scholarly journals Correlations of Inhibitor Kinetics forPneumocystis jiroveciiand Human Dihydrofolate Reductase with Structural Data for Human Active Site Mutant Enzyme Complexes†‡

Biochemistry ◽  
2009 ◽  
Vol 48 (8) ◽  
pp. 1702-1711 ◽  
Author(s):  
Vivian Cody ◽  
Jim Pace ◽  
Jennifer Makin ◽  
Jennifer Piraino ◽  
Sherry F. Queener ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Structural Data ◽  
Mutant Enzyme ◽  
Human Dihydrofolate Reductase ◽  
Enzyme Complexes ◽  
Inhibitor Kinetics

scholarly journals Crystallographic analysis reveals a novel second binding site for trimethoprim in active site double mutants of human dihydrofolate reductase

2011 ◽  
Vol 176 (1) ◽  
pp. 52-59 ◽  
Author(s):  
Vivian Cody ◽  
Jim Pace ◽  
Jennifer Piraino ◽  
Sherry F. Queener
Keyword(s):  
Binding Site ◽  
Dihydrofolate Reductase ◽  
Active Site ◽  
Crystallographic Analysis ◽  
Human Dihydrofolate Reductase

Conversion of arginine to lysine at position 70 of human dihydrofolate reductase: generation of a methotrexate-insensitive mutant enzyme

Biochemistry ◽  
1991 ◽  
Vol 30 (33) ◽  
pp. 8124-8130 ◽  
Author(s):  
Paul D. Thompson ◽  
James H. Freisheim
Keyword(s):  
Dihydrofolate Reductase ◽  
Mutant Enzyme ◽  
Human Dihydrofolate Reductase

Role of the conserved active site residue tryptophan-24 of human dihydrofolate reductase as revealed by mutagenesis

Biochemistry ◽  
1991 ◽  
Vol 30 (5) ◽  
pp. 1432-1440 ◽  
Author(s):  
William A. Beard ◽  
James R. Appleman ◽  
Shaoming Huang ◽  
Tavner J. Delcamp ◽  
James H. Freisheim ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Active Site Residue ◽  
Human Dihydrofolate Reductase

scholarly journals Inhibitors of Human Dihydrofolate Reductase: A Computational Design and Docking Studies Using Glide

2008 ◽  
Vol 5 (2) ◽  
pp. 263-270 ◽  
Author(s):  
Lingala Yamini ◽  
Manga Vijjulatha
Keyword(s):  
Dna Synthesis ◽  
Dihydrofolate Reductase ◽  
Active Site ◽  
Computational Design ◽  
Docking Studies ◽  
Vital Role ◽  
Essential Component ◽  
Tetrahydrofolic Acid ◽  
Human Dihydrofolate Reductase

Dihydrofolate reductase (DHFR) plays a vital role in the DNA synthesis by reducing dihydrofolic acid to tetrahydrofolic acid which is an essential component. Synthetic ligands like methotrexate (MTX), aminopterin (AMP) and their analogues act as potential anti metabolites by mimicking the coenzyme dihydrofolic acid (DHFA) they inhibit the activity of DHFR antagonistically. Several ligands which are similar to MTX analogues and 6, 8 substituted 2 – naphthyls (NAP) which can mimic the pteridyl group of DHFA have been computationally designed. These ligands were proposed to hinder the formation N5, N10 methylene tetrahydrofolic acid (THFA) coenzyme, which is essential for the DNA synthesis. The docking studies were done using grid, generated with the 0.9 Vander Waals scaling for non polar bonds in the active site of the receptor. These newly designed ligands such as 14 -21 ,23 and 28 have shown good docking scores and predicted activities when compared to already existing ligands MTX and its analogues.

scholarly journals Disruption of the crossover helix impairs dihydrofolate reductase activity in the bifunctional enzyme TS–DHFR from Cryptosporidium hominis

2009 ◽  
Vol 417 (3) ◽  
pp. 757-764 ◽  
Author(s):  
Melissa A. Vargo ◽  
W. Edward Martucci ◽  
Karen S. Anderson
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Reductase Activity ◽  
The Other ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Bifunctional Enzyme ◽  
Cryptosporidium Hominis ◽  
Single Polypeptide Chain ◽  
Species Specific

In contrast with most species, including humans, which have monofunctional forms of the folate biosynthetic enzymes TS (thymidylate synthase) and DHFR (dihydrofolate reductase), several pathogenic protozoal parasites, including Cryptosporidium hominis, contain a bifunctional form of the enzymes on a single polypeptide chain having both catalytic activities. The crystal structure of the bifunctional enzyme TS–DHFR C. hominis reveals a dimer with a ‘crossover helix’, a swap domain between DHFR domains, unique in that this helical region from one monomer makes extensive contacts with the DHFR active site of the other monomer. In the present study, we used site-directed mutagenesis to probe the role of this crossover helix in DHFR catalysis. Mutations were made to the crossover helix: an ‘alanine-face’ enzyme in which the residues on the face of the helix close to the DHFR active site of the other subunit were mutated to alanine, a ‘glycine-face’ enzyme in which the same residues were mutated to glycine, and an ‘all-alanine’ helix in which all residues of the helix were mutated to alanine. These mutant enzymes were studied using a rapid transient kinetic approach. The mutations caused a dramatic decrease in the DHFR activity. The DHFR catalytic activity of the alanine-face mutant enzyme was 30 s−1, the glycine-face mutant enzyme was 17 s−1, and the all-alanine helix enzyme was 16 s−1, all substantially impaired from the wild-type DHFR activity of 152 s−1. It is clear that loss of helix interactions results in a marked decrease in DHFR activity, supporting a role for this swap domain in DHFR catalysis. The crossover helix provides a unique structural feature of C. hominis bifunctional TS–DHFR that could be exploited as a target for species-specific non-active site inhibitors.

