Lactobacillus casei dihydrofolate reductase

Crystal structures of escherichia coli and lactobacillus casei dihydrofolate reductase refined at 1.7A resolution, J. Biol. Chem. 257 13663 (1982). 

Fig. 14. Nicotinamide-binding site of Lactobacillus casei dihydrofolate reductase. NADPH is indicated by its van der Waals surface. The oxygen atoms of isoleucin-12, threonine-45, and alanine-97 that are involved in enthalpically favorable oxygen-aromatic interactions with NADPH are labeled.

Bolin JT, Filman DJ, Matthews DA, Hamlin RC, Kraut J (1982) Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 A resolution. J Biol Chem 257 13650-13662... 

Hansch C, Li RL, Blaney JM, Langridge R. Comparison of the inhibition of Escherichia coli and Lactobacillus casei dihydrofolate reductase by 2,4-diamino-5-(substituted-benzyl)pyrimidines quantitative structure-activity relationships, x-ray crystallography, and computer graphics in structure-activity analysis. J Med Chem 1982 25 777-784. 

Way, J. L., Birdsall, B., Feeney, J., Roberts, G. . K., and Burgen, A. S. V. (1975). Biochemistry 14, 3470. A Nuclear Magnetic Resonance Study of Nicotinamide Adenine Dinucleotide Phosphate Binding to Lactobacillus casei Dihydrofolate Reductase. 

Bolin J T, D J Filman, D A Matthews, R C Hamlin and J Kraut 1982. Crystal Structures of Escherichia coli and Lactobacillus casei Dihydrofolate Reductase Refined at 1.7 Angstroms Resolution. I. Features and Binding of Methotrexate Journal of Biological Chemistry 257.13650-13662. 

Bohn, J.T. Filman, D.J. Matthews, D.A. Hanlin, R.C. Kraut, J. Crystal Structures of Escherichia coli and lactobacillus casei Dihydrofolate Reductase Refined at 1.7A Resolution. J. Biol. Chem. 1982,257, 13650-13762. 

Figure 1 Dynamic processes in the complex of trimethoprim with Lactobacillus casei dihydrofolate reductase measured at 298 K. Reproduced with permission from Searle MS etal. (1988) Proceedings of the National Academy of Sciences of the USA 85 3787-3791.

Matthews DA, Alden RA, Bolin JT, Filman DJ, Freer ST, Hamlin R, Hoi WG, Kisliuk RL, Pastore EJ, Plante FT, Xuong N, Kraut J. Dihydrofolate reductase from Lactobacillus casei. X-ray structure of the enzyme methotrexate-NADPH complex. J Biol Chem 1978 253 6946-54. 

Hoi, R. L. Kisliuk, E. J. Pastore, L. T. Plante, N. Xuong, J. Kraut, Dihydrofolate reductase from lactobacillus casei, J. Biol. Chem. 253 6946 (1978). 

The expansion in the power of computers and theoretical methods has made it possible to investigate the mechanism of action of enzymes by combinations of quantum-mechanical and molecular-mechanical calculations. A study of two possible mechanisms for dihydrofolate reductase catalysis was consistent with indirect proton transfer from aspartate to N-5 of the pterin as has been suggested for many years by crystallographic evidence <2003PCB14036>. This conclusion is also consistent with the outcome of a study that directly measured the of the active site aspartate in the Lactobacillus casei enzyme <1999B8038>. Observations of chemical shifts of... 

Another enzyme for which X-ray diffraction studies have aided in an analysis of the mode of action is the enzyme dihydrofolate reductase. This catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate, an essential coenzyme used in the synthesis of thymidylate, inosinate, and methionine. The antitumor agent methotrexate is a powerful inhibitor of dihydrofolate reductase, causing, on binding, a cellular deficiency of thymidylate (the cause of its antitumor activity). The crystal structures of the enzyme from two bacterial sources—Escherichia coli and Lactobacillus casei—and from chicken liver have been studied (88-90). Both the E. coli and L casei enzymes have been studied as complexes with methotrexate bound at the active site, and, in the case of the . casei enzyme, the cofactor, NADPH, was also present. 

The value of A, in turn, is exponentially related to the percentage of identical core residues so that a protein structure will conservatively provide a close general stmctural model for other proteins if the sequence similarity exceeds 50%. Consequently, there is an increasing use of this type of relationship to predict tertiary stmcture and secondary elements from primary sequences. Nevertheless, there are cases, such as the Lactobacillus casei and E. coli dihydrofolate reductases, where the primary sequence similarity is only 30% yet A is only 1.3 A (727). 

Resistance to End Product Repression, a) Antimetabolites. The use of toxic antimetabolites to select resistant cultures often yields mutants whose normal biosynthetic pathway enzymes are not repressed by the end product. For example, trifluoreoleucine selects mutants with derepressed levels (as much as 10-fold) of leucine biosynthetic enzymes (Calvo and Calvo, 1967). Certain canavanine-resistant mutants produce 30 times more of the arginine pathway enzymes than do their sensitive parents (Jacoby and Gorini, 1967). Mutants of Lactobacillus casei resistant to dichloroamethopterin are derepressed 80-fold in their ability to form thymidylate synthetase (Crusberg and Kisliuk, 1969). When Diplococcus pneumoniae mutants are selected on the basis of resistance to amethopterin, these cultures produce 100 times as much dihydrofolate reductase as the parent culture (Sirotnak et al, 1969). 

Fig. 36 a-c. Binding in dihydrofolate reductase [88] a binding of the pterin ring of methotrexate in crystals of the Lactobacillus casei enzyme... 

X-ray diffraction analysis of dihydrofolate reductase (DHFR), co-crystal-lized with methotrexate, has shed much light on the action of this inhibitor. This work, one of the earliest visualizations of a drug interacting with its receptor (Matthews etal., 1977), has since been refined to the remarkably clear resolution of 1.7 A (Bolin et al., 1982). A typical diagram of DHFR, its coenzyme (NADPH), and methotrexate is shown in Fig. 9.4. The enzyme depicted there is from Lactobacillus casei and the same authors also report on DHFR (with cocrystallized methotrexate) from the bacterium E. colt. However, they have not been able to co-crystallize methotrexate with DHFR from any vertebrate source. 

Dihydrofolate Reductase from Lactobacillus casei X-Ray Structure of the Enzyme Methotrexate.NADPH Complex. 

Because of the central role of dihydrofolate reductase (DHFR) in purine biosynthesis, DHFR inhibitors are important as antibacterial (trimethoprim), antimalarial, and antitumor agents (methotrexate). For a series of 5-(X-benzyl)-2,4-diaminopyrimidines 9 with several different groups X, QSAR equations were derived for the inhibition of Escherichia coli (equation 14 Xi app == experimental Inhibition constants) and of Lactobacillus casei (equation 15) in both equations the numbers 3, 4, and 5 refer to the X substituent positions at the benzyl group. " ... 

Dihydrofolate reductase catalyzes the reduction of dihydrofolate to tetra-hydrafolate using NADPH as coenzyme. As part of their continuing and intensive investigation of this enzyme (from Lactobacillus casei) by various... 

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