<i>Plasmodium</i>
            Parasite Malate-Quinone Oxidoreductase Functionally Complements a Yeast Deletion Mutant of Mitochondrial Malate Dehydrogenase

Ito, Takeshi; Kajita, Sayaka; Fujii, Megumi; Shinohara, Yasuo

doi:10.1128/spectrum.00168-23

Cited by 4 publications

(22 citation statements)

References 62 publications

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“…The result is similar to that of ΔMdh1 under the experimental conditions used. 15 While some mutations might also somewhat affect protein stability, the consistency with the growth test results rules out the possibility that the loss of activity would be entirely due to protein denaturation during the isolation of mitochondria and enzyme activity assay. In addition, under conditions with 10fold higher concentrations of the substrates (600 μM decylubiquinone, 100 mM malate), we found essentially no significant difference in MQO activity of the mutants compared with the results of Figure 5, i.e., their activity was similar to that of the ΔMdh1 strain alone.…”

Section: ■ Resultsmentioning

confidence: 91%

“…This suggests that the loss of activity in the mutants was due to a loss of functional ability rather than decreased affinity for the substrates (Figure S2). We should note that, under the assay conditions used in Figure S2, the Δ Mdh1 and mutant strains had moderate substrate-dependent DCIP-reducing activities, possibly due to malate oxidation by mitochondrial succinate dehydrogenase. ,, However, this nonspecific activity is nondetectable under conditions with 60 μM decylubiquinone and 10 mM malate . Finally, to rule out the possibility that our observations might reflect changes in protein expression levels, we analyzed mitochondria extracted from each strain by Western blotting using an anti-Flag antibody.…”

Section: Resultsmentioning

confidence: 99%

“…Malate-Quinone Oxidoreductase Activity. Malatedependent ubiquinone reduction in the mitochondrial fractions was measured spectrophotometrically at 37 °C in a quartz cuvette as described previously, 15 with some modifications when necessary. DCIP, which is reduced by the ubiquinol produced, was used as an indicator.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…Strains of Saccharomyces cerevisiae were grown as described previously . Briefly, cells were grown at 30 °C on synthetic minimal medium (0.67% yeast nitrogen base without amino acids; BD Difco) supplemented with 2% glucose, essential amino acids, and other nutrients to meet auxotrophic requirements (SD).…”

Section: Materials and Methodsmentioning

confidence: 99%

“…In Plasmodium parasites, which cause malaria, mitochondrial MDH is missing. ,− Thus, based on its catalytic properties, Plasmodium parasite MQO is thought to function in place of MDH as a TCA cycle enzyme in mitochondria, and to be coupled with the electron transport chain (ETC) through quinone reduction; ,− accordingly, Plasmodium MQO has received attention as a putative antimalarial target. − Recently, using a yeast expression system, we showed that P. falciparum MQO (PfMQO) can functionally replace yeast mitochondrial malate dehydrogenase (MDH1), clearly demonstrating that PfMQO contributes to the energy metabolism of the TCA cycle and the ETC . However, as no crystal structure is yet available for MQO, we are able to say little about the mechanism of its catalytic activity, although the biochemical properties of several eukaryotic and bacterial MQOs have been studied in detail. ,, …”

Section: Introductionmentioning

confidence: 99%

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Insights into the Mechanism of Catalytic Activity of Plasmodium Parasite Malate-Quinone Oxidoreductase

Ito,

Tojo,

Fujii

et al. 2024

ACS Omega

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Plasmodium malate-quinone oxidoreductase (MQO) is a membrane flavoprotein catalyzing the oxidation of malate to oxaloacetate and the reduction of quinone to quinol. Recently, using a yeast expression system, we demonstrated that MQO, expressed in place of mitochondrial malate dehydrogenase (MDH), contributes to the TCA cycle and the electron transport chain in mitochondria, making MQO attractive as a promising drug target in Plasmodium malaria parasites, which lack mitochondrial MDH. However, there is little information on the structure of MQO and its catalytic mechanism, information that will be required to develop novel drugs. Here, we investigated the catalytic site of P. falciparum MQO (PfMQO) using our yeast expression system. We generated a model structure for PfMQO with the AI tool AlphaFold and used protein footprinting by acetylation with acetic anhydride to analyze the surface topology of the model, confirming the computational prediction to be reasonably accurate. Moreover, a putative catalytic site, which includes a possible flavin-binding site, was identified by this combination of protein footprinting and structural prediction model. This active site was analyzed by site-directed mutagenesis. By measuring enzyme activity and protein expression levels in the PfMQO mutants, we showed that several residues at the active site are essential for enzyme function. In addition, a single substitution mutation near the catalytic site resulted in enhanced sensitivity to ferulenol, an inhibitor of PfMQO that competes with malate for binding to the enzyme. This strongly supports the notion that the substrate binds to the proposed catalytic site. Then, the location of the catalytic site was demonstrated by structural comparison with a homologous enzyme. Finally, we used our results to propose a mechanism for the catalytic activity of MQO by reference to the mechanism of action of structurally or functionally homologous enzymes.

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Section: ■ Resultsmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Section: Materials and Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Insights into the Mechanism of Catalytic Activity of Plasmodium Parasite Malate-Quinone Oxidoreductase

Ito,

Tojo,

Fujii

et al. 2024

ACS Omega

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Recent advances in microbially derived chlorinated antiparasitic compounds

Saklani,

Chaudhari,

Shukla

et al. 2024

Mol Divers

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Malate dehydrogenase in parasitic protozoans: roles in metabolism and potential therapeutic applications

Springer,

Agrawal,

Chang

2024

Essays in Biochemistry

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The role of malate dehydrogenase (MDH) in the metabolism of various medically significant protozoan parasites is reviewed. MDH is an NADH-dependent oxidoreductase that catalyzes interconversion between oxaloacetate and malate, provides metabolic intermediates for both catabolic and anabolic pathways, and can contribute to NAD+/NADH balance in multiple cellular compartments. MDH is present in nearly all organisms; isoforms of MDH from apicomplexans (Plasmodium falciparum, Toxoplasma gondii, Cryptosporidium spp.), trypanosomatids (Trypanosoma brucei, T. cruzi) and anaerobic protozoans (Trichomonas vaginalis, Giardia duodenalis) are presented here. Many parasitic species have complex life cycles and depend on the environment of their hosts for carbon sources and other nutrients. Metabolic plasticity is crucial to parasite transition between host environments; thus, the regulation of metabolic processes is an important area to explore for therapeutic intervention. Common themes in protozoan parasite metabolism include emphasis on glycolytic catabolism, substrate-level phosphorylation, non-traditional uses of common pathways like tricarboxylic acid cycle and adapted or reduced mitochondria-like organelles. We describe the roles of MDH isoforms in these pathways, discuss unusual structural or functional features of these isoforms relevant to activity or drug targeting, and review current studies exploring the therapeutic potential of MDH and related genes. These studies show that MDH activity has important roles in many metabolic pathways, and thus in the metabolic transitions of protozoan parasites needed for success as pathogens.

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Plasmodium Parasite Malate-Quinone Oxidoreductase Functionally Complements a Yeast Deletion Mutant of Mitochondrial Malate Dehydrogenase

Cited by 4 publications

References 62 publications

Insights into the Mechanism of Catalytic Activity of Plasmodium Parasite Malate-Quinone Oxidoreductase

Insights into the Mechanism of Catalytic Activity of Plasmodium Parasite Malate-Quinone Oxidoreductase

Recent advances in microbially derived chlorinated antiparasitic compounds

Malate dehydrogenase in parasitic protozoans: roles in metabolism and potential therapeutic applications

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