and the other at neutral pH. The results suggest that the yeast Δ 3-Δ 2-enoyl-CoA isomerase could occur as a trimer at low pH.

Studies Directed to the Optimization of Fermentation of Rhodococcus sp. DAP 96253 and Rhodococcus rhodochrous DAP 96622 by GENE KIRK DRAGO Under the Direction of George E. Pierce ABSTRACT Bench- and pilot plant scale fed-batch fermentations were performed in stirred-tank bioreactors (STBR) with Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 in an attempt to elucidate parameters that may affect the optimization of a fermentation process for high biomass production and high inducible expression of cobalt-high-molecular-mass nitrile hydratase (Co-H-NHase. The effects of these factors on amidase (AMDase) activity were also investigated. Biomass and NHase production were inhibited by a total addition of acetonitrile and acrylonitrile (AC / AN) at 500 ppm during a 48 h run. Biomass and enzyme activity were uncoupled when the inoculum mass was increased from 4 g (wet weight) to ¡Ý 19 g. Other factors that allowed for the uncoupling of biomass production from enzyme activity were the reduction of the AC / AN feed rate from a step-addition at 2500 ¦Ìl / min to a continuous addition at 80 ¨C 120 ¦Ìl / min, and the delay to 18 h post-inoculation the time of initial inducer addition. The inhibition of both biomass production and NHase activity was relieved when both the total concentration of AC / AN was reduced to ¡Ü 350 ppm and the AC / AN feedrate was reduced. The factors with the greatest influence were shown to be the inducer, the inducer concentration, inoculum mass and source as well as the major carbohydrate and nitrogen source. In addition, this lab is the first to report high AN-specific NHase induction by asparagine (1300 ppm) in a fed-batch fermentation system. Prior to this program, 250 mg of cells (wet weight) per liter could be provided in 4 ¨C 10 days with an activity of 1 U NHase per mg of cells (dry weight). Current production is > 50 g / L in 48 h with an NHase activity > 150 U / mg of dry cell weight. INDEX WORDS: Amidase, Asparagine, Biodetoxification, Fermentation, Nitrile, Nitrile Hydratase, Rhodococcus

Treatment of industrial wastewaters contaminated with toxic and hazardous organics can be a costly process. In the case of acrylonitrile production, due to highly volatile and toxic nature of the contaminant organics, production wastewaters are currently disposed by deepwell injection without treatment. Under the terms granting deepwell injection of the waste, alternative treatments must be investigated, and an effective treatment identified. Cells of two Gram-positive bacteria, Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 were evaluated for their potential as biocatalysts for detoxification of acrylonitrile production wastewaters. Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 when multiply induced, are capable of utilizing the hazardous nitrile and amide components present in the wastewater as sole carbon and/or nitrogen sources, employing a 2-step enzymatic system involving nitrile hydratase (NHase) and amidase enzymes. There is a significant potential for overproduction of NHase upon multiple induction. However, high-level multiple induction required the presence of highly toxic nitriles and/or amides in the growth medium. Asparagine and glutamine were identified as potent inducers with overexpression at 40% of total soluble cellular protein as NHase. In native form (either cell free enzymes or whole cells) the desired NHase is very labile. In order to develop a practical catalyst to detoxify acrylonitrile production wastewaters, it is necessary to significantly improve and enhance the stability of NHase. Stabilization of desired NHase activity was achieved over a broad range of thermal and pH conditions using simultaneous immobilization and chemical stabilization. Previously where 100% of NHase activity was lost in 24 hours in the non-stabilized cells, retention of 20% of initial activity was retained over 260 days when maintained at 50-55 C, and for over 570 days for selected catalyst formulations maintained at proposed temperature of the biodetoxification process. In addition, NHase and amidase enzymes from Rhodococcus sp. DAP 96253 were purified. Cell free NHase was characterized for its substrate range and effect of common enzyme inhibitors and was compared to available information for NHase from other organisms. As a result of this research a practical alternative to the deepwell injection of acrylonitrile production wastewaters is closer to reality.

