Characterization and stereochemistry of cofactor oxidation by a type II dihydrofolate reductase

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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., and 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., and 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. Addition of NADP+ to the enzyme results in the formation of an initial binary complex (conformation I) which interconverts to a more stable binary complex (conformation II). At 25.degree.C the apparent first-order rate constant for the interconversion between conformations I and II was determined to be approximately 1.0 .times. 10-4 s-1. Conformations I and II are characterized by upfield and downfield chemical shifts of the nicotinamide proton resonances from their positions in the free coenzyme, respectively. Changes in the 1H NMR chemical shifts of the upfield-shifted methyl resonances of RBG200 DHFR were also observed upon NADP+ binding and accompanying the interconversion between conformations I and II. The relevance of these two distinct binary conformations in coenzyme binding and catalysis remains to be determined.