NMR studies of the interaction of a type II dihydrofolate reductase with pyridine nucleotides reveal unexpected phosphatase and reductase activity.
The interaction of type II R67 dihydrofolate reductase (DHFR) with its cofactor nicotinamide adenine dinucleotide phosphate (NADP(+)) has been studied using nuclear magnetic resonance (NMR). Doubly labeled [U-(13)C,(15)N]DHFR was obtained from Escherichia coli grown on a medium containing [U-(13)C]-D-glucose and (15)NH(4)Cl, and the 16 disordered N-terminal amino acids were removed by treatment with chymotrypsin. Backbone and side chain NMR assignments were made using triple-resonance experiments. The degeneracy of the amide (1)H and (15)N shifts of the tetrameric DHFR was preserved upon addition of NADP(+), consistent with kinetic averaging among equivalent binding sites. Analysis of the more titration-sensitive DHFR amide resonances as a function of added NADP(+) gave a K(D) of 131 +/- 50 microM, consistent with previous determinations using other methodology. We have found that the (1)H spectrum of NADP(+) in the presence of the R67 DHFR changes as a function of time. Comparison with standard samples and mass spectrometric analysis indicates a slow conversion of NADP(+) to NAD(+), i.e., an apparent NADP(+) phosphatase activity. Studies of this activity in the presence of folate and a folate analogue support the conclusion that this activity results from an interaction with the DHFR rather than a contaminating phosphatase. (1)H NMR studies of a mixture of NADP(+) and NADPH in the presence of the enzyme reveal that a ternary complex forms in which the N-4A and N-4B nuclei of the NADPH are in the proximity of the N-4 and N-5 nuclei of NADP(+). Studies using the NADP(+) analogue acetylpyridine adenosine dinucleotide phosphate (APADP(+)) demonstrated a low level of enzyme-catalyzed hydride transfer from NADPH. Analysis of DHFR backbone dynamics revealed little change upon binding of NADP(+). These additional catalytic activities and dynamic behavior are in marked contrast to those of type I DHFR.
D000577:Amides / Q000737:chemistry; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009243:NAD / Q000032:analysis / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010088:Oxidoreductases / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*
D000577:Amides; D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; D010088:Oxidoreductases; C098921:dihydrofolate reductase type II; D013762:Tetrahydrofolate Dehydrogenase; D010744:Phosphoric Monoester Hydrolases