Hypoxia inducible factor-1α (HIF-1α) plays a critical role in the oxygen homeostasis of all metazoans. HIF-1α is a master transcriptional regulator which coordinates the adaptive response to low oxygen tension. Through activation of a plethora of downstream target genes, HIF-1α facilitates oxygenation by promoting angiogenesis and blood vessel dilation, in addition to modulating metabolic pathways to inhibit oxidative phosphorylation and promote glycolytic energy production. Given the critical roles of hypoxia, insufficient blood supply and perturbed energetics in the pathogenesis of cardiovascular disorders, notably ischaemic heart disease, therapeutic modulation of HIF-1α is of significant clinical interest. Previous studies have demonstrated an acute cardioprotective role for both endogenous and supraphysiological HIF-1α signalling in the context of myocardial ischaemia. In contrast, chronic supraphysiological HIF-1α activation in the unstressed heart has been shown to induce cardiac dysfunction. To address the effect of chronic endogenous HIF-1α activation post-myocardial infarction (MI), the present work employed a murine coronary artery ligation (CAL) model in conjunction with temporally-inducible, cardiac-specific deletion of Hif-1α. While CAL surgery successfully modelled myocardial infarction – eliciting substantial adverse cardiac remodelling and contractile dysfunction – there was no evidence of chronic HIF-1α activation by CAL in HIF knockout or control left ventricular samples. In keeping with this, chronic ablation of Hif-1α (from 2 weeks post-CAL) had no discernible additional effect upon cardiac function. Overall, these findings do not support a potential therapeutic role for inhibition of HIF-1α signalling in the chronic phase post-MI. The fundamental tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) converts fumarate to malate. FH deficiency is associated with smooth muscle and kidney tumours which exhibit normoxic HIF signalling due to fumarate accumulation. To investigate the potential for fumarate accumulation to elicit protective HIF signalling, a cardiac-specific Fh1 null mouse was developed through Cre-loxP recombination. Strikingly, despite interruption of the TCA cycle in a highly metabolically demanding organ, cardiac Fh1 null mice were viable, fertile and survived into adulthood, demonstrating the remarkable metabolic plasticity of the heart. However, by 3-4 months Fh1 null mice develop a lethal cardiomyopathy characterised by cardiac hypertrophy, ventricular dilatation and contractile dysfunction. Despite lack of a pseudohypoxic response, Fh1 null hearts did exhibit another phenomenon observed in FH-deficient cancers and also attributed to fumarate accumulation – activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) antioxidant pathway. Heterozygous, but not homozygous, somatic deletion of Nrf2 extended the life expectancy of cardiac Fh1 null mice. Exploration of redox status revealed a more reductive environment in Fh1 null hearts than controls. As a corollary, inhibition of the rate limiting enzyme of the pentose phosphate pathway – a major source of cellular reducing equivalents – with dehydroepiandrosterone conferred striking amelioration of the Fh1 null cardiomyopathy, suggesting a possible pathogenic role for reductive stress. While loss of mitochondrial Fh1 activity and subsequent TCA cycle dysfunction likely contribute to the Fh1 null phenotype, the importance of cytosolic FH was unclear. To clarify this, FH was expressed specifically in the cytosol in vivo. This was sufficient to substantially rescue the Fh1 null cardiomyopathy, supporting a role for cytosolic FH disruption in its pathogenesis. Taken together, these findings highlight the potential for reductive stress to contribute to cardiac dysfunction and suggest a function for cytosolic FH in cardiac metabolic homeostasis.

