ANÁLISE IN SILICO PARA DEFINIÇÃO DE COMMON CORE MICROBIANO ASSOCIADO À DEGRADAÇÃO DE SURFACTANTE ANIÔNICO ALQUILBENZENO LINEAR SULFONADO (LAS)

Introdução: O alquilbenzeno linear sulfonado é o surfactante aniônico de maior interesse econômico, correspondendo a 84% do mercado no Brasil, e 45% no mundo. Sua rota de biodegradação envolve reações de adição de fumarato, β-oxidação, clivagem do anel aromático e dessulfonação. Tratando-se de um processo complexo, o uso de consórcios microbianos possibilita usufruir de diferentes metabolismos para a degradação do LAS. Contudo, nota-se uma escassez na literatura acerca dos microrganismos que desempenham papel fundamental na degradação do surfactante em reatores biológicos. Portanto, o presente trabalho visa preencher esta lacuna por meio do estudo de sequências de gene 16S rRNA obtidas em amostras utilizadas para testar a degradação de LAS, em biorreatores de diferentes configurações e contendo inóculos de origens distintas, presentes na literatura. Objetivos: Definir o common core microbiano associado à degradação do surfactante aniônico Alquilbenzeno Linear Sulfonado (LAS) em biorreatores, por meio da avaliação da diversidade e riqueza microbiana do Domínio Archaea e Bacteria, e predição do perfil funcional das taxonomias obtidas. Material e métodos: Utilizaram-se 52 amostras de sequências de gene 16S rRNA, obtidas de 12 artigos presentes na literatura, que estudaram a remoção biológica de LAS em águas residuárias reais. Para a análise das sequências, utilizou-se a plataforma QIIME 2 e seus plug-ins, a ferramenta Tax4Fun e software R. Resultados: Os filos mais abundantes encontrados foram Proteobacteria e Bacteroidota, e gêneros Methanosaeta e C39. Dentre os common core, o gênero SJA-28 (filo Bacteroidota) foi o mais presente. A enzima fumarate reductase, relacionada à adição de fumarato, foi pouco abundante entre as amostras. Para a reação de β-oxidação, a enzima 3-oxoacyl-acyl- reductase foi predominante. As enzimas relacionadas ao processo de dessulfonação encontradas na literatura foram registradas no presente trabalho. Nenhuma enzima produzida durante a clivagem do anel aromático foi registrada durante a análise. Conclusão: Não foram encontrados gêneros em comum entre todas as amostras analisadas. O perfil funcional relacionado à reação de β-oxidação foi mais abundante, quando comparada às outras reações. Os resultados alcançados pelo presente estudo aumentam o conhecimento sobre a composição taxonômica e funcional existente e compartilhada entre diferentes ambientes associados à degradação do surfactante LAS.

Mycobacterium tuberculosis and M. bovis BCG Moreau Fumarate Reductase Operons Produce Different Polypeptides That May Be Related to Non-canonical Functions

Tuberculosis is a world widespread disease, caused by Mycobacterium tuberculosis (M.tb). Although considered an obligate aerobe, this organism can resist life-limiting conditions such as microaerophily mainly due to its set of enzymes responsible for energy production and coenzyme restoration under these conditions. One of these enzymes is fumarate reductase, an heterotetrameric complex composed of a catalytic (FrdA), an iron-sulfur cluster (FrdB) and two transmembrane (FrdC and FrdD) subunits involved in anaerobic respiration and important for the maintenance of membrane potential. In this work, aiming to further characterize this enzyme function in mycobacteria, we analyzed the expression of FrdB-containing proteins in M.tb and Mycobacterium bovis Bacillus Calmette–Guérin (BCG) Moreau, the Brazilian vaccine strain against tuberculosis. We identified three isoforms in both mycobacteria, two of them corresponding to the predicted encoded polypeptides of M.tb (27 kDa) and BCG Moreau (40 kDa) frd sequences, as due to an insertion on the latter’s operon a fused FrdBC protein is expected. The third 52 kDa band can be explained by a transcriptional slippage event, typically occurring when mutation arises in a repetitive region within a coding sequence, thought to reduce its impact allowing the production of both native and variant forms. Comparative modeling of the M.tb and BCG Moreau predicted protein complexes allowed the detection of subtle overall differences, showing a high degree of structure and maybe functional resemblance among them. Axenic growth and macrophage infection assays show that the frd locus is important for proper bacterial development in both scenarios, and that both M.tb’s and BCG Moreau’s alleles can partially revert the hampered phenotype of the knockout strain. Altogether, our results show that the frdABCD operon of Mycobacteria may have evolved to possess other yet non-described functions, such as those necessary during aerobic logarithmic growth and early stage steps of infection.

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei

A subset of flavoproteins has a covalently attached flavin prosthetic group enzymatically attached via phosphoester bonding. In prokaryotes, this is catalysed by ApbE flavin transferases. ApbE-like domains are present in few eukaryotic taxa, e.g. the N-terminal domain of fumarate reductase (FRD) of Trypanosoma, a parasitic protist known as a tropical pathogen causing African sleeping sickness. We use the versatile reverse genetic tools available for Trypanosoma to investigate the flavinylation of glycosomal FRD (FRDg) in vivo in the physiological and organellar context. Using direct in-gel fluorescence detection of covalently attached flavin as proxy for activity, we show that the ApbE-like domain of FRDg has flavin transferase activity in vivo. The ApbE domain is preceded by a consensus flavinylation target motif at the extreme N-terminus of FRDg, and serine 9 in this motif is essential as flavin acceptor. The preferred mode of flavinylation in the glycosome was addressed by stoichiometric expression and comparison of native and catalytically dead ApbE domains. In addition to the trans-flavinylation activity, the ApbE domain catalyses the intramolecular cis-flavinylation with at least 5-fold higher efficiency. We discuss how the higher efficiency due to unusual fusion of the ApbE domain to its substrate protein FRD may provide a selective advantage by faster FRD biogenesis during rapid metabolic adaptation of trypanosomes. The first 37 amino acids of FRDg, including the consensus motif, are sufficient as flavinylation target upon fusion to other proteins. We propose FRDg(1-37) as 4 kDa heat-stable, detergent-resistant fluorescent protein tag and suggest its use as a new tool to study glycosomal protein import.

Metabolic selection of a homologous recombination mediated loss of glycosomal fumarate reductase in Trypanosoma brucei

The genome of trypanosomatids is rearranged at the level of repeated sequences, where serve as platforms for amplification or deletion of genomic segments. We report here that the PEPCK gene knockout (Δpepck) leads to the selection of such a deletion event between the FRDg and FRDm2 genes to produce a chimeric FRDg-m2 gene in the Δpepck* cell line. FRDg is expressed in peroxisome-like organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is a non-functional cytosolic FRD. Re-expression of FRDg significantly impaired growth of the Δpepck* cells, while inhibition of FRDg-m2 expression had no effect, which indicated that this recombination event has been selected in the Δpepck* cells to eliminate FRDg. FRD activity was not involved in the FRDg-mediated negative effect, while its auto-flavinylation motif is required to impair growth. Considering that (i) FRDs are known to generate reactive oxygen species (ROS) by transferring electrons from their flavin moiety(ies) to oxygen, (ii) intracellular ROS production is essential for the differentiation of procyclic to epimastigote forms of the parasite and (iii) the fumarate reductase activity is not essential for the parasite, we propose that the main role of FRD is to produce part of the ROS necessary to complete the parasitic cycle in the tsetse fly. In this context, the negative effect of FRDg expression in the PEPCK null background is interpreted as an increased production of ROS from oxygen since fumarate, the natural electron acceptor of FRDg, is no longer produced in glycosomes.

