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Conformation of NADP+ bound to a type II dihydrofolate reductase
R M Brito1, F B Rudolph, P R Rosevear
1Department of Biochemistry, Rice University, Houston, Texas 77005.
Abstract:
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 confering 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, KD = 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 (X = 94 +/- 26 degrees) and an anti conformation about the adenine-ribose (X = -92 +/- 32 degrees) glycosidic bonds.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Type II dihydrofolate reductases (DHFRs) confer trimethoprim resistance. Researchers characterized RBG200, an R388 DHFR derivative, and determined the NADP+ binding conformation using transferred NOEs.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Type II dihydrofolate reductases (DHFRs) from R67 and R388 plasmids confer trimethoprim resistance.
- These plasmid-derived DHFRs differ structurally and sequentially from chromosomal DHFRs.
Purpose of the Study:
- To characterize the physical properties of RBG200, a derivative of R388 DHFR.
- To determine the conformation of NADP+ bound to RBG200 DHFR.
Main Methods:
- Cloning and characterization of RBG200.
- Analysis of RBG200.NADP+ binary complexes using transferred Nuclear Overhauser Effect (NOE) spectroscopy.
- Distance geometry calculations to model NADP+ conformation.
Main Results:
- RBG200 transfers the pro-R hydrogen of NADPH to dihydrofolate, classifying it as A-stereospecific.
- Two distinct RBG200.NADP+ binary complexes were identified, with slow interconversion.
- Weak binding affinity of NADP+ to RBG200 (KD = 1.9 +/- 0.4 mM).
- Transferred NOEs revealed syn conformation at the nicotinamide-ribose bond and anti conformation at the adenine-ribose bond of bound NADP+.
Conclusions:
- The study elucidates the structural basis of NADP+ binding to a plasmid-derived DHFR.
- Understanding these interactions is crucial for developing new antimicrobial strategies targeting DHFR enzymes.