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

  • ️Wed Jan 01 2020

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

Purna Bahadur Chetri et al. Sci Rep. 2020.

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1

Evolutionary sequence analysis. (A) Multiple sequence alignment of FgMDH with MDH from other organisms (Fasciola hepatica (THD24885.1), Echinostoma caproni (VDP72865.1), Opisthorchis viverrini (OON21665.1), Clonorchis sinensis (AAT46071.1), Schistosoma margrebowiei (VDO56091.1), Schistosoma matthaei (VDP64368.1), Schistosoma bovis (RTG88342.1), Schistosoma mansoni (XP_018647879.1), Schistosoma japonicum (CAX722031.1), and Homo sapiens (NP_005908.1). The alignment was generated by ClustalW algorithm. The red boxes show identical amino acids; yellow boxes show similar amino acids, while the amino acids with different properties have no boxes. The blue box indicates the active site residues. (B) Phylogenetic relation of the FgMDH. The Neighbour Joining method was used to construct the phylogenetic tree by comparing amino acid sequences by using MEGA V10.0 software. The values represent the evolutionary distance among different species.

Figure 2
Figure 2

Overexpression, purification, and oligomeric status of recombinant FgMDH. (A) SDS-PAGE gel showing the overexpressed and purified protein. Lanes 1–3 represent molecular weight markers, uninduced cell lysate, induced cell lysate, and lane 4–5 represent purified FgMDH, respectively. (B) GFC profile showing the oligomeric status and molecular mass of FgMDH. The column calibration was performed with the gel filtration calibration kit containing aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), carbonic anhydrase (29 kDa), ribonuclease (13.7 kDa), and aprotinin (6.5 kDa).

Figure 3
Figure 3

Enzymatic reactions and Michaelis–Menten kinetic plots. (A) Schematic diagram of the forward and reverse reactions catalyzed by FgMDH. (B–E) Michaelis–Menten Kinetic plots for (B) malate, (C) NAD+, (D) oxaloacetate, and (E) NADH. Enzyme activities were taken in both forward and reverse directions, keeping one substrate/cofactor constant while varying the other.

Figure 4
Figure 4

pH and temperature dependent catalytic activity of FgMDH. (A) Effect of pH on the catalytic activity of FgMDH. (B) Effect of temperature on the catalytic activity of FgMDH.

Figure 5
Figure 5

Structural features of recombinant FgMDH. (A) Intrinsic tryptophan fluorescence spectrum. (B) Far-UV CD spectrum.

Figure 6
Figure 6

Structural comparison of FgMDH and HsMDH. Structural alignment of FgMDH (cyan) with HsMDH (tan) generates an RMSD of 0.241 Å for 326 Cα atoms. High RMSD value indicates lower structural similarity. The inset shows the comparison of the conformation of the active site loop region of FgMDH and HsMDH.

Figure 7
Figure 7

Molecular dynamics simulations analyses of FgMDH and FgMDH–malate complex. (A) RMSD of the backbone Cα atoms. (B) RMSF of Cα atoms of last 50 ns MD trajectories. (C) Number of hydrogen bonds for the last 50 ns time period. (D) The radius of gyration vs. time. (E) The first 50 PCs vs. eigenvectors are shown. (F) Solvent accessible surface area.

Figure 8
Figure 8

Interaction of substrates and cofactors with FgMDH. The dotted line represents the H-bond with (A) Malate, (B) Oxaloacetate, (C) NAD+, and (D) NADH.

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