Investigating the possible activity regulators of Leishmania major malic enzymes

Modak, Debasmita (2023) Investigating the possible activity regulators of Leishmania major malic enzymes. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (Ms dissertation of Debasmita Modak (18MS145)) Thesis_18MS145.pdf - Submitted Version Restricted to Repository staff only Download (3MB)

Abstract

Malic enzymes (MEs) catalyze the reversible oxidative decarboxylation of malate and produce pyruvate in the presence of NADP+/ NADPH co-factors and divalent metal ions like Mn+/Mg+. This oxidoreductase class of enzyme is ubiquitously present throughout all life forms and plays vital roles in metabolic pathways like fatty acid biosynthesis, redox homeostasis, TCA cycle, gluconeogenesis, etc. The reported physiological functions of MEs have been studied mostly in higher eukaryotic (like plants, mammals) and some lower eukaryotic organisms (like fungi). Thus, the lower eukaryotic MEs, like that of protists, as well as the prokaryotic MEs remain to be explored. We are studying the MEs of Leishmania major, a protozoan parasite of the Kingdom Protista. Here we have the malic enzymes from Leishmania major, which is a notorious protozoan parasite, causing the group of diseases called Leishmaniasis, to understand the role of MEs in parasite physiology. L. major encodes two isoforms of MEs, denoted as LmME1 and LmME2. LmME1 is present in mitochondria, and recent studies from our lab have found LmME2 to be present in the organism’s cytosol (unpublished). Previously our group reported that pyruvate carboxylation is the dominant activity of LmMEs inside L. major. However, our data reveal that none of the purified LmMEs follows this pattern in in vivo assays, from where we speculate that this difference might be due to the presence of activity regulators in the cellular scenario. So to check whether this alteration happens due to any activity regulations of these isoforms in the cellular environment, we performed activity assays with purified LmMEs in the presence of some shortlisted regulators like; oxaloacetate, ATP, and glucose. Interestingly we found that oxaloacetate, fumarate, and ATP inhibit both LmMEs, unlike human mitochondrial ME, which gets upregulated by fumarate. Also, our data shows that fumarate and ATP inhibit the mitochondrial isoform LmME1 in greater folds than the cytosolic LmME2. The fluorescence quenching assay confirms that the observed activity regulations are due to binding with the respective enzymes and gives us preliminary insight into the binding affinity of those regulators in the context of both the LmMEs. Such findings hint towards structural variations of LmMEs among themselves and with human MEs, which might be helpful for identifying new drug targets as well as in designing species-specific therapeutics against leishmaniasis.

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