Dihydrofolate reductase system

Fig. 4.25 Reaction route (RR) graph for the dihydrofolate reductase system. (From I. Fishtik, C A. Callaghan, R. Datta, Reaction route graphs. II. examples of enzyme and surface catalyzed single overall reactions, J. Rhys. Chem. B, 108 (2004) 5683-5697. Copyright 2004 American Chemical Society).

The sulfa dmgs are stiH important as antimicrobials, although they have been replaced in many systemic infections by the natural and semisynthetic antibiotics. They are of great value in third world countries where problems of storage and lack of medical personnel make appropriate use of antibiotics difficult. They are especially useful in urinary tract infections, particularly the combination of sulfamethoxazole with trimethoprim. Their effectiveness has been enhanced by co-adniinistration with dihydrofolate reductase inhibitors, and the combination of sulfamethoxazole with trimethoprim is of value in treatment of a number of specific microbial infections. The introduction of this combination (cotrimoxazole) in the late 1960s (1973 in the United States) resulted in increased use of sulfonamides. 

A review is given of the application of Molecular Dynamics (MD) computer simulation to complex molecular systems. Three topics are treated in particular the computation of free energy from simulations, applied to the prediction of the binding constant of an inhibitor to the enzyme dihydrofolate reductase the use of MD simulations in structural refinements based on two-dimensional high-resolution nuclear magnetic resonance data, applied to the lac repressor headpiece the simulation of a hydrated lipid bilayer in atomic detail. The latter shows a rather diffuse structure of the hydrophilic head group layer with considerable local compensation of charge density. 

A wheat germ, cell-free, translation extract was fractionated into three concentrated parts using ammonium sulfate the 0 - 40 % saturated fraction, the 40 - 60 % saturated fraction, and the ribosome fraction. These fractions were tested for their ability to enhance the translational activity of the wheat germ, cell-free extract for dihydrofolate reductase. The fortified cell-free system supplemented with the 0 - 40 % ammonium sulfate fraction enhanced the efficiency of protein synthesis by 50 %. 

Fig. 3. Autoradiograph of SDS-PAGE of in vitro translated dihydrofolate reductase (DHFR) in the wheat germ cell-free protein synthesis systems with (n) 4 pi of ribosome fiaction, (III) 4 pi of 0 -40 % ammonium sulfate fraction, or (IV) 4 pi of 40 - 60% ammonium sulfate fraction, respectively. Lane I is control dihydrofolate reductase produced in the normal wheat germ cell-free protein synthesis system.

Bajorath, J., D. H. Kitson, G. Fitzgerald, J. Andzelm, J. Kraut, and A. T. Hagler. 1991. Local Density Functional Calculations on a Protein System Folate and Escherichia Coli dihydrofolate reductase. Electron Redistribution on Binding of a Substrate to an Enzyme. PROTEINS 9, 217. 

Classical methodology was used to prepare the dibenz[b,f]azepine derivative 21 (R = substituted pyrido[2,3-d]pyrimidine) utilising amide ion formation from dibenz[b,f]azepine itself with sodium hydride and then iV-alkylation with 2,4-diamino-6-bromomethylpyrido[2,3-d]pyrimidine. The bulky bis-fused azepine moiety was required to introduce steric bulk in the system and to study the effect of this on inhibition of the enzyme dihydrofolate reductase <00JHC921>. 

Hagler, Structure and energetics of ligand binding to proteins Escherichia coli dihydrofolate reductase-trimethoprim, a drug-receptor system, Proteins 4 31 (1988). 

A. Liposomal amphotericin B was approved by the US. Food and Drug Administration to treat visceral leishmaniasis. Pentavalent antimony compounds, pentamidine, amphotericin B, and aminosi-dine (paromomycin) have all been demonstrated efficacious here. The liposomal amphotericin appears to be better taken up by the reticuloendothelial system, where the parasite resides, and partitions less in the kidney, where amphotericin B traditionally manifests its toxicity. In addition to being better tolerated by patients, it has proved to be very effective in India, where resistance to antimony drugs is widespread. This patient appears to have acquired his infection there, where many infected patients develop darkening of the skin, hence the name kala-azar, or black sickness. Albendazole, an anthelmintic, has no role here. Atovaquone, a naphthoquinone, is used to treat malaria, babesiosis, and pneumocystosis. Pyrimethamine-sulfadoxine is used to treat malaria and toxoplasmosis. Proguanil inhibits the dihydrofolate reductase of malaria parasites and is used in combination with atovaquone. 

The cell-free system prepared from washed embryos has much higher translational activity than the conventional system (compare Fig. 3A and B). When 5 -capped dihydrofolate reductase (DHFR) mRNA containing 549 nt of 3 UTR with a pA tail was incubated with newly prepared as well as conventional extract, there was almost linear kinetics in DHFR synthesis over 4 h, compared with the regular system, which ceased to function after 1.5 h. Further, when washed extract in the reaction volume was increased to 48%, amino acid incorporation occurred initially at a rate twice that of 24% extract, and then stopped after 1 h. However, this pause was caused by a shortage of substrates rather than an irreversible inactivation of ribosomes or factors necessary for translation addition of amino acids, ATP, and GTP after cessation of the reaction (arrow) restarted... 

More recently Michnick and co-workers have introduced a dihydrofolate reductase complementation system, which seems to be particularly robust [61 - 65], They attribute the success of this system to the fact that the N-terminal (1 - 105) and C-terminal (106 - 186) DHFR fragments do not fold until they are dimerized. In addition to the obvious selection for essential metabolites dependent on the reduction of dihydrofolate to tetrahydrofolate, protein-protein interactions are detected based on the retention of a fluorescein-methotrexate conjugate. Several other enzymes have been employed for the design of complementation assays, including green fluorescent protein, which allows screens based on fluorescence or FRET [66 - 68]. As with the bacterial transcription assays, these complementation systems are new. It will be interesting to see if, as the selections are optimized, these systems prove competitive with the Y2H assay. 

While providing valuable insight towards a better understanding of protein evolution, the bisection of proteins into functional heterodimers has also found practical applications to study protein-protein interactions. Heterodimeric variants of dihydrofolate reductase (DHFR), green fluorescent protein (GFP), GAR transformylase, and phosphotransferases were constructed to work in two-hybrid systems [64]. 

The Michnick group has also developed an elegant split-reporter system, based on reconstitution of dihydrofolate reductase (DHFR) (41). In this system, cell survival requires functional DHFR thus, a successful protein-protein interaction can be identified readily by selection. This system has been applied to the identification of peptide sequences that bind the Ras-binding domain of Raf (42). 

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