Investigations of Amino Acids in the 5-Formyltetrahydrofolate Binding Site of 5,10-Methenyltetrahydrofolate Synthetase from Mycoplasma pneumonia


5,10-Methenyltetrahydrofolate synthetase plays a significant role in folate metabolism by catalyzing the conversion of 5-formyltetrahydrofolate into 5,10-methenyltetrahydrofolate. The enzyme is important in some forms of chemotherapy, and it has been implicated in resistance to antifolate antibiotics. A co-crystal structure of the enzyme (1U3G) and primary sequence analysis were used to select highly conserved amino acids in close proximity to bound 5-formyltetrahydrofolate. The amino acids were then investigated using site directed mutagenesis and kinetics. Y123, E55, and F118 were concluded to be important for binding 5-formyltetrahydrofolate in the active site and/or for substrate turnover of the enzyme. Replacement of E55 or Y123 with alanine resulted in no detectable activity. The more subtle replacement of E55 with glutamine was also inactive suggesting an ionic interaction with 5-formyltetrahydrofolate. Mutations to F118 resulted in substantial increases in apparent Km for both 5-formyltetrahydrofolate and ATP, but did not substantially affect catalytic turnover. Outside the active site, the replacement of Q144 with alanine yielded an enzyme that bound the substrates of ATP and 5-formyltetrahydrofolate with higher apparent Km values than the wild-type enzyme, but demonstrated a 3.1 fold increase in kcat.

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Deoxyribonucleic acid


5,10-Methenyltetrahydrofolate synthetase




Serine hydroxymethyltransferase








Circular dichroism spectroscopy


Sodium dodecylsulfate polyacrylamide gel electrophoresis


Para-aminobenzoic acid


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We would like to thank Drs. William (Skip) Brenzovich, Gary Hollis, and Catherine A. Sarisky for helpful discussions. We are also thankful to the Jeffress Memorial Trust (J788) for funding of this project.


This study was funded by the Jeffress Memorial Trust (J788).

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Correspondence to Timothy W. Johann.

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Cooper, C., Bryant, M., Hogan, N. et al. Investigations of Amino Acids in the 5-Formyltetrahydrofolate Binding Site of 5,10-Methenyltetrahydrofolate Synthetase from Mycoplasma pneumonia. Protein J 38, 409–418 (2019).

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  • 5-Formyltetrahydrofolate
  • Folinic acid
  • ATP
  • π Interactions
  • Mycoplasma pneumoniae