Characterization and stereochemistry of cofactor oxidation by a type II dihydrofolate reductase.
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)
Brito
R M
RM
Department of Biochemistry, Rice University, Houston, Texas 77005.
Reddick
R
R
Bennett
G N
GN
Rudolph
F B
FB
Rosevear
P R
PR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011815:R Factors; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D014296:Trimethoprim Resistance
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase