Crystal Structures of Escherichia coli Dihydrofolate Reductase Complexed with 5-Formyltetrahydrofolate (Folinic Acid) in Two Space Groups:  Evidence for Enolization of Pteridine O4

The crystal structure of Escherichia coli dihydrofolate reductase (ecDHFR, EC 1.5.1.3) as a binary complex with folinic acid (5-formyl-5,6,7,8-tetrahydrofolate; also called leucovorin or citrovorum factor) has been solved in two space groups, P61 and P65, with, respectively, two molecules and one molecule per asymmetric unit. The crystal structures have been refined to an R-factor of 14.2% at resolutions of 2.0 and 1.9 Å. The P61 structure is isomorphous with several previously reported ecDHFR binary complexes [Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650−13662; Reyes, V. M., Sawaya, M. R., Brown, K. A., & Kraut, J. (1995) Biochemistry 34, 2710−2723]; enzyme and ligand conformations are very similar to the P61 5,10-dideazatetrahydrofolate complex. While the two enzyme subdomains of the P61 structure are nearly in the closed conformation, exemplified by the methotrexate P61 binary complex, in the P65 structure they are in an intermediate conformation, halfway between the closed and the fully open conformation of the apoenzyme [Bystroff, C., Oatley, S. J., & Kraut, J. (1990) Biochemistry 29, 3263−3277]. Thus crystal packing strongly influences this aspect of the enzyme structure. In contrast to the P61 structure, in which the Met-20 loop (residues 9−23) is turned away from the substrate binding pocket, in the P65 structure the Met-20 loop blocks the pocket and protrudes into the cofactor binding site. In this respect, the P65 structure is unique. Additionally, positioning of a Ca2+ ion (a component of the crystallization medium) is different in the two crystal packings: in the P61 structure it lies at the boundary between the two molecules of the asymmetric unit, while in P65 it coordinates two water molecules, the hydroxyl group of an ethanol molecule, and the backbone carbonyl oxygens of Glu-17, Asn-18, and Met-20. The Ca2+ ion thus stabilizes a single turn of 310 helix (residues 16−18 in the Met-20 loop), a second unique feature of the P65 crystal structure. The disposition of the N5-formyl group in these structures indicates formation, at least half of the time, of an intramolecular hydrogen bond between the formyl oxygen and O4 of the tetrahydropterin ring. This observation is consistent with the existence of an enol−keto equilibrium in which the enolic tautomer is favored when a hydrogen-bond acceptor is present between O4 and N5. Such would be the case whenever a water molecule occupies tha