Nitrilases are well known for their unique property to effectively convert nitriles into corresponding carboxylic acids and ammonia. They can also form amides as by-products. In contrast to nitrile hydratases they do not require cofactors or prosthetic groups. The research in this work is focused on nitrilase from filamentous fungus Arthroderma benhamiae and cyanide hydratase from Aspergillus niger K10. Genes of these enzymes were expressed using pET-30a(+) plasmid in the bacterium Escherichia coli strain BL21-Gold (DE3). The products obtained were purified by a series of ion exchange chromatography and gel filtration and subsequently characterized with respect to oligomeric state of the protein and its usability for protein crystallography. To obtain information regarding the structural arrangement of the individual proteins, electrophoretic separation in polyacrylamide gel, gel filtration, analytical ultracentrifugation, mass spectrometry, dynamic light scattering and drop coating deposition Raman spectroscopy were used. Keywords: nitrilase, cyanide hydratase, Aspergillus niger, Arthroderma benhamiae, liquid chromatography (In Czech)

Fumarato hidratases ou fumarases (FH) catalisam a reação estereoespecífica reversível de hidratação do fumarato em L-malato. Essas enzimas se apresentam em todas as classes de organismos, desde procariotos a eucariotos, e podem ser encontradas nas formas mitocondrial e citosólica. A enzima tem papel importante na produção de energia pois participa do ciclo do ácido cítrico, na resposta ao dano do DNA e como supressor tumoral. A fumarase humana (HsFH), que pertence à classe II, é codificada pelo gene 1q42.1, possui 467 aminoácidos em cada monômero com peso molecular de 50,2 kDa cada. Estudos associaram mutações no gene da FH com diversas doenças humanas como acidúria fumárica, leiomiomatoses de útero e pele (MCUL), que quando associadas com um agressivo carcinoma múltiplo de células é conhecido como leiomiomatose hereditária e câncer renal (HLRCC). Apesar da grande importância da fumarase humana no metabolismo energético, ainda há pouca informação em relação ao mecanismo catalítico adotado pela enzima e o efeito estrutural e cinético causado pelas mutações envolvidas com essas doenças. Diante disso, nosso trabalho utilizou uma abordagem híbrida que envolve a caracterização biofísica, bioquímica e estrutural da enzima HsFH, e seus mutantes: N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, G282VHsFH, E362QHsFH, S365GHsFH e N373DHsFH, identificados em pacientes. Estudos cinéticos foram realizados em sete diferentes pHs e, pela primeira vez para fumarases, o ensaio foi realizado com os dois substratos presentes na mesma mistura reacional, confirmando a contribuição da reação reversa para a velocidade global da enzima. De acordo com os estudos de termoflúor a proteína é estabilizada em pHs alcalinos e através da ligação de compostos no sítio ativo. A estrutura da enzima HsFH nativa foi resolvida a 1,8 Å e identificou a presença de moléculas de HEPES complexadas na região C-terminal da enzima. Os estudos cinéticos demonstraram um aumento da eficiência catalítica na presença do HEPES, sugerindo um possível papel alostérico de seu sítio de ligação para a atividade catalítica. Foram determinadas as estruturas para os mutantes N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, E362QHsFH, S365GHsFH e N373DHsFH. As mutações Q185R, E362Q, S365G e N373D foram identificadas no sítio ativo afetando diretamente a capacidade da proteína em ligar os substratos, enquanto que a mutação H180R foi localizada no sítio B, que conduz os substratos e produtos para dentro e fora do sítio ativo. Já a mutação K230R está localizada no domínio central, mas os resultados de termoflúor demonstram um efeito direto na capacidade da enzima em acomodar o substrato. A mutação N107T, localizada longe do sítio ativo foi a única que permaneceu ativa e teve seus parâmetros cinéticos residuais determinados. O presente trabalho contribui para o entendimento das bases estruturais que correlacionam mutações na HsFH, deficiência enzimática e patologia. / Fumarate hydratases or fumarases (FH) catalyze the reversible stereospecific hydration of fumarate to L-malate. They are present in all classes of organisms, from prokaryotes to eukaryotes, and can be found in the mitochondrial and cytosolic