Academic literature on the topic 'Methylcitrate cycle'

ABSTRACT Mycobacterium tuberculosis is predicted to subsist on alternative carbon sources during persistence within the human host. Catabolism of odd- and branched-chain fatty acids, branched-chain amino acids, and cholesterol generates propionyl-coenzyme A (CoA) as a terminal, three-carbon (C3) product. Propionate constitutes a key precursor in lipid biosynthesis but is toxic if accumulated, potentially implicating its metabolism in M. tuberculosis pathogenesis. In addition to the well-characterized methylcitrate cycle, the M. tuberculosis genome contains a complete methylmalonyl pathway, including a mutAB-encoded methylmalonyl-CoA mutase (MCM) that requires a vitamin B12-derived cofactor for activity. Here, we demonstrate the ability of M. tuberculosis to utilize propionate as the sole carbon source in the absence of a functional methylcitrate cycle, provided that vitamin B12 is supplied exogenously. We show that this ability is dependent on mutAB and, furthermore, that an active methylmalonyl pathway allows the bypass of the glyoxylate cycle during growth on propionate in vitro. Importantly, although the glyoxylate and methylcitrate cycles supported robust growth of M. tuberculosis on the C17 fatty acid heptadecanoate, growth on valerate (C5) was significantly enhanced through vitamin B12 supplementation. Moreover, both wild-type and methylcitrate cycle mutant strains grew on B12-supplemented valerate in the presence of 3-nitropropionate, an inhibitor of the glyoxylate cycle enzyme isocitrate lyase, indicating an anaplerotic role for the methylmalonyl pathway. The demonstrated functionality of MCM reinforces the potential relevance of vitamin B12 to mycobacterial pathogenesis and suggests that vitamin B12 availability in vivo might resolve the paradoxical dispensability of the methylcitrate cycle for the growth and persistence of M. tuberculosis in mice.

ABSTRACT We previously identified the prpBCDE operon, which encodes catabolic functions required for propionate catabolism inSalmonella typhimurium. Results from13C-labeling experiments have identified the route of propionate breakdown and determined the biochemical role of each Prp enzyme in this pathway. The identification of catabolites accumulating in wild-type and mutant strains was consistent with propionate breakdown through the 2-methylcitric acid cycle. Our experiments demonstrate that the α-carbon of propionate is oxidized to yield pyruvate. The reactions are catalyzed by propionyl coenzyme A (propionyl-CoA) synthetase (PrpE), 2-methylcitrate synthase (PrpC), 2-methylcitrate dehydratase (probably PrpD), 2-methylisocitrate hydratase (probably PrpD), and 2-methylisocitrate lyase (PrpB). In support of this conclusion, the PrpC enzyme was purified to homogeneity and shown to have 2-methylcitrate synthase activity in vitro.1H nuclear magnetic resonance spectroscopy and negative-ion electrospray ionization mass spectrometry identified 2-methylcitrate as the product of the PrpC reaction. Although PrpC could use acetyl-CoA as a substrate to synthesize citrate, kinetic analysis demonstrated that propionyl-CoA is the preferred substrate.

Propionyl-CoA is an inhibitor of both primary and secondary metabolism in Aspergillus species and a functional methylcitrate cycle is essential for the efficient removal of this potentially toxic metabolite. Although the genomes of most sequenced fungal species appear to contain genes coding for enzymes of the methylcitrate cycle, experimental confirmation of pathway activity in filamentous fungi has only been provided for Aspergillus nidulans and Aspergillus fumigatus. In this study we demonstrate that pathogenic Fusarium species also possess a functional methylcitrate cycle. Fusarium solani appears highly adapted to saprophytic growth as it utilized propionate with high efficiency, whereas Fusarium verticillioides grew poorly on this carbon source. In order to elucidate the mechanisms of propionyl-CoA detoxification, we first identified the genes coding for methylcitrate synthase from both species. Despite sharing 96 % amino acid sequence identity, analysis of the two purified enzymes demonstrated that their biochemical properties differed in several respects. Both methylcitrate synthases exhibited low K m values for propionyl-CoA, but that of F. verticillioides displayed significantly higher citrate synthase activity and greater thermal stability. Activity determinations from cell-free extracts of F. solani revealed a strong methylcitrate synthase activity during growth on propionate and to a lesser extent on Casamino acids, whereas activity by F. verticillioides was highest on Casamino acids. Further phenotypic analysis confirmed that these biochemical differences were reflected in the different growth behaviour of the two species on propionyl-CoA-generating carbon sources.

