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Characterization and stereochemistry of cofactor oxidation by a type II dihydrofolate reductase
R M Brito1, R Reddick, G N Bennett
1Department of Biochemistry, Rice University, Houston, Texas 77005.
Abstract:
Type II dihydrofolate reductases (DHFRs) encoded by the R67 and R388 plasmids are different both in sequence and in structure 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 overproduced [Vermersch, P. S., Klass, M. R., & Bennett, G. N. (1986) Gene 41, 289]. With this cloned and overproduced protein, a rapid purification procedure has been developed that yields milligram quantities of apparently homogeneous RBG200 DHFR with a specific activity 1.5-fold greater than that previously reported for the purified R388 protein [Amyes, S. G. B., & Smith, J. T. (1976) Eur. J. Biochem. 61, 597]. The pH versus activity profile and the native molecular weight of RBG200 DHFR were found to be similar to those previously reported for other type II DHFRs but different from those of the known chromosomal DHFRs. Stereospecifically labeled [4(S)-2H,4(R)-1H]NADPH was synthesized and used to determine the stereospecificity of NADPH oxidation by RBG200 DHFR. RBG200 DHFR was found to specifically transfer the pro-R hydrogen of NADPH to dihydrofolate, making it a member of the A-stereospecific class of dehydrogenases. Thus, although RBG200 DHFR is different both in sequence and in structure from known chromosomal enzymes, both enzymes catalyze identical hydrogen-transfer reactions. Two distinct binary RBG200 DHFR-NADP+ complexes were detected by monitoring the 1H NMR chemical shifts and line widths of the coenzyme in the presence of RBG200 DHFR.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Plasmid-derived dihydrofolate reductases (DHFRs) confer trimethoprim resistance. This study details the purification and characterization of RBG200 DHFR, revealing its A-stereospecific dehydrogenase activity, similar to chromosomal DHFRs despite structural differences.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Type II dihydrofolate reductases (DHFRs) from R67 and R388 plasmids confer trimethoprim resistance.
- These plasmid DHFRs exhibit distinct sequences and structures compared to chromosomal DHFRs.
Purpose of the Study:
- To clone, overproduce, and purify the R388 DHFR derivative, RBG200.
- To characterize the biochemical properties and stereospecificity of RBG200 DHFR.
- To investigate the interaction between RBG200 DHFR and its coenzyme.
Main Methods:
- Cloning and overproduction of RBG200 DHFR.
- Development of a rapid purification procedure.
- Determination of pH versus activity profile and native molecular weight.
- Synthesis of stereospecifically labeled NADPH for stereospecificity studies.
- 1H NMR spectroscopy to detect enzyme-coenzyme complexes.
Main Results:
- A rapid purification yielded homogeneous RBG200 DHFR with high specific activity.
- RBG200 DHFR exhibits properties (pH optimum, molecular weight) similar to other type II DHFRs.
- RBG200 DHFR specifically transfers the pro-R hydrogen of NADPH to dihydrofolate, classifying it as A-stereospecific.
- Two distinct RBG200 DHFR-NADP+ binary complexes were identified using 1H NMR.
Conclusions:
- RBG200 DHFR, despite structural divergence from chromosomal DHFRs, catalyzes identical hydrogen transfer reactions.
- The enzyme's A-stereospecificity is conserved across different DHFR classes.
- NMR studies provide insights into the binary complex formation between RBG200 DHFR and NADP+.