A new water-soluble bacterial NADH: fumarate oxidoreductase

ABSTRACT The cytoplasmic fumarate reductase of Klebsiella pneumoniae (FRD) is a monomeric protein which contains three prosthetic groups: noncovalently bound FMN and FAD plus a covalently bound FMN. In the present work, NADH is revealed to be an inherent electron donor for this enzyme. We found that the fumarate reductase activity of FRD significantly exceeds its NADH dehydrogenase activity. During the catalysis of NADH:fumarate oxidoreductase reaction, FRD turnover is limited by a very low rate (∼10/s) of electron transfer between the noncovalently and covalently bound FMN moieties. Induction of FRD synthesis in K. pneumoniae cells was observed only under anaerobic conditions in the presence of fumarate or malate. Enzymes with the FRD-like domain architecture are widely distributed among various bacteria and apparently comprise a new type of water-soluble NADH:fumarate oxidoreductases.

Identification of a Novel Fumarate Reductase Potentially Involved in Electron Bifurcation

In the succinic acid-producing bacterium Pseudoclostridium thermosuccinogenes the fumarate reductase (FRD) genes reside in an operon together with those encoding an electron bifurcating complex (FlxABCD-HdrABC) that shuttles electrons from NADH to ferredoxin and a disulfide bond. Based on phylogeny and genomic co-occurrence we propose two hypothetical mechanisms via which the FRD is involved in electron bifurcation: (I) A disulfide bond from a hitherto unknown cofactor is reduced by the electron-bifurcating FlxABCD-HdrABC complex, using NADH to generate two thiol groups, while facilitating the unfavourable reduction of ferredoxin by NADH. The disulfide bond is subsequently regenerated via the reduction of fumarate by the FRD using the previously formed thiol groups. (II) The FRD forms an integral part of the FlxABCD-HdrABC complex, and NADH is used to reduce ferredoxin and fumarate directly, without an intermediate disulfide-forming cofactor. Either way enables the conservation of additional energy by a soluble FRD, analogous to fumarate respiration.

Studi in silico potensi anthelmintik rambusa (Passiflora foetida) sebagai inhibitor produksi ATP pada Ascaris suum

Resistensi terhadap anthelmintika memacu upaya penemuan anthelmintika baru yang bekerja melalui penghambatan produksi energi ATP anaerobik pada sel parasit. Parasit melakukan metabolisme menggunakan enzim fumarat reduktase yang berfungsi untuk mengubah fumarat menjadi suksinat. Reaksi fumarat reduktase terjadi di mitokondria dengan bantuan rhodoquinone. Mitochondrial rhodoquinol-fumarate reductase dianggap sebagai target reseptor yang baik untuk pengembangan anthelmintik baru. Penelitian ini bertujuan untuk memprediksi interaksi antara senyawa Passiflora foetida dengan reseptor mitochondrial rhodoquinol-fumarate reductase dari cacing Ascaris suum dengan metode in silico. Hasil penelitian menunjukkan bahwa senyawa bioaktif ermanin dan deidadlin dari Passiflora foetida memiliki potensi sebagai senyawa obat anthelmintik. Aktivitas senyawa ermanin dan deidaclin memiliki ernegi afinitias berturut turut-turut sebesar -8,2 kcal/mol dan -7,8 kcal/mol, bahkan lebih rendah dari senyawa Atpenin (-7,6kcal/mol)

Crystal Structure of the Active Site Mutant Form of Soluble Fumarate Reductase, Osm1

Soluble fumarate reductase is essential for survival under anaerobic conditions. This enzyme can maintain the redox balance in the cell by catalyzing the reduction of fumarate to succinate. Although the overall reaction mechanism of soluble fumarate reductase in yeast, Osm1, has been proposed by a previous structural study, the details of the underlying mechanism are not completely elucidated. The present study provides the structural information regarding the active site mutant form of Osm1 (R326A), thus, revealing that R326A mutation does not affect the substrate binding. Structural alterations of the residues surrounding the active site, and the missing 2nd flavin adenine dinucleotide (FAD) in the previously defined 2nd FAD binding site, were observed as characteristic features of the Osm1 R326A crystal structure. Based on these findings, we provided a clue that can explain the loss of activity of Osm1 R326A.