The methylcitrate cycle metabolizes propionyl-CoA, a toxic metabolite, into pyruvate. Pyricularia oryzae (syn. Magnaporthe oryzae) is a phytopathogenic fungus that causes a destructive blast disease in rice and wheat. We characterized the essential roles of the methylcitrate cycle in the development and virulence of P. oryzae using functional genomics. In P. oryzae, the transcript levels of MCS1 and MCL1, which encode a 2-methylcitrate synthase and a 2-methylisocitrate lyase, respectively, were upregulated during appressorium formation and when grown on propionyl-CoA-producing carbon sources. We found that deletion of MCS1 and MCL1 inhibited fungal growth on media containing both glucose and propionate, and media using propionate or propionyl-CoA-producing amino acids (valine, isoleucine, methionine, and threonine) as the sole carbon or nitrogen sources. The Δmcs1 mutant formed sparse aerial hyphae and did not produce conidia on complete medium (CM), while the Δmcl1 mutant showed decreased conidiation. The aerial mycelium of Δmcs1 displayed a lowered NAD+/NADH ratio, reduced nitric oxide content, and downregulated transcription of hydrophobin genes. Δmcl1 showed reduced appressorium turgor, severely delayed plant penetration, and weakened virulence. Addition of acetate recovered the growth of the wild type and Δmcs1 on medium containing both glucose and propionate and recovered the conidiation of both Δmcs1 and Δmcl1 on CM by reducing propionyl-CoA formation. Deletion of MCL1 together with ICL1, an isocitrate lyase gene in the glyoxylate cycle, greatly reduced the mutant’s virulence as compared with the single-gene deletion mutants (Δicl1 and Δmcl1). This experimental evidence provides important information about the role of the methylcitrate cycle in development and virulence of P. oryzae by detoxification of propionyl-CoA and 2-methylisocitrate.

ABSTRACT Genome sequencing revealed that the Corynebacterium glutamicum genome contained, besides gltA, two additional citrate synthase homologous genes (prpC) located in two different prpDBC gene clusters, which were designated prpD1B1C1 and prpD2B2C2. The coding regions of the two gene clusters as well as the predicted gene products showed sequence identities of about 70 to 80%. Significant sequence similarities were found also to the prpBCDE operons of Escherichia coli and Salmonella enterica, which are known to encode enzymes of the propionate-degrading 2-methylcitrate pathway. Homologous and heterologous overexpression of the C. glutamicum prpC1 and prpC2 genes revealed that their gene products were active as citrate synthases and 2-methylcitrate synthases. Growth tests showed that C. glutamicum used propionate as a single or partial carbon source, although the beginning of the exponential growth phase was strongly delayed by propionate for up to 7 days. Compared to growth on acetate, the specific 2-methylcitrate synthase activity increased about 50-fold when propionate was provided as the sole carbon source, suggesting that in C. glutamicum the oxidation of propionate to pyruvate occurred via the 2-methylcitrate pathway. Additionally, two-dimensional gel electrophoresis experiments combined with mass spectrometry showed strong induction of the expression of the C. glutamicum prpD2B2C2 genes by propionate as an additional carbon source. Mutational analyses revealed that only the prpD2B2C2 genes were essential for the growth of C. glutamicum on propionate as a sole carbon source, while the function of the prpD1B1C1 genes remains obscure.

Abstract Talaromyces marneffei (T. marneffei), which used to be known as Penicillium marneffei, is the causative agent of the fatal systemic mycosis known as talaromycosis. For the purpose of understanding the role of methylcitrate cycle in the virulence of T. marneffei, we generated MCD deletion (ΔMCD) and complementation (ΔMCD+) mutants of T. marneffei. Growth in different carbon sources showed that ΔMCD cannot grow on propionate media and grew slowly on the valerate, valine, methionine, isoleucine, cholesterol, and YNB (carbon free) media. The macrophage killing assay showed that ΔMCD was attenuated in macrophages of mice in vitro, especially at the presence of propionate. Finally, virulence studies in a murine infection experiment revealed attenuated virulence of the ΔMCD, which indicates MCD is essential for T. marneffei virulence in the host. This experiment laid the foundation for the further study of the specific mechanisms underlying the methylcitrate cycle of T. marneffei and may provide suitable targets for new antifungals.