forms. The enzyme has an important role in energy production as part of the well-known Citric Acid Cycle, in DNA damage response and as tumor suppressor. Human fumarase (HsFH) belongs to class II and is encoded by 1q42.1 gene. HsFH is tetrameric and has 467 amino acids per monomer, with predicted molecular weight of 50.2 kDa. Several studies associated FH gene mutations with some human diseases such as fumaric aciduria, multiple cutaneous and uterine leiomyomatosis (MCUL), which when associated with an aggressive form of multiple cell carcinoma is known as hereditary leiomyomatosis and renal cancer (HLRCC) syndrome. Although the major role of HsFH in energetic metabolism, there are still little structural and kinetic information about the mutants involved in these diseases. Thus, this study aims, through a hybrid approach, composed by biophysics, biochemical and structural characterization of mutants N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, G282VHsFH, E362QHsFH, S365GHsFH and N373DHsFH identified from patients. Steady-state kinetics studies were performed in seven different pHs and, for the first time, the contribution of both substrates was analyzed simultaneously in a single kinetic assay and allowed to quantify the contribution of the reverse reaction for kinetics. According to thermofluor studies, structural stability can be achieved at alkaline pHs and suggests that ligand binding can modulate the protein stability. HsFH crystal structure was solved at 1.8 Å resolution and identified HEPES molecules complexed with the enzyme C-terminal region. Kinetics studies with HEPES showed an increase of the catalytic efficiency and suggests that HEPES binding site might have an allosteric role. Crystal structures for the mutants N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, E362QHsFH, S365GHsFH and N373DHsFH were determined. The mutations Q185R, E362Q, S365G and N373D were identified in the active site and affect the substrate binding capacity directly, while mutation H180R was localized in the B site, which conducts the substrates and products in and out the active site. The mutation K230R is localized in the central domain, but thermofluor results demonstrate a direct effect on the ability of the enzyme to accommodate the substrate. The N107T mutation located far from the active site was the only one that remained active and had its residual kinetic parameters determined. The present work contributes to the understanding of the structural bases that correlate mutations in HsFH, enzymatic deficiency and pathology.

<p>In eukaryotic cells, DNA is packaged into a structure named chromatin which contains DNA and proteins. Nucleosomes are building blocks of chromatin and contain DNA wrapped around a histone octamer. Chromatin modifications (histone post-translational modifications and histone variants) play central roles in various cellular processes including gene expression and DNA damage response. Chromatin modifying enzymes use metabolites as co-substrates and co-factors, and changes in metabolic pathways and metabolite availability affects chromatin modifications and chromatin-associated functions. Moreover, recent studies have uncovered direct roles of metabolic enzymes in chromatin-associated functions. Fumarase, a TCA cycle enzyme that catalyzes the reversible conversion of fumarate to malate in mitochondria (a hydration reaction), is an example of an enzyme with dual functions in metabolism and genome integrity. Cytoplasmic fraction of yeast fumarase, Fum1p, localizes to the nucleus and promotes growth upon DNA damage. Fum1p promotes homologous recombination by enhancing DNA end resection. Human fumarase is involved in DNA repair by non-homologous end joining. Here, we provide evidence that yeast Fum1p and the histone variant Htz1p are also involved in DNA replication stress response and DNA repair by non-homologous end joining (NHEJ). Using mutants lacking the histone variant <i>HTZ1</i>, we show that high cellular levels of fumarate, by deletion of <i>FUM1</i> or addition of exogenous fumarate, suppressed the sensitivity to DNA replication stress by modulation of activity of Jhd2p. This suppression required sensors and mediators of the intra-S phase checkpoint, but not factors involved in the processing of replication intermediates. These results imply that