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

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.

Download Full-text

Free Energy Simulations of Active-Site Mutants of Dihydrofolate Reductase

The Journal of Physical Chemistry B ◽

10.1021/jp5059963 ◽

2014 ◽

Vol 119 (3) ◽

pp. 906-916 ◽

Cited By ~ 14

Author(s):

Dvir Doron ◽

Vanja Stojković ◽

Lokesh Gakhar ◽

Alexandra Vardi-Kilshtain ◽

Amnon Kohen ◽

...

Keyword(s):

Free Energy ◽

Dihydrofolate Reductase ◽

Active Site ◽

Energy Simulations ◽

Active Site Mutants

Download Full-text

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

Biochemistry ◽

10.1021/bi00219a038 ◽

1991 ◽

Vol 30 (5) ◽

pp. 1432-1440 ◽

Cited By ~ 14

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

Download Full-text

Probing the role of two hydrophobic active site residues in the human dihydrofolate reductase by site-directed mutagenesis

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)47131-7 ◽

1989 ◽

Vol 264 (34) ◽

pp. 20786-20795

Author(s):

B.I. Schweitzer ◽

S Srimatkandada ◽

H Gritsman ◽

R Sheridan ◽

R Venkataraghavan ◽

...

Keyword(s):

Dihydrofolate Reductase ◽

Active Site ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Active Site Residues ◽

Human Dihydrofolate Reductase

Download Full-text

Determination of the functional role of phenylalanine-31 of recombinant human dihydrofolate reductase by site-directed mutagenesis

Zurich, Switzerland, September 3–8, 1989 ◽

10.1515/9783110889000-148 ◽

1990 ◽

pp. 765-768

Keyword(s):

Dihydrofolate Reductase ◽

Functional Role ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Human Dihydrofolate Reductase

Download Full-text

The Loop of the TPR1 Subdomain of Phi29 DNA Polymerase Plays a Pivotal Role in Primer-Terminus Stabilization at the Polymerization Active Site

Biomolecules ◽

10.3390/biom9110648 ◽

2019 ◽

Vol 9 (11) ◽

pp. 648

Author(s):

del Prado ◽

Santos ◽

Lázaro ◽

Salas ◽

de Vega

Keyword(s):

Dna Polymerase ◽

Active Site ◽

Structural Changes ◽

Binding Capacity ◽

Dna Polymerases ◽

Crystallographic Structure ◽

Genome Replication ◽

Terminal Protein ◽

Phi29 Dna Polymerase

Bacteriophage Phi29 DNA polymerase belongs to the protein-primed subgroup of family B DNA polymerases that use a terminal protein (TP) as a primer to initiate genome replication. The resolution of the crystallographic structure showed that it consists of an N-terminal domain with the exonuclease activity and a C-terminal polymerization domain. It also has two subdomains specific of the protein-primed DNA polymerases; the TP Regions 1 (TPR1) that interacts with TP and DNA, and 2 (TPR2), that couples both processivity and strand displacement to the enzyme. The superimposition of the structures of the apo polymerase and the polymerase in the polymerase/TP heterodimer shows that the structural changes are restricted almost to the TPR1 loop (residues 304–314). In order to study the role of this loop in binding the DNA and the TP, we changed the residues Arg306, Arg308, Phe309, Tyr310, and Lys311 into alanine, and also made the deletion mutant Δ6 lacking residues Arg306–Lys311. The results show a defective TP binding capacity in mutants R306A, F309A, Y310A, and Δ6. The additional impaired primer-terminus stabilization at the polymerization active site in mutants Y310A and Δ6 allows us to propose a role for the Phi29 DNA polymerase TPR1 loop in the proper positioning of the DNA and TP-priming 3’-OH termini at the preinsertion site of the polymerase to enable efficient initiation and further elongation steps during Phi29 TP-DNA replication.

Download Full-text