Biochemical Studies of Mitochondrial Malate: Quinone Oxidoreductase from Toxoplasma gondii

Abstract

Toxoplasma gondii is a protozoan parasite that causes toxoplasmosis and infects almost one-third of the global human population. A lack of effective drugs and vaccines and the emergence of drug resistant parasites highlight the need for the development of new drugs. The mitochondrial electron transport chain (ETC) is an essential pathway for energy metabolism and the survival of T. gondii. In apicomplexan parasites, malate:quinone oxidoreductase (MQO) is a monotopic membrane protein belonging to the ETC and a key member of the tricarboxylic acid cycle, and has recently been suggested to play a role in the fumarate cycle, which is required for the cytosolic purine salvage pathway. In T. gondii, a putative MQO (TgMQO) is expressed in tachyzoite and bradyzoite stages and is considered to be a potential drug target since its orthologue is not conserved in mammalian hosts. As a first step towards the evaluation of TgMQO as a drug target candidate, in this study, we developed a new expression system for TgMQO in FN102(DE3)TAO, a strain deficient in respiratory cytochromes and dependent on an alternative oxidase. This system allowed, for the first time, the expression and purification of a mitochondrial MQO family enzyme, which was used for steady-state kinetics and substrate specificity analyses. Ferulenol, the only known MQO inhibitor, also inhibited TgMQO at IC50 of 0.822 μM, and displayed different inhibition kinetics compared to Plasmodium falciparum MQO. Furthermore, our analysis indicated the presence of a third binding site for ferulenol that is distinct from the ubiquinone and malate sites.

Biochemical characterization and identification of ferulenol and embelin as potent inhibitors of malate:quinone oxidoreductase from Campylobacter jejuni

“…These α values suggest that the two inhibitors had the potential to bind to both free CjMQO as well as CjMQO/substrates complex with slightly high preference for free CjMQO in case of inhibitor versus malate. Although this mechanism is consistent with previously reported inhibition mechanisms for ferulenol on TgMQO (Acharjee et al, 2021), it contrasts with the inhibition mechanism reported for PfMQO (Hartuti et al, 2018). The noncompetitive and mixed-type of inhibition suggest that 1) the concentration of the substrate does not influence the binding capacity of the two inhibitors, and 2) they 3) and using graphical methods (Supplementary Figures 12, 13), further supports that embelin and ferulenol were noncompetitive inhibitors versus quinone and mixed-type versus malate.…”

“…The optimal temperature for the activity of the purified CjMQO was 40 °C; however, the enzyme retained 60% and 93% of its maximal activity at ambient (25 °C) and human body (37 °C) temperatures, respectively (Figure 2A). This optimal temperature was lower than values published for MQOs from T. gondii (50 °C), thermophilic (Ohshima and Tanaka, 1993;Kabashima et al, 2013;Acharjee et al, 2021). It was superior to the temperature reported for P. taetrolens MQO (30 °C) and P. falciparum MQO (37 °C) (Hartuti et al, 2018;Oh et al, 2020).…”

“…The reaction mechanism observed for MQOs are unusual amongst other ETC dehydrogenases, such as dihydroorotate dehydrogenase and Type 2 NADH dehydrogenase, which show a ping-pong mechanism of catalysis (Takashima et al, 2002;Yano et al, 2014). Considering the α-value higher than 1 for the observed mixed-type inhibition of ferulenol and embelin versus malate, and the noncompetitive nature versus quinone, this would suggest the binding of malate occurs first followed by the binding of quinones, which is the opposite binding sequence to what was observed for mitochondrial MQOs (Hartuti et al, 2018;Acharjee et al, 2021). In such case, the two electrons from malate are first transferred to the FAD, producing FADH 2 , and then to the quinone, with bound FAD functioning as the redox centre.…”

Parasite powerhouse: A review of the Toxoplasma gondii mitochondrion

“…MQO, found in many bacteria and single-celled eukaryotes, catalyzes the conversion of malate to oxaloacetate as part of the TCA cycle. While MQO catalyzes this conversion in an irreversible reaction and reduces CoQ, the canonical mammalian malate dehydrogenase (MDH) catalyzes the same reaction reversibly and ultimately reduces NAD+ (Acharjee et al, 2021). T. gondii also expresses MDH and localization studies found that MQO and MDH both localize to the mitochondrion (Fleige et al, 2008).…”

“…Furthermore, a study revealed that these molecules also target dihydroorotate dehydrogenase (DHODH) and it is likely that the simultaneous inhibition of NDH2 and DHODH by HDQs impairs parasite growth (Hegewald et al, 2013). Finally, a recently published study demonstrated that the apicomplexan MQO, a mitochondrial enzyme with no orthologues in mammals, could represent a potential drug target (Acharjee et al, 2021). In T. gondii, MQO activity can be inhibited with ferulenol, and in P. falciparum inhibition is more pronounced in combination with atovaquone (Hartuti et al, 2018).…”