Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 A resolution. II. Environment of bound NADPH and implications for catalysis.
New details of NADPH binding to Lactobacillus casei dihydrofolate reductase have become visible as a result of crystallographic refinement to an R factor of 0.152 at 1.7 A resolution. Conformational torsion angles for bound NADPH have been extensively revised and specific interatomic contacts responsible for cofactor binding have been identified. In addition, several structurally conserved water molecules are seen to mediate the protein-ligand interaction. In the nicotinamide binding site three oxygen atoms of the enzyme lie in the plane of the pyridine ring and close to ring carbons 2, 4, and 6. The placement of these polar groups suggests that the enzyme stabilizes a C4-carbonium electronic isomer of oxidized nicotinamide in the transition state. Pyramidalization of ring nitrogen N1 in the transition state might be promoted by a fixed water molecule positioned to donate a hydrogen bond to the N1 lone pair orbital. Pyramidalization could also relieve an unfavorable steric contact due to the observed rotation of the nicotinamide's carboxamide group by 180 degrees from its most stable conformation.