2-Tier Bacterial and In Vitro Selection of Active and Methotrexate-Resistant Variants of Human Dihydrofolate Reductase

2008 ◽  
Vol 13 (6) ◽  
pp. 504-514 ◽  
Author(s):  
Elena Fossati ◽  
Jordan P. Volpato ◽  
Lucie Poulin ◽  
Vanessa Guerrero ◽  
David-Antoine Dugas ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Drug Target ◽  
In Vitro Selection ◽  
Fold Increase ◽  
Active Site Residues ◽  
20 Nm ◽  
Human Dihydrofolate Reductase ◽  
Selection Of

We report a rapid and reliable 2-tier selection and screen for detection of activity as well as drug-resistance in mutated variants of a clinically-relevant drug-target enzyme. Human dihydrofolate reductase point-mutant libraries were subjected to a 1st-tier bacterial complementation assay, such that bacterial propagation served as an indicator of enzyme activity. Alternatively, when selection was performed in the presence of the inhibitor methotrexate (MTX), propagation indicated MTX resistance. The selected variants were then subjected to a 2nd-tier in vitro screen in 96-well plate format using crude bacterial lysate. Conditions were defined to establish a threshold for activity or for MTX resistance. The 2nd-tier assay allowed rapid detection of the best variants among the leads and provided reliable estimates of relative reactivity, ( kcat) and IC50MTX. Screening saturation libraries of active-site positions 7, 15, 24, 70, and 115 revealed a variety of novel mutations compatible with reactivity as well as 2 novel MTX-resistant variants: V115A and V115C. Both variants displayed KiMTX = 20 nM, a 600-fold increase relative to the wild-type. We also present preliminary results from screening against further antifolates following simple modifications of the protocol. The flexibility and robustness of this method will provide new insights into interactions between ligands and active-site residues of this clinically relevant human enzyme. ( Journal of Biomolecular Screening 2008:504-514)

scholarly journals Multiple Conformers in Active Site of Human Dihydrofolate Reductase F31R/Q35E Double Mutant Suggest Structural Basis for Methotrexate Resistance

2009 ◽  
Vol 284 (30) ◽  
pp. 20079-20089 ◽  
Author(s):  
Jordan P. Volpato ◽  
Brahm J. Yachnin ◽  
Jonathan Blanchet ◽  
Vanessa Guerrero ◽  
Lucie Poulin ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Double Mutant ◽  
Structural Basis ◽  
Methotrexate Resistance ◽  
Human Dihydrofolate Reductase ◽  
Multiple Conformers

The Function of the Active Site Glutamate in Human Dihydrofolate Reductase

Pteridines ◽  
1991 ◽  
Vol 3 (1-2) ◽  
pp. 135-136
Author(s):  
R. L. Blakley ◽  
J. R. Appleman ◽  
J. T. Tsay ◽  
W. A. Beard ◽  
J. H. Freisheim ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Human Dihydrofolate Reductase

scholarly journals Computer modeling studies of the structural role of NADPH binding to active site mutants of human dihydrofolate reductase in complex with piritrexim.

2001 ◽  
Vol 48 (4) ◽  
pp. 903-916 ◽  
Author(s):  
W Nowak ◽  
V Cody ◽  
A Wojtczak
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Structural Changes ◽  
Energy Substitution ◽  
Energy Simulations ◽  
Detailed Computer ◽  
Human Dihydrofolate Reductase ◽  
Folate Cycle ◽  
Active Site Mutants

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is one of the enzymes active in the folate cycle which plays an important role in DNA synthesis. Inhibition of DHFR is a key element in the treatment of many diseases, including cancer and AIDS related infections. A search for new selective inhibitors is motivated by the resistance to common drugs observed in the course of treatment. In this paper, results of a detailed computer analysis of human DHFR interactions with the lipophilic inhibitor piritrexim (PTX) are presented. It was found that the NADPH cofactor contributes 30% of the total PTX-enzyme interaction energy. Substitution of the highly conserved Glu30 with alanine does not lead to the release of the inhibitor from the hDHFR pocket. The important L22F point mutation does affect PTX orientation but does not changethe binding energy. Simulations of the dynamics of binary hDHFR-PTX complexes were performed with the use of Extensible Systematic Force Field (ESFF) and the results indicate structural changes in the enzyme induced by NADPH binding.

Increasing Methotrexate Resistance by Combination of Active-site Mutations in Human Dihydrofolate Reductase

2007 ◽  
Vol 373 (3) ◽  
pp. 599-611 ◽  
Author(s):  
Jordan P. Volpato ◽  
Elena Fossati ◽  
Joelle N. Pelletier
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Methotrexate Resistance ◽  
Human Dihydrofolate Reductase ◽  
Active Site Mutations
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