Skip to main content
SpringerLink
Log in

Protein Motions, Dynamic Effects and Thermal Stability in Dihydrofolate Reductase from the Hyperthermophile Thermotoga maritima

  • Conference paper
Physical Biology of Proteins and Peptides

  • 911 Accesses

Abstract

Dihydrofolate reductase (DHFR) has long been used as a model system in studies of the relationship between enzyme structure and catalysis. DHFR from the hyperthermophilic bacterium Thermotoga maritima (TmDHFR) is substantially different to other chromosomal DHFRs. It is dimeric where most others are monomeric, it lacks the conformational behaviour of monomeric DHFRs, and the kinetics of the catalysed reaction are significantly different. Experimental and computational studies of TmDHFR and comparison to other DHFRs have yielded deep insights into the role of enzyme motions and dynamics in catalysis. Mutational studies and formation of hybrids between TmDHFR and a monomeric homologue have demonstrated that dimerisation is required for extreme thermostability, but also leads to an inability to adequately close the active site with detrimental effects for the speed of the catalysed reaction. However, in common with other DHFRs there is no involvement of large-scale enzyme motions in the chemical reaction itself and dynamic coupling to the reaction coordinate is efficiently minimised. Studies of DHFRs from hyperthermophilic organisms and comparisons to their mesophilic counterparts remain a rich source of information on the fundamental nature of enzyme catalysis.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Boehr DD, McElheny D, Dyson HJ, Wright PE (2006) The dynamic energy landscape of dihydrofolate reductase catalysis. Science 313(5793):1638–1642

    Article  CAS  PubMed  Google Scholar 

  2. Henzler-Wildman K, Lei M, Thai V, Kerns SJ, Karplus M, Kern D (2007) A hierarchy of timescales in protein dynamics is linked to enzyme catalysis. Nature 450(7171):913

    Article  CAS  PubMed  Google Scholar 

  3. Weikl TR, Boehr DD (2012) Conformational selection and induced changes along the catalytic cycle of Escherichia coli dihydrofolate reductase. Proteins Struct Funct Bioinf 80(10):2369–2383

    Article  CAS  Google Scholar 

  4. Wysoczanski P, Mart RJ, Loveridge EJ, Williams C, Whittaker SBM, Crump MP, Allemann RK (2012) NMR solution structure of a photoswitchable apoptosis activating Bak peptide bound to Bcl-xL. J Am Chem Soc 134(18):7644–7647

    Article  CAS  PubMed  Google Scholar 

  5. Knapp MJ, Klinman JP (2002) Environmentally coupled hydrogen tunneling—linking catalysis to dynamics. Eur J Biochem 269(13):3113–3121

    Article  CAS  PubMed  Google Scholar 

  6. Mincer JS, Schwartz SD (2004) Rate-promoting vibrations and coupled hydrogen-electron transfer reactions in the condensed phase: a model for enzyme catalysis. J Chem Phys 120:7755–7760

    Article  CAS  PubMed  Google Scholar 

  7. Antoniou D, Schwartz SD (2006) Protein dynamics and enzymatic chemical barrier passage. Philos Trans R Soc B 361:1433–1438

    Article  Google Scholar 

  8. Schwartz SD, Schramm VL (2009) Enzymatic transition states and dynamic motion in barrier crossing. Nat Chem Biol 5(8):552–559. doi:10.1038/nchembio.202

    Article  Google Scholar 

  9. Pudney CR, Hay S, Levy C, Pang J, Sutcliffe MJ, Leys D, Scrutton NS (2009) Evidence to support the hypothesis that promoting vibrations enhance the rate of an enzyme catalyzed H-tunneling reaction. J Am Chem Soc 131(47):17072–17073

    Article  CAS  PubMed  Google Scholar 

  10. Klinman JP (2009) An integrated model for enzyme catalysis emerges from studies of hydrogen tunneling. Chem Phys Lett 471:179–193

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Hay S, Scrutton NS (2012) Good vibrations in enzyme-catalysed reactions. Nat Chem 4:161–168

    Article  CAS  PubMed  Google Scholar 

  12. Pisliakov AV, Cao J, Kamerlin SCL, Warshel A (2009) Enzyme millisecond conformational dynamics do not catalyze the chemical step. Proc Natl Acad Sci U S A 106:17359–17364

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Loveridge EJ, Tey L-H, Allemann RK (2010) Solvent effects on catalysis by Escherichia coli dihydrofolate reductase. J Am Chem Soc 132(3):1137–1143

