Identification and characterization of glyceraldehyde 3-phosphate dehydrogenase from Fasciola gigantica

  • Purna B. Chetri
  • Rohit Shukla
  • Timir TripathiEmail author
Helminthology - Original Paper


Fasciola gigantica is an important food-borne trematode responsible for the hepatobiliary disease, commonly known as fascioliasis. In F. gigantica, the glyceraldehyde 3-phosphate dehydrogenase (FgGAPDH) is a key enzyme of the glycolytic pathway and catalyzes the reversible oxidative phosphorylation of d-glyceraldehyde-3-phosphate (G-3-P) to 1,3-bisphosphoglycerate (1,3-BPG), with the simultaneous reduction of NAD+ to NADH. In the present study, we analyzed the sequence of FgGAPDH and investigated its structural, binding, and catalytic properties. Sequence alignment of FgGAPDH showed 100% identity with the sister fluke Fasciola hepatica GAPDH. The gapdh gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified. The purified FgGAPDH exists as a homo-tetramer, composed of a ~ 37-kDa subunit under non-dissociating conditions at 300 mM salt concentration indicating that higher salt stabilizes the tetrameric state. The binding of the cofactor NAD+ caused a conformational rearrangement in the enzyme structure, leading to the stabilization of the enzyme. A homology model of FgGAPDH was constructed, the cofactor (NAD+) and substrate (G-3-P) were docked, and the binding sites were identified in a single chain. The inter-subunit cleft of GAPDH that has been exploited for structure-based drug design in certain protozoan parasites is closed in the case of FgGAPDH, similar to the human GAPDH. Thus, the conformation of FgGAPDH in this region is similar to the human enzyme. Therefore, GAPDH may not be a suitable target for drug discovery against fascioliasis. Still, the analysis of the structural and functional attributes of GAPDH will be significant in understanding the various roles of this enzyme in the parasite as well as provide new insights into the biochemistry of flukes.


Fasciola gigantica Liver fluke Glyceraldehyde 3-phosphate dehydrogenase Activity Modeling Docking Quenching 



Glyceraldehyde 3-phosphate dehydrogenase


F. gigantica glyceraldehyde 3-phosphate dehydrogenase


Size exclusion chromatography






(4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid)


Authors’ contributions

PBC and RS carried out all the experiments. PBC, RS, and TT analyzed the data, conceived the study, and participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Competing interests

The authors declare that there are no competing interests.

Supplementary material

436_2019_6225_MOESM1_ESM.docx (2 mb)
ESM 1 (DOCX 2028 kb)


