CONFORMATION OF NADP+ BOUND TO A TYPE-II DIHYDROFOLATE-REDUCTASE

Type II dihydrofolate reductases (DHFRs) encoded by the R67 and R388 plasmids are sequence and structurally different from known chromosomal DHFRs. These plasmid-derived DHFRs are responsible for conferring trimethoprim resistance to the host strain. A derivative of R388 DHFR, RBG200, has been cloned and its physical properties have been characterized. This enzyme has been shown to transfer the pro-R hydrogen of NADPH to its substrate, dihydrofolate, making it a member of the A-stereospecific class of dehydrogenases [Brito, R. M. M., Reddick, R., Bennett, G. N., Rudolph, F. B., & Rosevear, P. R. (1990) Biochemistry 29, 9825]. Two distinct binary RBG200.NADP+ complexes were detected. Addition of NADP+ to RBG200 DHFR results in formation of an initial binary complex, conformation I, which slowly interconverts to a second more stable binary complex, conformation II. The binding of NADP+ to RBG200 DHFR in the second binary complex was found to be weak, K(D) = 1.9 +/- 0.4 mM. Transferred NOEs were used to determine the conformation of NADP+ bound to RBG200 DHFR. The initial slope of the NOE buildup curves, measured from the intensity of the cross-peaks as a function of the mixing time in NOESY spectra, allowed interproton distances on enzyme-bound NADP+ to be estimated. The experimentally measured distances were used to define upper and lower bound distance constraints between proton pairs in distance geometry calculations. All NADP+ structures consistent with the experimental distance bounds were found to have a syn conformation about the nicotinamide-ribose (chi = 94 +/- 26-degrees) and an anti conformation about the adenine-ribose (chi = -92 +/- 32-degrees) glycosidic bonds. The conformation of NADP+ bound to RBG200 DHFR in the initial binary complex was qualitatively evaluated at 5-degrees-C, to decrease the rate of interconversion to conformation II. The ratio of cross-peak intensities as well as the pattern of observed NOEs was only consistent with syn and anti conformations about the nicotinamide-ribose and adenine-ribose bonds, respectively, in the initial complex, conformation I. From the known stereochemistry of hydride transfer and the conformation of the enzyme-bound cofactor, a model is proposed for the orientation of cofactor and substrate at the active site of RBG200 DHFR. In this model, the NMNH moiety of NADPH is bound in a syn conformation with the pteridine portion of dihydrofolate located below the plane of the nicotinamide ring and rotated 180-degrees with respect to dihydrofolate binding in chromosomal DHFRs.