    Article  CAS  PubMed  Google Scholar 

  14. Adamczyk AJ, Cao J, Kamerlin SCL, Warshel A (2011) Catalysis by dihydrofolate reductase and other enzymes arises from electrostatic preorganization, not conformational motions. Proc Natl Acad Sci U S A 108:14115–14120

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Boekelheide N, Salomón-Ferrer R, Miller TF (2011) Dynamics and dissipation in enzyme catalysis. Proc Natl Acad Sci U S A 108(39):16159–16163. doi:10.1073/pnas.1106397108

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Loveridge EJ, Tey L-H, Behiry EM, Dawson WM, Evans RM, Whittaker SB-M, Gunther UL, Williams C, Crump MP, Allemann RK (2011) The role of large-scale motion in catalysis by dihydrofolate reductase. J Am Chem Soc 133:20561–20570

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Loveridge EJ, Behiry EM, Guo J, Allemann RK (2012) Evidence that a ‘dynamic knockout’ in Escherichia coli dihydrofolate reductase does not affect the chemical step of catalysis. Nat Chem 4(4):292–297

    Article  CAS  PubMed  Google Scholar 

  18. Luk LYP, Loveridge EJ, Allemann RK (2015) Protein motions, dynamic effects and enzyme catalysis. Phys Chem Chem Phys. doi:10.1039/C5CP00794A

  19. Cameron CE, Benkovic SJ (1997) Evidence for a functional role of the dynamics of Glycine-121 of Escherichia coli dihydrofolate reductase obtained from kinetic analysis of a site-directed mutant. Biochemistry 36:15792–15800

    Article  CAS  PubMed  Google Scholar 

  20. Radkiewicz JL, Brooks CL III (2000) Protein dynamics in enzymatic catalysis: exploration of dihydrofolate reductase. J Am Chem Soc 122(2):225–231

    Article  CAS  Google Scholar 

  21. Agarwal PK, Billeter SR, Rajagopalan PTR, Benkovic SJ, Hammes-Schiffer S (2002) Network of coupled promoting motions in enzyme catalysis. Proc Natl Acad Sci U S A 99(5):2794–2799

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Garcia-Viloca M, Truhlar DG, Gao J (2003) Reaction-path energetics and kinetics of the hydride transfer reaction catalyzed by dihydrofolate reductase. Biochemistry 42(46):13558–13575

    Article  CAS  PubMed  Google Scholar 

  23. Rod TH, Radkiewicz JL, Brooks CL III (2003) Correlated motions and the effect of distal mutation in dihydrofolate reductase catalysis. Proc Natl Acad Sci U S A 100:6980–6985

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Watney LB, Agarwal PK, Hammes-Schiffer S (2003) Effect of mutation on enzyme motion in dihydrofolate reductase. J Am Chem Soc 125:3745–3750

    Article  CAS  PubMed  Google Scholar 

  25. Pu J, Ma S, Gao J, Truhlar DG (2005) Small temperature dependence of the kinetic isotope effect for the hydride transfer reaction catalyzed by Escherichia coli dihydrofolate reductase. J Phys Chem B 109(18):8551–8556

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Wang L, Tharp S, Selzer T, Benkovic SJ, Kohen A (2006) Effects of a distal mutation on active site chemistry. Biochemistry 45(5):1383–1392. doi:10.1021/bi0518242

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Liu H, Warshel A (2007) Origin of the temperature dependence of isotope effects in enzymatic reactions: the case of dihydrofolate reductase. J Phys Chem B 111(27):7852–7861

    Article  CAS  PubMed  Google Scholar 

  28. Loveridge EJ, Allemann RK (2009) Direct methods for the analysis of quantum-mechanical tunnelling: dihydrofolate reductase. In: Quantum tunnelling in enzyme-catalysed reactions. The Royal Society of Chemistry, London, pp 179–198

    Google Scholar 

  29. Stojković V, Perissinotti LL, Lee J, Benkovic SJ, Kohen A (2010) The effect of active-site isoleucine to alanine mutation on the DHFR catalyzed H-transfer. Chem Commun 46:8974–8976

    Article  Google Scholar 

  30. Bhabha G, Lee J, Ekiert DC, Gam J, Wilson IA, Dyson HJ, Benkovic SJ, Wright PE (2011) A dynamic knockout reveals that conformational fluctuation influence the chemical step of enzyme catalysis. Science 332:234–238

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  31. Fan Y, Cembran A, Ma S, Gao J (2013) Connecting protein conformational dynamics with catalytic function as illustrated in dihydrofolate reductase. Biochemistry 52(12):2036–2049