ABSTRACT The glyoxylate and methylcitrate cycles are involved in the metabolism of two- or three-carbon compounds in fungi. To elucidate the role(s) of these pathways in Gibberella zeae, which causes head blight in cereal crops, we focused on the functions of G. zeae orthologs (GzICL 1 and GzMCL1) of the genes that encode isocitrate lyase (ICL) and methylisocitrate lyase (MCL), respectively, key enzymes in each cycle. The deletion of GzICL1 (ΔGzICL1) caused defects in growth on acetate and in perithecium (sexual fruiting body) formation but not in virulence on barley and wheat, indicating that GzICL1 acts as the ICL of the glyoxylate cycle and is essential for self-fertility in G. zeae. In contrast, the ΔGzMCL1 strains failed to grow on propionate but exhibited no major changes in other traits, suggesting that GzMCL1 is required for the methylcitrate cycle in G. zeae. Interestingly, double deletion of both GzICL1 and GzMCL1 caused significantly reduced virulence on host plants, indicating that both GzICL1 and GzMCL1 have redundant functions for plant infection in G. zeae. Thus, both GzICL1 and GzMCL1 may play important roles in determining major mycological and pathological traits of G. zeae by participating in different metabolic pathways for the use of fatty acids.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Pathogens can find different carbon sources in host niches generating propionyl-CoA, among them propionate. This compound is toxic to the organism if accumulated within the cell, and can be generated in the host tissue by the metabolism of amino acids isoleucine, valine and methionine, or by the metabolism of odd-chain fatty acids. Therefore, during infection, the propionyl-CoA metabolism to nontoxic nutrient and usable energetically is of great relevance. Nonetheless, there are no studies about the propionyl-CoA metabolization pathway in fungi of the genus Paracoccidioides, causer of paracoccidioidomycosis, a systemic mycosis of high incidence in Latin America. Thus, to characterize of which metabolic pathway this fungus utilizes to propionyl-CoA metabolization, it was made a search the genes coding to enzymes of methylcitrate cycle in genome of Paracoccidioides spp. and were identified genes coding to the three exclusive enzymes of this cycle, which are methylcitrate synthase, methylcitrate dehydrogenase and methylcitrate lyase. After analysis of growth and viability, which demonstrated that Paracoccidioides lutzii utilizes propionate as carbon source, it was made gene expression analysis of enzymes of methylcitrate cycle and was observed that are regulated in response to propionate. Additionally, the enzymatic activity of the MCS showed that this enzyme is active inside of fungal cells and also when is secreted, as well as its dual capacity of to act with a citrate synthase and metylcitrate synthase. Finally, the proteomic profile of P. lutzii in propionate showed enzymes induction of methylcitrate cycle, glyoxylate cycle, amino acid metabolism and gluconeogenesis, and the repression of glycolytic pathway, fermentation and fatty acid synthesis, which demonstrated of metabolic rearrangement to supply the cellular energetic demand, metabolizing propionate. In this sense, to understand the mechanism of propionyl-CoA metabolism in P. lutzii provides data for visualization of metabolic adaptation that this fungus makes use in different colonization of niches.
Micro-organismos patogênicos podem encontrar em nichos do hospedeiro diferentes fontes de carbono geradoras de propionil-CoA, dentre elas, o propionato. Este composto é tóxico para a célula se acumulado no seu interior, e pode ser gerado nos tecidos do hospedeiro pelo metabolismo dos aminoácidos isoleucina, valina e metionina, ou pelo metabolismo de ácidos graxos de cadeia ímpar. Portanto, durante a infecção, o metabolismo de propionil-CoA a um nutriente não tóxico e aproveitável energeticamente se faz de grande relevância. Contudo, não há estudos sobre a via de metabolização de propionil-CoA em fungo do gênero Paracoccidioides, causador da paracoccidioidomicose, uma micose sistêmica de alta incidência na América Latina. Sendo assim, para caracterização de qual via metabólica este organismo utiliza para metabolização de propionil-CoA, foi feita uma busca dos genes codificantes para enzimas do ciclo do metilcitrato no genoma de Paracoccidioides spp. e foram identificados genes codificantes para as três enzimas exclusivas desse ciclo, as quais são metilcitrato sintase, metilcitrato desidrogenase e metilcitrato liase. Após análise de crescimento e viabilidade, a qual demonstrou que Paracoccidioides lutzii utiliza propionato como fonte de carbono, foi feita a análise de expressão gênica das enzimas do ciclo do metilcitrato e observou-se que são reguladas em resposta ao propionato. Adicionalmente, a atividade enzimática da MCS demonstrou que essa enzima é ativa no interior da célula fúngica ou mesmo quando é secretada. Além disso, foi demostrada sua capacidade de atuar também como uma citrato sintase. Por fim, o perfil proteômico de P. lutzii em propionato mostrou a indução do ciclo do metilcitrato, ciclo do glioxilato, metabolismo de aminoácidos e gliconeogênese, e a repressão de vias como glicólise, fermentação e síntese de ácidos graxos, os quais demonstram o rearranjo metabólico para suprir a demanda energética celular metabolizando propionato. Neste sentido, entender os mecanismos pelo qual P. lutzii lança mão para metabolizar propionil-CoA permite a compreensão dos mecanismos de adaptação metabólica que esse fungo lança mão em diferentes nichos de colonização.