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Type II dihydrofolate reductases: Probing the natural diversity of enzyme active sites

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Title: Type II dihydrofolate reductases: Probing the natural diversity of enzyme active sites
Author: De Brito, Rui Manuel Pontes Meireles Ferreira
Abstract: Type II dihydrofolate reductases (DHFRs) encoded by the R67 and R388 plasmids are sequence and structurally different from known chromosomal DHFRs, and are responsible for conferring trimethoprim resistance to the host bacterial strain. An overproduced derivative of R388 DHFR, RBG200 DHFR, was purified to apparent homogeneity. The pH versus activity profile of RBG200 DHFR was found to be similar to that previously reported for R388 DHFR, but different from that of chromosomal DHFRs. Gel filtration and equilibrium ultracentrifugation experiments suggested that at pH 6, where the enzyme is most active, RBG200 DHFR is in equilibrium between tetramer and other protein species. Gel filtration studies suggested that the loss of enzyme activity between pH 6 and pH 5 is not due to tetramer dissociation. Gel filtration experiments with R67 DHFR produced similar results. RBG200 DHFR was found to catalyze the transfer of the pro-R hydrogen of NADPH to dihydrofolate, making it a type-A dehydrogenase, along with the chromosomal DHFRs. Addition of NADP$\sp+$ to RBG200 DHFR results in the formation of an initial binary complex which slowly interconverts to a second binary complex, with an apparent first order rate constant of 1.0 $\times$ 10$\sp{-4}$ s$\sp{-1}$, at 25$\sp\circ$C. The binding of NADP$\sp+$ to RBG200 DHFR in the second binary complex was found to be weak, K$\sb{\rm D}$ = 1.9 $\pm$ 0.4 mM. The stoichiometry for coenzyme binding was found to be approximately one coenzyme per tetramer of RBG200 DHFR. Transferred NOEs were used to estimate internuclear distances, and it was found that NADP$\sp+$ binds to RBG200 DHFR in the initial and final RBG200 DHFR$\cdot$NADP$\sp+$ binary complexes, as well as in the ternary complex with folate, with a syn conformation about the nicotinamide-ribose glycosidic bond and an anti conformation about the adenine-ribose glycosidic bond. From the stereochemistry of hydride transfer and the conformation of the enzyme-bound cofactor, a model was proposed for the orientation of coenzyme and substrate as the active site of RBG200 DHFR, which differs from that observed at the active site of chromosomal DHFRs, and may be responsible for the resistance of type II DHFRs to several antifolates.
Citation: De Brito, Rui Manuel Pontes Meireles Ferreira. (1992) "Type II dihydrofolate reductases: Probing the natural diversity of enzyme active sites." Doctoral Thesis, Rice University. http://hdl.handle.net/1911/16509.
URI: http://hdl.handle.net/1911/16509
Date: 1992

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