    Article  CAS  PubMed  Google Scholar 

  32. Luk LYP, Ruiz-Pernia JJ, Dawson WM, Roca M, Loveridge EJ, Glowacki DR, Harvey JN, Mulholland AJ, Tuñón I, Moliner V, Allemann RK (2013) Unraveling the role of protein dynamics in dihydrofolate reductase catalysis. Proc Natl Acad Sci U S A 110:16344–16349

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. Ruiz-Pernia JJ, Luk LYP, García-Meseguer R, Martí S, Loveridge EJ, Tuñón I, Moliner V, Allemann RK (2013) Increased dynamic effects in a catalytically compromised variant of Escherichia coli dihydrofolate reductase. J Am Chem Soc 135(49):18689–18696

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Roston D, Kohen A, Doron D, Major DT (2014) Simulations of remote mutants of dihydrofolate reductase reveal the nature of a network of residues coupled to hydride transfer. J Comput Chem 35(19):1411

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Singh P, Sen A, Francis K, Kohen A (2014) Extension and limits of the network of coupled motions correlated to hydride transfer in dihydrofolate reductase. J Am Chem Soc 136(6):2575–2582

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Wang Z, Singh PN, Czekster CM, Kohen A, Schramm VL (2014) Protein mass-modulated effects in the catalytic mechanism of dihydrofolate reductase: beyond promoting vibrations. J Am Chem Soc 136(23):8333

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  37. Blakley RL (1984) Folates and pterins. Wiley, New York

    Google Scholar 

  38. Dams T, Bohm G, Auerbach G, Bader G, Schuring H, Jaenicke R (1998) Homo-dimeric recombinant dihydrofolate reductase from Thermotoga maritima shows extreme intrinsic stability. Biol Chem 379(3):367–371

    CAS  PubMed  Google Scholar 

  39. Dams T, Jaenicke R (1999) Stability and folding of dihydrofolate reductase from the hyperthermophilic bacterium Thermotoga maritima. Biochemistry 38(28):9169–9178

    Article  CAS  PubMed  Google Scholar 

  40. Dams T, Auerbach G, Bader G, Jacob U, Ploom T, Huber R, Jaenicke R (2000) The crystal structure of dihydrofolate reductase from Thermotoga maritima: molecular features of thermostability. J Mol Biol 297(3):659–672

    Article  CAS  PubMed  Google Scholar 

  41. Sawaya MR, Kraut J (1997) Loop and subdomain movements in the mechanism of Escherichia coli dihydrofolate reductase: crystallographic evidence. Biochemistry 36(3):586–603

    Google Scholar 

  42. Fierke CA, Johnson KA, Benkovic SJ (1987) Construction and evaluation of the kinetic scheme associated with dihydrofolate reductase from Escherichia coli. Biochemistry 26(13):4085–4092

    Article  CAS  PubMed  Google Scholar 

  43. Andrews J, Fierke CA, Birdsall B, Ostler G, Feeney J, Roberts GCK, Benkovic SJ (1989) A kinetic study of wild-type and mutant dihydrofolate reductases from Lactobacillus casei. Biochemistry 28(14):5743–5750. doi:10.1021/bi00440a007

    Article  CAS  PubMed  Google Scholar 

  44. Appleman JR, Beard WA, Delcamp TJ, Prendergast NJ, Freisheim JH, Blakley RL (1990) Unusual transient-state and steady-state kinetic behavior is predicted by the kinetic scheme operational for recombinant human dihydrofolate reductase. J Biol Chem 265(5):2740–2748

    CAS  PubMed  Google Scholar 

  45. Bhabha G, Ekiert DC, Jennewein M, Zmasek CM, Tuttle LM, Kroon G, Dyson HJ, Godzik A, Wilson IA, Wright PE (2013) Divergent evolution of protein conformational dynamics in dihydrofolate reductase. Nat Struct Mol Biol 20(11):1243–1249

    Article  CAS  PubMed  Google Scholar 

  46. Behiry EM, Luk LYP, Matthews SM, Loveridge EJ, Allemann RK (2014) Role of the occluded conformation in bacterial dihydrofolate reductases. Biochemistry 53(29):4761–4768

    Article  CAS  PubMed  Google Scholar 

  47. Hay S, Evans RM, Levy C, Loveridge EJ, Wang X, Leys D, Allemann RK, Scrutton NS (2009) Are the catalytic properties of enzymes from piezophilic organisms pressure adapted? Chembiochem 10(14):2348–2353

