Dihydrofolate Reductase (DHFR) Ruixiao ‘Ray’ Gao Department of chemistry Illinois state university 10/30/2014

DHFR is a key enzyme in folate metabolism DHFR is a key enzyme in folate metabolism. It contributes to the de novo mitochondrial thymidylate biosynthesis pathway. Catalyzes an essential reaction for de novo glycine and purine synthesis, and for DNA precursor synthesis. Binds its own mRNA and that of DHFRL1. The DHFR gene is located on long (q) arm of chromosome 5 between positions 11.2 and 13.2. More precisely, the DHFR gene is located from base pair 80,626,225 to base 80,654,980 on chromosome 5 . DHFR catalyzes the transfer of a hydride from NADPH to dihydrofolate with an accompanying protonation to produce tetrahydrofolate. In the end, dihydrofolate is reduced to tetrahydrofolate and NADPH is oxidized to NAPD+.  Background Found in all organisms: Bacteria Human Virus Encoded by DHFR gene that exists in chromosome 5 Homo Dimer Bind substrates Catalyze enzyme DNA precursor synthesis De novo mitochondrial thymidylate biosynthesis pathway

structure This figure shows the structure of DHFR isoform, staphylococcus aureus, by using Rasmol through NCBI with PDB ID: 3FRA. Rasmol displayed 3D saDHFR structure is in cartoon diagram with alpha helices in pink and beta sheets in yellow. There are three alpha helices positions in this amino acids sequence shown above, helix 12, helix 11, and helix 16. This string is defined from residue 1-125 with 1984 atoms selected. Each particular helix has couple of interactions in theory stabilize the alpha helix such as serine 24 bound with glycine 39, glycine 102 and glutamic acid 112, serine 81 and glycine 9. However, the helix with side chain interaction may also destabilize the overall structure, such as helix 8 with arginine 45 and glycine 52. These amino acid side chains can’t form either hydrogen bond, or attract each other, therefore destabilize overall helix

Amino Acid Sequence Alignment Residues “Met20” or “loop 1” and, along with other loops, are part of the major subdomain that surround the active site.The active site is situated in the N-terminal half of the sequence, which includes a conserved Pro-Trp （PW) dipeptide; the tryptophan has been shown to be involved in the binding of substrate by the enzyme. Met20 Green: Major AA to bind substrates in active site Red: Residue Met20/Loop 1 as one of major subdomains

Light Grey: Beta Sheets Stick on Loop: Met20 Blue: Ser24 & Gly39 Pink: alpha helice Light Grey: Beta Sheets Stick on Loop: Met20 Blue: Ser24 & Gly39 Sticks: Gly102 & Glu112 Ser81 & Gly9 Blue & sticks on helices are used to stablize alpha helices through hydrogen bonding 26.04.2017

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Anticancer Properties Antimalarial drugs for their anticancer potential: DHFR inhibitors resist the growth of several human cancer lines Bind with pyrimethamine and P218 Cluster analysis clustered tighly the synthetic peroxides and DHFR inhibitors. Artemisinins & paclitaxel with DHFR inhibitors kill cancer cells by inducing apoptosis 26.04.2017

DHFR Mutants (A) Dimer structure of TS-DHFR. TS and DHFR domains are labeled. Crossover helix and Helix B are also labeled in the DHFR domains. The DHFR ligands, NADP+ and H2F are shown in sticks. (B) Close-up of the crossover helix region. Residues on the crossover helix (light grey) are displayed as well as residues on the active site helix (dark grey). (C) Space filling representation highlighting the close interactions of the crossover helix (light grey) and helix B (dark grey) residues. DHFR active site ligands are shown in sticks. Plots show the rate constant of single DHFR turnover reactions, results indicate that binding with mutated DHFR proteins will significantly decrease the protein catalyst reactivity because the amino acid side chains will become nonpolar with no charge which can’t form either hydrogen bond, or attract any other substrates/enzymes.

Thank you 26.04.2017