Identification and Biochemical Characterisation of a novel Malic enzyme from Leishmania major

Mandal, Ardhendu (2019) Identification and Biochemical Characterisation of a novel Malic enzyme from Leishmania major. Masters thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

Leishmaniasis is a broad range of diseases caused by protozoan parasite called Leishmania. They impose major health and socioeconomic problem in human threatening around 350 million people worldwide, mainly effecting the deprived sections of world. However not much is known about the essential metabolic enzymes of the parasite, which may benefit the parasite in maintaining its redox homeostasis balance. With exception to the L.major, the reported sequenced genomes of Leishmania, have two different genes encrypting putative malic enzymes (MEs) having an identity of about 55% between them. In L.major genome the number of malic enzyme is one. Derived by the curiosity of discovering the unreported isoform of metabolically essential and novel malic enzyme in Leishmania major, we have unravel the existence of the enzyme in Leishmania major genome by simple primer designing. This indicates current genome assembly for Leishmania major perhaps misassembled during its whole genome sequencing. Further by cDNA synthesis we have confirmed the expression of the enzyme at RNA level. To further characterise the biochemical and physiological properties of the enzyme we have cloned the gene encoding sequence in bacterial and leishmanial expression system. The purified enzyme was specific for NADPH production and didn’t show interaction towards NADH even at micro molar level. Moreover, the enzyme vary from higher eukaryotic enzymatic organisation and exhibits dimeric instead of tetrameric molecular assembly. However, in both isozymes, the residues exhibited a good degree of conservation. Antibody against the purified enzyme was generated to predict the localisation and endogenous physiological fuction of the enzyme. Our findings has vindicated the existence of a novel malic enzyme isoform in L.major and shed light into its biochemical and molecular properties. The novel leishmanial ME2 isoform could potentially regulate parasite survival in the host and possibly can be a potential drug target against leishmaniasis.

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