The Protein Journal

, 27:303

Investigations of the Roles of Arginine 115 and Lysine 120 in the Active Site of 5,10-Methenyltetrahydrofolate Synthetase from Mycoplasma pneumoniae

  • Amber N. Hancock
  • R. Shane Coleman
  • Richard T. Johnson
  • Catherine A. Sarisky
  • Timothy W. Johann


5,10-Methenyltetrahydrofolate synthetase (MTHFS) catalyzes the conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate coupled to the hydrolysis of ATP. A co-crystal structure of MTHFS bound to its substrates has been published (Chen et al., Proteins 56:839–843, 2005) that provides insights into the mechanism of this reaction. To further investigate this mechanism, we have replaced the arginine at position 115 and the lysine at position 120 with alanine (R115A and K120A, respectively). Circular dichroism spectra for both mutants are consistent with folded proteins. R115A shows no activity, suggesting that R115 plays a critical role in the activity of the enzyme. The K120A mutation increases the Michaelis constant (Km) for ATP from 76 to 1,200 μM and the Km for 5-formylTHF from 2.5 to 7.1 μM. The weaker binding of substrates by K120A may be due to movement of a loop consisting of residues 117 though 120, which makes several hydrogen bonds to ATP and may be held in position by K120.


5-Formyltetrahydrofolate Site-directed mutagenesis 5,10-Methenyltetrahydrofolate synthetase ATP Kinetics 







Adenosine diphosphate


Adenosine triphosphate


Circular dichroism spectroscopy


N-Cyclohexyl-2-aminoethanesulfonic acid


Michaelis constant




2-(N-Morpholino)ethanesulfonic acid


5,10-Methenyltetrahydrofolate synthetase


Polymerase chain reaction


Phosphate buffered saline






  1. 1.
    Jolivet J, Dayan A, Beauchemin M, Chahla D, Mamo A, Bertrand R (1996) Stem Cells 14:33–40Google Scholar
  2. 2.
    Mason JB (1995) In: Bailey LB (ed) Folate in health and disease. Marcel Dekker, Inc., New York, pp 329–360Google Scholar
  3. 3.
    Bailey LB, Gregory JF (1999) J Nutr 129:919–922Google Scholar
  4. 4.
    Scott JM, Weir DG, Kirke PN (1995). In: Bailey LB (ed) Folate in health and disease. Marcel Dekker, Inc., New York, pp 329–360Google Scholar
  5. 5.
    Kruschwitz HL, McDonald D, Cossins EA, Schirch V (1994) J Biol Chem 269:28757–28763Google Scholar
  6. 6.
    Huang T, Schirch V (1995) J Biol Chem 270:22296–22300CrossRefGoogle Scholar
  7. 7.
    Kounga K, Vander Velde DG, Himes RH (1995) FEBS Lett 364:215–217CrossRefGoogle Scholar
  8. 8.
    Field MS, Szebenyi DME, Perry CA, Stover PJ (2007) Arch Biochem Biophys 458:194–201CrossRefGoogle Scholar
  9. 9.
    Chen S, Yakunin AF, Proudfoot M, Kim R, Kim S (2005) Proteins 61:433–443CrossRefGoogle Scholar
  10. 10.
    Chen S, Shin D, Purfan R, Kim R, Kim S (2004) Proteins 56:839–843CrossRefGoogle Scholar
  11. 11.
    Meier C, Carter LG, Winter G, Owens RJ, Stuart DI, Esnouf RM (2007) Acta Cryst F63:168–172Google Scholar
  12. 12.
    Koradi R, Billeter M, Wüthrich K (1996) J Mol Graph 14:51–55CrossRefGoogle Scholar
  13. 13.
    Kim R, Sandler SJ, Goldman S, Yokota H, Clark AJ, Kim S (1998) Biotechnol Lett 20:207–210CrossRefGoogle Scholar
  14. 14.
    Inoue H, Nojima H, Okayama H (1990) Gene 96:23–28CrossRefGoogle Scholar
  15. 15.
    Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor pp A8.46–A8.47Google Scholar
  16. 16.
    Jolivet J (1997) Meth Enzymol 281:162–170CrossRefGoogle Scholar
  17. 17.
    Fersht A (1985) Enzyme structure and mechanism, 2nd edn. W. H. Freeman and Company, New YorkGoogle Scholar
  18. 18.
    Gorry PA (1990) Anal Chem 62:570–573CrossRefGoogle Scholar
  19. 19.
    Sreerama N, Woody RW (2000) Anal Biochem 287:252–260CrossRefGoogle Scholar
  20. 20.
    Johnson WC (1999) Proteins: Str Func Genet 35:307–312CrossRefGoogle Scholar
  21. 21.
    Provencher SW, Glockner J (1981) Biochemistry 20:33–37CrossRefGoogle Scholar
  22. 22.
    Sreerama N, Venyaminov SY, Woody RW (1999) Protein Sci 8:370–380Google Scholar
  23. 23.
    Anantharaman V, Aravind L (2006) J Mol Biol 356:823–842CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Amber N. Hancock
    • 1
  • R. Shane Coleman
    • 2
  • Richard T. Johnson
    • 2
  • Catherine A. Sarisky
    • 3
  • Timothy W. Johann
    • 2
  1. 1.Department of ChemistryVirginia TechBlacksburgUSA
  2. 2.Department of Chemistry and PhysicsRadford UniversityRadfordUSA
  3. 3.Department of ChemistryRoanoke CollegeSalemUSA

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