1H, 15N and 13C resonance assignments, secondary structure, and the conformation of substrate in the binary folate complex of Escherichia coli dihydrofolate reductase.
By using fully 15N- and 15N/13C-labeled Escherichia coli dihydrofolate reductase, the sequence-specific 1H and 15N NMR assignments were achieved for 95% of the backbone resonances and for 90% of the 13C alpha resonances in the binary folate complex. These assignments were made through a variety of three-dimensional proton-detected 15N and 13C experiments. A smaller but significant subset of side-chain 1H and 13C assignments were also determined. In this complex, only one 15N or 13C resonance was detected per 15N or 13C protein nucleus, which indicated a single conformation. Proton-detected 13C experiments were also performed with unlabeled DHFR, complexed with 13C-7/13C-9 folate to probe for multiple conformations of the substrate in its binary complex. As was found for the protein resonances, only a single bound resonance corresponding to a productive conformation could be detected for C-7. These results are consistent with an earlier report based on 1H NMR data [Falzone, C.J. et al. (1990) Biochemistry, 29, 9667-9677] and suggest that the E. coli enzyme is not involved in any catalytically unproductive binding modes in the binary complex. This feature of the E. coli enzyme seems to be unique among the bacterial forms of DHFR that have been studied to date.
Pennsylvania State University, Department of Chemistry, University Park 16802.
C J
CJ
Falzone
J
J
Cavanagh
M
M
Cowart
A G
AG
Palmer
C R
CR
Matthews
S J
SJ
Benkovic
P E
PE
Wright
D000595:Amino Acid Sequence; D001665:Binding Sites; D002247:Carbon Isotopes; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000201:enzymology*; D005492:Folic Acid / Q000378:metabolism*; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D006859:Hydrogen; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase