Abstract
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.
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Abbreviations
- 5-formylTHF:
-
5-Formyltetrahydrofolate
- 5,10-methenylTHF:
-
5,10-Methenyltetrahydrofolate
- ADP:
-
Adenosine diphosphate
- ATP:
-
Adenosine triphosphate
- CD:
-
Circular dichroism spectroscopy
- HEPES:
-
N-Cyclohexyl-2-aminoethanesulfonic acid
- Km :
-
Michaelis constant
- LB:
-
Luria-Bertani
- MES:
-
2-(N-Morpholino)ethanesulfonic acid
- MTHFS:
-
5,10-Methenyltetrahydrofolate synthetase
- PCR:
-
Polymerase chain reaction
- PBS:
-
Phosphate buffered saline
- THF:
-
Tetrahydrofolate
- UV:
-
Ultraviolet
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Acknowledgments
We would like to thank Mark Bouley (Radford Univeristy) for help with purification protocols, Rosalind Kim (Structural Genomics Center) for advice in expression and purification of wild type MTHFS, and Sung-Hou Kim (Structural Genomics Center) for the generous gifts of the plasmid containing the MTHFS gene and the BL21(DE3)/pSJS1244 expression line for the protein. This work was funded through the Jeffress Memorial Trust (J-788) and Radford University internal grants. DNA sequencing was supported through the LI-COR Biosciences Genomics Education Matching Fund Program.
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Hancock, A.N., Coleman, R.S., Johnson, R.T. et al. Investigations of the Roles of Arginine 115 and Lysine 120 in the Active Site of 5,10-Methenyltetrahydrofolate Synthetase from Mycoplasma pneumoniae . Protein J 27, 303–308 (2008). https://doi.org/10.1007/s10930-008-9138-z
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DOI: https://doi.org/10.1007/s10930-008-9138-z