    Article  CAS  PubMed  Google Scholar 

  48. Maglia G, Javed MH, Allemann RK (2003) Hydride transfer during catalysis by dihydrofolate reductase from Thermotoga maritima. Biochem J 374:529–535

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  49. Maglia G, Allemann RK (2003) Evidence for environmentally coupled hydrogen tunneling during dihydrofolate reductase catalysis. J Am Chem Soc 125(44):13372–13373

    Article  CAS  PubMed  Google Scholar 

  50. Loveridge EJ, Rodriguez RJ, Swanwick RS, Allemann RK (2009) Effect of dimerization on the stability and catalytic activity of dihydrofolate reductase from the hyperthermophile Thermotoga maritima. Biochemistry 48(25):5922–5933

    Article  CAS  PubMed  Google Scholar 

  51. Guo J, Loveridge EJ, Luk LYP, Allemann RK (2013) Effect of dimerization on dihydrofolate reductase catalysis. Biochemistry 52(22):3881–3887

    Article  CAS  PubMed  Google Scholar 

  52. Tey LH, Loveridge EJ, Swanwick RS, Flitsch SL, Allemann RK (2010) Highly site-selective stability increases by glycosylation of dihydrofolate reductase. FEBS J 277(9):2171–2179

    Article  CAS  PubMed  Google Scholar 

  53. Loveridge EJ, Allemann RK (2010) The temperature dependence of the kinetic isotope effects of dihydrofolate reductase from Thermotoga maritima is influenced by intersubunit interactions. Biochemistry 49(25):5390–5396

    Article  CAS  PubMed  Google Scholar 

  54. Pang J, Allemann RK (2007) Molecular dynamics simulation of thermal unfolding of Thermatoga maritima DHFR. Phys Chem Chem Phys 9(6):711–718

    Article  CAS  PubMed  Google Scholar 

  55. Pang JY, Pu JZ, Gao JL, Truhlar DG, Allemann RK (2006) Hydride transfer reaction catalyzed by hyperthermophilic dihydrofolate reductase is dominated by quantum mechanical tunneling and is promoted by both inter- and intramonomeric correlated motions. J Am Chem Soc 128(24):8015–8023

    Article  CAS  PubMed  Google Scholar 

  56. Li L, Falzone CJ, Wright PE, Benkovic SJ (1992) Functional role of mobile loop of Escherichia coli dihydrofolate reductase in transition-state stabilization. Biochemistry 31:7826–7833

    Article  CAS  PubMed  Google Scholar 

  57. Loveridge EJ, Maglia G, Allemann RK (2009) The role of arginine 28 in catalysis by dihydrofolate reductase from the hyperthermophile Thermotoga maritima. Chembiochem 10(16):2624–2627

    Article  CAS  PubMed  Google Scholar 

  58. Kim HS, Damo SM, Lee SY, Wemmer D, Klinman JP (2005) Structure and hydride transfer mechanism of a moderate thermophilic dihydrofolate reductase from Bacillus stearothermophilus and comparison to its mesophilic and hyperthermophilic homologues. Biochemistry 44(34):11428–11439

    Article  CAS  PubMed  Google Scholar 

  59. Guo J, Luk LYP, Loveridge EJ, Allemann RK (2014) Thermal adaptation of dihydrofolate reductase from the moderate thermophile Geobacillus stearothermophilus. Biochemistry 53:2855–2863

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  60. Warshel A (1984) Dynamics of enzymatic reactions. Proc Natl Acad Sci U S A 81(2):444–448

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  61. Warshel A, Sharma PK, Kato M, Xiang Y, Liu H, Olsson MHM (2006) Electrostatic basis for enzyme catalysis. Chem Rev 106(8):3210–3235

    Article  CAS  PubMed  Google Scholar 

  62. Walser R, van Gunsteren WF (2001) Viscosity dependence of protein dynamics. Proteins Struct Funct Bioinf 42(3):414–421

    Article  CAS  Google Scholar 

  63. Affleck R, Haynes CA, Clark DS (1992) Solvent dielectric effects on protein dynamics. Proc Natl Acad Sci U S A 89(11):5167–5170

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  64. Fenimore PW, Frauenfelder H, McMahon BH, Parak FG (2002) Slaving: Solvent fluctuations dominate protein dynamics and functions. Proc Natl Acad Sci U S A 99(25):16047–16051

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  65. Loveridge EJ, Evans RM, Allemann RK (2008) Solvent effects on environmentally coupled hydrogen tunnelling during catalysis by dihydrofolate reductase from Thermotoga maritima. Chem Eur J 14(34):10782–10788