high cellular levels of fumarate can confer resistance to DNA replication stress by bypassing or complementing the defects caused by loss of <i>HTZ1</i> and replication fork processing factors. We also show that upon induction of DSBs, exogenous fumarate conferred resistance to mutants with defects in NHEJ, early steps of homologous recombination (DNA end resection pathway) or late steps of homologous recombination (strand invasion and exchange). Taken together, these results link the metabolic enzyme fumarase and the metabolite fumarate to DNA damage response and show that modulation of DNA damage response by regulating activity of chromatin modifying enzymes is a plausible pathway linking metabolism and nutrient availability to chromatin-associated functions like genome integrity.<br><a></a></p>

Objetivo: este estudo objetivou avaliar a presença do anticorpo anti-alfaenolase (AAAE) IgM na doença de Behçet (DB) e suas possíveis associações com as manifestações clínicas e atividade da doença. Métodos: noventa e sete pacientes com DB foram comparados a 36 pacientes com enteroartrite (EA) [24 com doença de Crohn (DC) e 12 com retocolite ulcerativa (RCU)], além de 87 controles saudáveis. Os testes para detecção do AAAE IgM foram realizados por Immunoblotting. A atividade de doença foi avaliada por índices padronizados, como o Formulário de Atividade Atual da Doença de Behçet (BR-BDCAF) para os pacientes com DB e o Índice de Harvey-Bradshaw (HBI) para os pacientes com DC e RCU. Uma segunda avaliação foi realizada somente nos pacientes com DB (n=56) para a detecção do AAAE IgM, avaliação de atividade de doença e dosagem de proteína-C-reativa (PCR). Resultados: maior prevalência do AAAE IgM foi encontrada na DB (17,7%) comparativamente à EA (2,8%) e aos controles saudáveis (2,3%), p < 0,001. A frequência do AAAE IgM foi maior na DB ativa quando comparada à DB inativa (30,2% vs. 7,4%, p=0,006). Este achado foi confirmado em uma segunda avaliação de 56 pacientes do grupo com DB (45,5% vs. 13,3%, p=0,02). A média do BR-BDCAF foi maior no grupo com AAAE IgM positivo, em ambas avaliações (9,1 ± 5,4 vs. 4,9 ± 4,9, p=0,002; 5,0 ± 4,9 vs. 2,2 ± 2,9, p=0,01, respectivamente). Os pacientes com DB em atividade mucocutânea e articular apresentaram maior incidência do AAAE IgM, tanto na primeira quanto na segunda avaliação (64,7% vs. 27,5%, p=0,005; 36,4% vs. 7,1%, p=0,039, respectivamente). Conclusões: os presentes dados corroboram que a alfa-enolase é um antígeno alvo na DB, particularmente associada à atividade mucocutânea e articular da doença. Além disso, o AAAE IgM pode distinguir a DB da EA, especialmente em pacientes com alta atividade de doença / Objective: this study aimed to assess IgM AAEA in systemic Behçet\'s disease (BD) and its possible association with clinical manifestations and disease activity. Methods: ninety-seven consecutively selected BD patients were compared to 36 enteropathic spondyloarthritis (ESpA) [24 Crohn\'s disease (CD) and 12 ulcerative colitis (UC)] patients and 87 healthy controls. IgM AAEA was detected by Immunoblotting. Disease activity was assessed by standardized indexes, Brazilian BD Current Activity Form (BR-BDCAF) for BD and Harvey-Bradshaw Index (HBI) for CD and UC patients. A second evaluation was performed in BD patients (n=56), regarding IgM AAEA presence, disease activity scores and C-reactive protein (CRP). Results: higher IgM AAEA prevalence was found in BD (17.7%) compared to ESpA (2.8%) and healthy controls (2.3%), p < 0.001. IgM AAEA frequency was higher in active BD compared to inactive BD (30.2% vs. 7.4%, p=0.006), a finding confirmed in the second cross-sectional evaluation of 56 of these BD patients (45.5% vs. 13.3%, p=0.02). Mean BR-BDCAF scores were higher in IgM AAEA positive group on both evaluations (9.1 ± 5.4 vs. 4.9 ± 4.9, p=0.002; 5.0 ± 4.9 vs. 2.2 ± 2.9, p=0.01, respectively). BD patients with mucocutaneous and articular symptoms presented higher IgM AAEA positivity in the first and second evaluations (64.7% vs. 27.5%, p=0.005; 36.4% vs. 7.1%, p=0.039 respectively). Conclusions: these data support the notion that alpha-enolase is a target antigen in BD, particularly associated with disease activity, mucocutaneous and articular involvement. In addition, IgM AAEA may distinguish BD from ESpA, especially in patients with high disease activity