Molecular Characterization of Trypanosoma evansi Mevalonate Kinase (TeMVK)

Abstract

The mevalonate pathway is an essential part of isoprenoid biosynthesis leading to production of a diverse class of >30,000 biomolecules including cholesterol, heme, and all steroid hormones. In trypanosomatids, the mevalonate pathway also generates dolichols, which play an essential role in construction of glycosylphosphatidylinositol (GPI) molecules that anchor variable surface proteins (VSGs) to the plasma membrane. Isoprenoid biosynthesis involves one of the most highly regulated enzymes in nature, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), which catalyzes the conversion of HMG-CoA to mevalonic acid. The enzyme mevalonate kinase (MVK) subsequently converts mevalonic acid to 5-phosphomevalonic acid. Trypanosoma evansi is a flagellate protozoan parasite that causes the disease “Surra” in domesticated large mammals, with great economic impact. T. evansi has only a trypomastigote bloodstream form and requires constant modification of the variant surface glycoprotein (VSG) coat for protection against the host immune system. We identified MVK of T. evansi (termed TeMVK) and performed a preliminary characterization at molecular, biochemical, and cellular levels. TeMVK from parasite extract displayed molecular weight ~36 kDa, colocalized with aldolase (a glycosomal marker enzyme) in glycosomes, and is structurally similar to Leishmania major MVK. Interestingly, the active form of TeMVK is the tetrameric oligomer form, in contrast to other MVKs in which the dimeric form is active. Despite lacking organized mitochondria, T. evansi synthesizes both HMGCR transcripts and protein. Both MVK and HMGCR are expressed in T. evansi during the course of infection in animals, and therefore are potential targets for therapeutic drug design.

Mechanistic insight into the role of mevalonate kinase by a natural fatty acid-mediated killing of Leishmania donovani

“…The pyruvate kinase–lactate dehydrogenase coupled assay system was used for the measurement of the activity of MevK where a decrease in absorbance of NADH at 340 nm is correlated with increased enzyme activity 28 , 29 . Recombinant MevK activity was represented as fold change in NADH oxidation 30 in presence of increasing concentration (0.5–8 mM) of R-mevalonate (Fig. 7 B).…”

“…The MevK assay was carried out with slight modification as described earlier 30 . The activity of the recombinant LD-MevK assay was assessed in a buffer containing 100 mM glycine, 25 mM KCl, 4 U LDH, 4 U pyruvate dehydrogenase, 1 mM phosphoenol pyruvate, 5 mM ATP, 5 mM MgCl 2 , R-mevalonate (0.5–8 mM), 160 µM β-NADH and 30–50 μM Ld-MevK at 37 °C for 10 min.…”

Leishmania donovani Secretory Mevalonate Kinase Regulates Host Immune Response and Facilitates Phagocytosis

“…Enzymatic activity of r-LdMVK was measured by methods described earlier ( Ferreira et al., 2016 ; Duarte et al., 2018 ); by relating ADP release with oxidation of NADH by pyruvate kinase and lactate dehydrogenase. With increasing concentration of MVK, change in absorbance with respect to time increased linearly ( Figure 2I ).…”

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“…Enzymatic activity of r-LdMVK was measured by methods described earlier (Ferreira et al, 2016;Duarte et al, 2018); by relating ADP release with oxidation of NADH by pyruvate kinase and lactate dehydrogenase. With increasing concentration of MVK, change in absorbance with respect to time increased linearly (Figure 2I).…”