  1. Anderson N, Luong TT, Vo NG, Bui KL, Smooker PM, Spithill TW (1999) The sensitivity and specificity of two methods for detecting Fasciola infections in cattle. Vet Parasitol 83(1):15–24CrossRefGoogle Scholar
  2. Aronov AM, Suresh S, Buckner FS, van Voorhis WC, Verlinde CLMJ, Opperdoes FR, Hol WGJ, Gelb MH (1999) Structure-based design of submicromolar, biologically active inhibitors of trypanosomatid glyceraldehyde-3-phosphate dehydrogenase. Proc Natl Acad Sci U S A 96(8):4273–4278CrossRefGoogle Scholar
  3. Ashmarina LI, Muronetz VI, Nagradova NK (1981) Immobilized D-glyceraldehyde-3-phosphate dehydrogenase can exist as a trimer. FEBS Lett 128(1):22–26CrossRefGoogle Scholar
  4. Baker BY, Shi W, Wang B, Palczewski K (2014) High-resolution crystal structures of the photoreceptor glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with three and four-bound NAD molecules. Protein Sci 23(11):1629–1639CrossRefGoogle Scholar
  5. Baxi MD, Vishwanatha JK (1995) Uracil DNA-glycosylase/glyceraldehyde-3-phosphate dehydrogenase is an Ap4A binding protein. Biochemistry 34(30):9700–9707CrossRefGoogle Scholar
  6. Bero J, Beaufay C, Hannaert V, Herent MF, Michels PA, Quetin-Leclercq J (2013) Antitrypanosomal compounds from the essential oil and extracts of Keetia leucantha leaves with inhibitor activity on Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase. Phytomedicine 20(3–4):270–274. CrossRefGoogle Scholar
  7. Biu AA, Ahmed MI, Mshelia SS (2006) Economic assessment of losses due to parasitic diseases common at the Maiduguri abattoir, Nigeria. vol 7, p 143–145Google Scholar
  8. Bowie JU, Luthy R, Eisenberg D (1991) A method to identify protein sequences that fold into a known three-dimensional structure. Science 253(5016):164–170CrossRefGoogle Scholar
  9. Carlile GW, Chalmers-Redman RM, Tatton NA, Pong A, Borden KE, Tatton WG (2000) Reduced apoptosis after nerve growth factor and serum withdrawal: conversion of tetrameric glyceraldehyde-3-phosphate dehydrogenase to a dimer. Mol Pharmacol 57(1):2–12Google Scholar
  10. Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31(13):3497–3500CrossRefGoogle Scholar
  11. Colovos C, Yeates TO (1993) Verification of protein structures: patterns of nonbonded atomic interactions. Protein Sci 2(9):1511–1519CrossRefGoogle Scholar
  12. Cywinska A (2005) Epidemiology of fascioliasis in human endemic areas. J Helminthol 79(3):207–216CrossRefGoogle Scholar
  13. Dell'Antone P (2009) Targets of 3-bromopyruvate, a new, energy depleting, anticancer agent. Med Chem 5(6):491–496CrossRefGoogle Scholar
  14. Elshraway NT, Mahmoud WG (2017) Prevalence of fascioliasis (liver flukes) infection in cattle and buffaloes slaughtered at the municipal abattoir of El-Kharga, Egypt. Vet World 10(8):914–917. CrossRefGoogle Scholar
  15. Ferreira-da-Silva F, Pereira PJB, Gales L, Roessle M, Svergun DI, Moradas-Ferreira P, Damas AM (2006) The crystal and solution structures of glyceraldehyde-3-phosphate dehydrogenase reveal different quaternary structures. J Biol Chem 281(44):33433–33440CrossRefGoogle Scholar
  16. Fothergill-Gilmore LA, Michels PA (1993) Evolution of glycolysis. Prog Biophys Mol Biol 59(2):105–235CrossRefGoogle Scholar
  17. France RM, Grossman SH (2000) Acrylamide quenching of apo- and holo-alpha-lactalbumin in guanidine hydrochloride. Biochem Biophys Res Commun 269(0006-291X (Print)):709–712CrossRefGoogle Scholar
  18. Frayne J, Taylor A, Cameron G, Hadfield AT (2009) Structure of insoluble rat sperm glyceraldehyde-3-phosphate dehydrogenase (GAPDH) via heterotetramer formation with Escherichia coli GAPDH reveals target for contraceptive design. J Biol Chem 284(34):22703–22712CrossRefGoogle Scholar
  19. Ganapathy-Kanniappan S et al (2009) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is pyruvylated during 3-bromopyruvate mediated cancer cell death. Anticancer Res 29(12):4909–4918Google Scholar
  20. Ganapathy-Kanniappan S, Kunjithapatham R, Geschwind JF (2013) Anticancer efficacy of the metabolic blocker 3-bromopyruvate: specific molecular targeting. Anticancer Res 33(1):13–20Google Scholar
  21. Goodsell DS, Morris GM, Olson AJ (1996) Automated docking of flexible ligands: applications of AutoDock. J Mol Recognit 9(1):1–5CrossRefGoogle Scholar
  22. Harris JI, Waters M (1976) 1 Glyceraldehyde-3-phosphate dehydrogenase. Enzymes 13:1–49CrossRefGoogle Scholar
  23. Hoagland VD Jr, Teller DC (1969) Influence of substrates on the dissociation of rabbit muscle D-glyceraldehyde 3-phosphate dehydrogenase. Biochemistry 8(2):594–602CrossRefGoogle Scholar
  24. Jenkins JL, Tanner JJ (2006) High-resolution structure of human D-glyceraldehyde-3-phosphate dehydrogenase. Acta Crystallogr D Biol Crystallogr 62(Pt 3):290–301CrossRefGoogle Scholar
  25. Krebs EG (1955) Glyceraldehyde-3-phosphate dehydrogenase from yeast. Methods Enzymol 1:407–411CrossRefGoogle Scholar
  26. Lakatos S, Zavodsky P (1976) The effect of substrates on the association equilibrium of mammalian D-glyceraldehyde 3-phosphate dehydrogenase. FEBS Lett 63(1):145–148CrossRefGoogle Scholar
