Functional characterization and subcellular localization of the three malate dehydrogenase isozymes in Leishmania spp.

Leroux, Alejandro E.; Fleming-Canepa, Ximena; Aranda, Alejandro; Maugeri, Dante; Cazzulo, Juán José; Sánchez, Marco A.; Nowicki, Cristina

doi:10.1016/j.molbiopara.2006.04.010

Cited by 22 publications

(17 citation statements)

References 31 publications

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“…This comparison between K m values suggests that both LmFHs have affinities for both substrates, fumarate and malate, lower than the other class I FHs. The low affinity for malate has also been reported for malate dehydrogenase isozymes in L. major [34], suggesting that the concentrations of these metabolites in Leishmania spp. can be relatively high.…”

Section: Kinetic Characterization Of the Lmfh Isoformsmentioning

confidence: 77%

Fumarate hydratase isoforms of Leishmania major: Subcellular localization, structural and kinetic properties

Feliciano

Gupta

Dyszy

et al. 2012

International Journal of Biological Macromolecules

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Fumarate hydratases (FHs; EC 4.2.1.2) are enzymes that catalyze the reversible hydration of fumarate to S-malate. Parasitic protists that belong to the genus Leishmania and are responsible for a complex of vector-borne diseases named leishmaniases possess two genes that encode distinct putative FH enzymes. Genome sequence analysis of Leishmania major Friedlin reveals the existence of genes LmjF24.0320 and LmjF29.1960 encoding the putative enzymes LmFH-1 and LmFH-2, respectively. In the present work, the FH activity of both L. major enzymes has been confirmed. Circular dichroism studies suggest important differences in terms of secondary structure content when comparing LmFH isoforms and even larger differences when comparing them to the homologous human enzyme. CD melting experiments revealed that both LmFH isoforms are thermolabile enzymes. The catalytic efficiency under aerobic and anaerobic environments suggests that they are both highly sensitive to oxidation and damaged by oxygen. Intracellular localization studies located LmFH-1 in the mitochondrion, whereas LmFH-2 was found predominantly in the cytosol with possibly also some in glycosomes. The high degree of sequence conservation in different Leishmania species, together with the relevance of FH activity for the energy metabolism in these parasites suggest that FHs might be exploited as targets for broad-spectrum antileishmanial drugs.

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Section: Kinetic Characterization Of the Lmfh Isoformsmentioning

confidence: 77%

Fumarate hydratase isoforms of Leishmania major: Subcellular localization, structural and kinetic properties

Feliciano

Gupta

Dyszy

et al. 2012

International Journal of Biological Macromolecules

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“…cruzi AHADH is structurally related to cMDH it has no activity with oxaloacetate as a substrate whereas the L . major cMDH has a high specificity for oxaloacetate indicating that it functions as a malate dehydrogenase [ 53 , 54 ]. The release of aromatic lactates by the three species ( Table 4 , S8 Table ) shows that that an AHADH activity must be present in Leishmania , although it may require special conditions for function.…”

Section: Discussionmentioning

confidence: 99%

Metabolomic Analyses of Leishmania Reveal Multiple Species Differences and Large Differences in Amino Acid Metabolism

et al. 2015

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Comparative genomic analyses of Leishmania species have revealed relatively minor heterogeneity amongst recognised housekeeping genes and yet the species cause distinct infections and pathogenesis in their mammalian hosts. To gain greater information on the biochemical variation between species, and insights into possible metabolic mechanisms underpinning visceral and cutaneous leishmaniasis, we have undertaken in this study a comparative analysis of the metabolomes of promastigotes of L. donovani, L. major and L. mexicana. The analysis revealed 64 metabolites with confirmed identity differing 3-fold or more between the cell extracts of species, with 161 putatively identified metabolites differing similarly. Analysis of the media from cultures revealed an at least 3-fold difference in use or excretion of 43 metabolites of confirmed identity and 87 putatively identified metabolites that differed to a similar extent. Strikingly large differences were detected in their extent of amino acid use and metabolism, especially for tryptophan, aspartate, arginine and proline. Major pathways of tryptophan and arginine catabolism were shown to be to indole-3-lactate and arginic acid, respectively, which were excreted. The data presented provide clear evidence on the value of global metabolomic analyses in detecting species-specific metabolic features, thus application of this technology should be a major contributor to gaining greater understanding of how pathogens are adapted to infecting their hosts.

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“…Rabbit antiserum raised against the L. major cytosolic malate dehydrogenase (MDH) isozyme (1:20) was used as the cytosolic marker in the co‐localization assays (Leroux et al. ). After 1 h incubation at room temperature, slides were washed three times with PBS.…”

Section: Methodsmentioning

confidence: 99%

“…Glass coverslips were treated with polyclonal antisera raised against T. cruzi CGL (1:50) and L. major CS (1:20), which were diluted in PBS supplemented with 20% FCS (v/v). Rabbit antiserum raised against the L. major cytosolic malate dehydrogenase (MDH) isozyme (1:20) was used as the cytosolic marker in the co-localization assays (Leroux et al 2006). After 1 h incubation at room temperature, slides were washed three times with PBS.…”

Section: Subcellular Distribution Of Cgl and Csmentioning

confidence: 99%

Cystathionine γ‐lyase, an Enzyme Related to the Reverse Transsulfuration Pathway, is Functional in Leishmania spp.

Giordana

Mantilla

Santana

et al. 2014

J Eukaryotic Microbiology

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Leishmania parasites seem capable of producing cysteine by de novo biosynthesis, similarly to bacteria, some pathogenic protists, and plants. In Leishmania spp., cysteine synthase (CS) and cystathionine β-synthase (CBS) are expected to participate in this metabolic process. Moreover, the reverse transsulfuration pathway (RTP) is also predicted to be operative in this trypanosomatid because CBS also catalyzes the condensation of serine with homocysteine, and a gene encoding a putative cystathionine γ-lyase (CGL) is present in all the sequenced genomes. Our results show that indeed, Leishmania major CGL is able to rescue the wild-type phenotype of a Saccharomyces cerevisiae CGL-null mutant and is susceptible to inhibition by an irreversible CGL inhibitor, DL-propargylglycine (PAG). In Leishmania promastigotes, CGL and CS are cytosolic enzymes. The coexistence of de novo synthesis with the RTP is extremely rare in most living organisms; however, despite this potentially high redundancy in cysteine production, PAG arrests the proliferation of L. major promastigotes with an IC50 of approximately 65 μM. These findings raise new questions regarding the biological role of CGL in these pathogens and indicate the need for understanding the molecular mechanism of PAG action in vivo to identify the potential targets affected by this drug.

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Functional characterization and subcellular localization of the three malate dehydrogenase isozymes in Leishmania spp.

Cited by 22 publications

References 31 publications

Fumarate hydratase isoforms of Leishmania major: Subcellular localization, structural and kinetic properties

Fumarate hydratase isoforms of Leishmania major: Subcellular localization, structural and kinetic properties

Metabolomic Analyses of Leishmania Reveal Multiple Species Differences and Large Differences in Amino Acid Metabolism

Cystathionine γ‐lyase, an Enzyme Related to the Reverse Transsulfuration Pathway, is Functional in Leishmania spp.

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