Cation binding and thermostability of FTHFS monovalent cation binding sites and thermostability of N-10-formyltetrahydrofolate synthetase from Moorella thermoacetica

Formyltetrahydrofolate synthetase (FTHFS) from the thermophilic homoacetoge n, Moorella thermoacetica, has an optimum temperature for activity of 55-60 degreesC and requires monovalent cations for both optimal activity and sta bilization of tetrameric structure at higher temperatures. The crystal stru ctures of complexes of FTHFS with cesium and potassium ions were examined a nd monovalent cation binding positions identified. Unexpectedly, NH4+ and K +, both of which are strongly activating ions, bind at a different site tha n a moderately activating ion, Cs+, does. Neither binding site is located i n the active site. The sites are 7 Angstrom apart, but in each of them, the side chain of Glu 98, which is conserved in all known bacterial FTHFS sequ ences, participates in metal ion binding. Other ligands in the Cs+ binding site are four oxygen atoms of main chain carbonyls and water molecules. The K+ and NH4+ binding site includes the carboxylate of Asp132 in addition to Glu98. Mutant FTHFS's (E98Q,E98D, and E98S) were obtained and analyzed usi ng differential scanning calorimetry to examine the effect of these mutatio ns on the thermostability of the enzyme with and without added K+ ions. The addition of 0.2 M K+ ions to the wild-type enzyme resulted in a 10 degrees C increase in the thermal denaturation temperature. No significant increase was observed in E98D or E98S. The lack of a significant effect of monovale nt cations on the stability of E98D and E98S indicates that this alteration of the binding site eliminates cation binding. The thermal denaturation te mperature of E98Q was 3 degreesC higher than that of the wild-type enzyme i n the absence of the cation, indicating that the removal of the unbalanced, buried charge of Glu98 stabilizes the enzyme. These results confirm that G lu98 is a crucial residue in the interaction of monovalent cations with FTH FS.