  27. Laskowski RA, Hutchinson EG, Michie AD, Wallace AC, Jones ML, Thornton JM (1997) PDBsum: a web-based database of summaries and analyses of all PDB structures. Trends Biochem Sci 22(12):488–490CrossRefGoogle Scholar
  28. Luthy R, Bowie JU, Eisenberg D (1992) Assessment of protein models with three-dimensional profiles. Nature 356(6364):83–85. CrossRefGoogle Scholar
  29. Mas-Coma S, Funatsu IR, Bargues MD (2001) Fasciola hepatica and lymnaeid snails occurring at very high altitude in South America. Parasitology 123(Suppl):S115–S127Google Scholar
  30. Mazzola JL, Sirover MA (2001) Reduction of glyceraldehyde-3-phosphate dehydrogenase activity in Alzheimer’s disease and in Huntington’s disease fibroblasts. J Neurochem 76(2):442–449CrossRefGoogle Scholar
  31. Möller M, Denicola A (2002) Protein tryptophan accessibility studied by fluorescence quenching. Biochem Mol Biol Educ 30(3):175–178. CrossRefGoogle Scholar
  32. Nelson D, Goldstein JM, Boatright K, Harty DWS, Cook SL, Hickman PJ, Potempa J, Travis J, Mayo JA (2001) pH-regulated secretion of a glyceraldehyde-3-phosphate dehydrogenase from Streptococcus gordonii FSS2: purification, characterization, and cloning of the gene encoding this enzyme. J Dent Res 80(1):371–377CrossRefGoogle Scholar
  33. Osborne HH, Hollaway MR (1976) An investigation of the nicotinamide-adenine dinucleotide-induced ‘tightening’ of the structure of glyceraldehyde 3-phosphate dehydrogenase. Biochem J 157(1):255–259CrossRefGoogle Scholar
  34. Pathak RK, Baunthiyal M, Shukla R, Pandey D, Taj G, Kumar A (2017) In silico identification of mimicking molecules as defense inducers triggering jasmonic acid mediated immunity against Alternaria blight disease in Brassica species. Front Plant Sci 8:609. CrossRefGoogle Scholar
  35. Pawlowski M, Gajda MJ, Matlak R, Bujnicki JM (2008) MetaMQAP: a meta-server for the quality assessment of protein models. BMC Bioinformatics 9:403. CrossRefGoogle Scholar
  36. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25(13):1605–1612CrossRefGoogle Scholar
  37. Robert X, Gouet P (2014) Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 42(Web Server issue):W320–W324. CrossRefGoogle Scholar
  38. Saunders PA, Chen RW, Chuang DM (1999) Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase isoforms during neuronal apoptosis. J Neurochem 72(3):925–932CrossRefGoogle Scholar
  39. Schultz DE, Hardin CC, Lemon SM (1996) Specific interaction of glyceraldehyde 3-phosphate dehydrogenase with the 5′-nontranslated RNA of hepatitis A virus. J Biol Chem 271(24):14134–14142CrossRefGoogle Scholar
  40. Shukla H, Shukla R, Sonkar A, Pandey T, Tripathi T (2017a) Distant Phe345 mutation compromises the stability and activity of Mycobacterium tuberculosis isocitrate lyase by modulating its structural flexibility. Sci Rep 7(1):1058. CrossRefGoogle Scholar
  41. Shukla H, Shukla R, Sonkar A, Tripathi T (2017b) Alterations in conformational topology and interaction dynamics caused by L418A mutation leads to activity loss of Mycobacterium tuberculosis isocitrate lyase. Biochem Biophys Res Commun 490:276–282. CrossRefGoogle Scholar
  42. Shukla R, Shukla H, Tripathi T (2018) Activity loss by H46A mutation in Mycobacterium tuberculosis isocitrate lyase is due to decrease in structural plasticity and collective motions of the active site. Tuberculosis (Edinb) 108:143–150CrossRefGoogle Scholar
  43. Singh R, Green MR (1993) Sequence-specific binding of transfer RNA by glyceraldehyde-3-phosphate dehydrogenase. Science 259(5093):365–368CrossRefGoogle Scholar
  44. Sirover MA (1999) New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase. Biochim Biophys Acta 1432(2):159–184CrossRefGoogle Scholar
  45. Soares FA, Sesti-Costa R, da Silva JS, de Souza MCBV, Ferreira VF, da C. Santos F, Monteiro PAU, Leitão A, Montanari CA (2013) Molecular design, synthesis and biological evaluation of 1,4-dihydro-4-oxoquinoline ribonucleosides as TcGAPDH inhibitors with trypanocidal activity. Bioorg Med Chem Lett 23(16):4597–4601. CrossRefGoogle Scholar
  46. Van de Weert M, Stella L (2011) Fluorescence quenching and ligand binding: a critical discussion of a popular methodology. J Mol Struct 998(1–3):144–150. CrossRefGoogle Scholar
  47. Webb B, Sali A (2014) Comparative protein structure modeling using MODELLER. Curr Protoc Bioinformatics 47:5 6 1–5 632. CrossRefGoogle Scholar
  48. WHO (2015) WHO estimates of the global burden of foodborne diseases: foodborne disease burden epidemiology reference group 2007–2015. World Health OrganizationGoogle Scholar
  49. Wiederstein M, Sippl MJ (2007) ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res 35(Web Server):W407–W410. CrossRefGoogle Scholar
  50. Zinsser VL, Hoey EM, Trudgett A, Timson DJ (2014) Biochemical characterisation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from the liver fluke, Fasciola hepatica. Biochim Biophys Acta 1844(4):744–749. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Molecular and Structural Biophysics Laboratory, Department of BiochemistryNorth-Eastern Hill UniversityShillongIndia

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