Identification and characterization of cytosolic malate dehydrogenase from the liver fluke Fasciola gigantica

Abstract

The liver fluke zoonoses, Fasciola spp. are parasitic helminths infecting humans and animals globally. Recent sequencing of the genome of Fasciola gigantica has provided a basis to understand the biochemistry of this parasite. Here, we identified the cytosolic malate dehydrogenase in F. gigantica (FgMDH) and characterized the enzyme biochemically and structurally. F. gigantica encodes a single cytosolic MDH, a key enzyme of the citric acid cycle. It catalyzes the reversible oxidation of malate to oxaloacetate using NAD +. The Fgmdh gene was amplified and cloned for expression of the recombinant protein. The purified protein showed a molecular weight of ~ 36 kDa that existed in a dimeric form in solution. The recombinant enzyme was catalytically active as it catalyzed both forward and reverse reactions efficiently. The kinetic parameters were determined for both directions. The structure of FgMDH and human MDH were modeled and validated. The superimposition of both the model structures showed overall structural similarity in the active site loop region, however, the conformation of the residues was different. Molecular docking elucidated the binding sites and affinities of the substrates and cofactors to the enzyme. Simulation of molecular dynamics and principal component analysis indicated the stability of the systems and collective motions, respectively. Understanding the structural and functional properties of MDH is important to better understand the roles of this enzyme in the biochemistry of the parasite. Two parasitic liver flukes, Fasciola gigantica and Fasciola hepatica are responsible for fascioliasis-a severe zoonotic disease that is financially detrimental to the global livestock industry 1. WHO classifies fascioliasis as a neglected tropical disease of economic importance 2. It is a major foodborne disease caused by the consumption of food/water contaminated with the parasites in their larval stage. It is primarily a veterinary disease that infects a variety of livestock, especially goat, sheep, and cattle 3-5 ; globally, over 600 million animals are affected by these flukes, resulting in loss of > US$3 billion p.a. F. gigantica is prevalent in tropical regions, including Africa and South and Southeast Asia, while F. hepatica is mostly found in temperate areas, including Europe, North and South America, and Australia 6. F. gigantica infects nearly 25-100% cattle annually in Africa, the Middle East, Southeast Asia, and other temperate countries 7. The global prevalence of fascioliasis is increasing in humans. As reported by WHO, nearly 180 million people are at risk, while 2.4-17 million people are infected globally 8. Human fascioliasis is primarily defined to Africa, Europe, the Middle East (including Egypt), Southeast Asia, and Latin America with the highest prevalence in Bolivian Plateau 9,10. The common symptoms of fascioliasis are enlarged liver and spleen, arthralgias, facial swelling, fever, abdominal pain, eosinophilia, and eggs in stool 11. The only drug of choice recommended by WHO is tricla...

Kinetic characterization and thermostability ofC. eleganscytoplasmic and mitochondrial malate dehydrogenases

“…More divergent results were found for recombinant cytosolic MDH from the liver fluke Fasciola gigantica. This enzyme had a K M for oxaloacetate and NADH of 276 μM and 141 μM, respectively (5). Other MDH enzymes from invertebrates had a wide range of K M and specific activity values, but for most of these studies, the results couldn't be directly compared to our results because the reaction conditions were too different.…”

“…We attempted to do enzyme kinetics with malate and NAD + with recombinant MDH-1 and MDH-2 under a variety of reaction conditions. Specifically, we tried higher concentrations of each reagent and higher pH values, because these conditions have worked for some MDH enzymes (5,6,62). However, we were not able to observe any enzyme activity with malate and NAD + under any of the conditions we tried (data not shown).…”

“…MDH from many different organisms ranging from archaea to plants to mammals have been studied previously (reviewed in (1)(2)(3)). Studies of MDH enzymes from invertebrates have been done, and most of these involved parasitic tapeworms and flukes (4)(5)(6)(7)(8)(9). In this study, we characterized Caenorhabditis elegans MDH enzymes to determine how the enzymes may be optimized to function in an organism that can withstand intermittent harsh environmental conditions.…”

A global phosphoproteomics analysis of adult Fasciola gigantica by LC–MS/MS

“…According to KEGG annotation, nearly 7% of phosphoproteins indicating multiple roles in different biological processes were predicted to be annotated in more than 5 KEGG pathways (Supplementary Table 9 ). Therein, malate dehydrogenase (MDH) of F. gigantica was a single cytosolic enzyme to catalyze the reversible oxidation of malate to oxaloacetate using NAD + (Chetri et al 2020 ). Been as an isoenzyme, the pattern of FgMDH was the same as FhMDH due to similar relative mobilities (Sarkari et al 2016 ).…”

“…Been as an isoenzyme, the pattern of FgMDH was the same as FhMDH due to similar relative mobilities (Sarkari et al 2016 ). The superimposition structure model of FgMDH and human MDH showed overall structural similarity in the active site loop region, while the conformation of the residues was different (Chetri et al 2020 ). Combining the structure model and our annotations in FgMDH in metabolic pathways such as pyruvate metabolism, cysteine and methionine metabolism, and glyoxylate and dicarboxylate metabolism, the roles of FgMDH can be used to better understand the biochemistry of F. gigantica .…”