    Article  CAS  PubMed  Google Scholar 

  66. Silva RG, Murkin AS, Schramm VL (2011) Femtosecond dynamics coupled to chemical barrier crossing in a Born-Oppenheimer enzyme. Proc Natl Acad Sci U S A 108:18661–18665

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  67. Kipp DR, Silva RG, Schramm VL (2011) Mass-dependent bond vibrational dynamics influence catalysis by HIV-1 protease. J Am Chem Soc 133:19358–19361

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  68. Pudney CR, Guerriero A, Baxter NJ, Johannissen LO, Waltho JP, Hay S, Scrutton NS (2013) Fast protein motions are coupled to enzyme H-transfer reactions. J Am Chem Soc 135(7):2512–2517

    Article  CAS  PubMed  Google Scholar 

  69. Toney MD, Castro JN, Addington TA (2013) Heavy-enzyme kinetic isotope effects on proton transfer in alanine racemase. J Am Chem Soc 135(7):2509–2511

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  70. Świderek K, Javier Ruiz-Pernía J, Moliner V, Tuñón I (2014) Heavy enzymes: experimental and computational insights in enzyme dynamics. Curr Opin Chem Biol 21:11–18

    Article  PubMed  Google Scholar 

  71. Luk LYP, Loveridge EJ, Allemann RK (2014) Different dynamical effects in mesophilic and hyperthermophilic dihydrofolate reductases. J Am Chem Soc 136(19):6862

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  72. Luk LYP, Ruiz-Pernía JJ, Dawson WM, Loveridge EJ, Tuñón I, Moliner V, Allemann RK (2014) Protein isotope effects in dihydrofolate reductase from Geobacillus stearothermophilus show entropic–enthalpic compensatory effects on the rate constant. J Am Chem Soc 136(49):17317. doi:10.1021/ja5102536

    Article  CAS  PubMed  Google Scholar 

  73. Meinhold L, Clement D, Tehei M, Daniel R, Finney JL, Smith JC (2008) Protein dynamics and stability: The distribution of atomic fluctuations in thermophilic and mesophilic dihydrofolate reductase derived using elastic incoherent neutron scattering. Biochem J 94(12):4812–4818

    CAS  Google Scholar 

  74. Oyeyemi OA, Sours KM, Lee T, Resing KA, Ahn NG, Klinman JP (2010) Temperature dependence of protein motions in a thermophilic dihydrofolate reductase and its relationship to catalytic efficiency. Proc Natl Acad Sci U S A 107(22):10074–10079

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  75. Myllykallio H, Leduc D, Filee J, Liebl U (2003) Life without dihydrofolate reductase FoIA. Trends Microbiol 11(5):220–223

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to acknowledge the many contributions made by the members of the Allemann group.

This work was supported by grants BB/L020394/1 and BB/J005266/1 from the UK Biotechnology and Biological Sciences Research Council (BBSRC) and EP/L027240/1 from the UK Engineering and Physical Sciences Research Council (EPSRC). We acknowledge the continuing support by Cardiff University.

Author information

Authors and Affiliations

  1. School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff, CF10 3AT, UK

    Rudolf K. Allemann, E. Joel Loveridge & Louis Y. P. Luk

Authors
  1. Rudolf K. Allemann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. E. Joel Loveridge
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Louis Y. P. Luk
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rudolf K. Allemann .

Editor information

Editors and Affiliations

  1. Autonomous University of Mexico City, Mexico City, Distrito Federal, Mexico

    Luis Olivares-Quiroz

  2. (Iztapalapa), Autonomous Metropolitan University, Mexico City, Distrito Federal, Mexico

    Orlando Guzmán-López

  3. (Lerma), Lerma de Villada, Autonomous Metropolitan University, Mexico City, Estado de México, Mexico

    Hector Eduardo Jardón-Valadez

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer International Publishing Switzerland

About this paper

Cite this paper

Allemann, R.K., Loveridge, E.J., Luk, L.Y.P. (2015). Protein Motions, Dynamic Effects and Thermal Stability in Dihydrofolate Reductase from the Hyperthermophile Thermotoga maritima . In: Olivares-Quiroz, L., Guzmán-López, O., Jardón-Valadez, H. (eds) Physical Biology of Proteins and Peptides. Springer, Cham. https://doi.org/10.1007/978-3-319-21687-4_6

Download citation

Publish with us

Policies and ethics

Search

Navigation