Title

The catalytic activity of Bacillus Stearothermophilus dihydrofolate reductase (BsDHFR) wildtype and mutant D27N

Presentation Type

Event

Start Date

27-4-2019 8:45 AM

End Date

27-4-2019 9:24 AM

Abstract

Tetrahydrofolate is a molecule essential for DNA synthesis. This molecule comes from a reaction dependent on dihydrofolate reductase or DHFR. Dihydrofolate (DHF) is converted into tetrahydrofolate (THF) by DHFR, with the help of cofactor NADPH. The 27th position of BsDHFR has been studied in other organisms in which it plays an important role, as part of the active site. To study the role of this residue in catalysis, the position in Bacillus Stearothermophilus DHFR was mutated into residue D27N, which lies in the active site of the enzyme. This mutant was expressed and purified. Once the purity and concentration were established, catalytic tests were performed. We determined the catalytic activity (kcat) for the D27N mutant and compared it to the value for the wildtype enzyme.

This is the first step in finding the catalytic efficiency (kcat/km). Once we know how efficient the enzyme is, we would like to test temperature dependency on wildtype DHFR since Bacillus Stearothermophilus is a thermophile. These studies should determine the overall importance of aspartic acid at the 27th position for catalytic activity, which we can predict to be extremely important since the same aspartic acid is present in E.Coli DHFR and has been established as necessary for catalysis.

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Apr 27th, 8:45 AM Apr 27th, 9:24 AM

The catalytic activity of Bacillus Stearothermophilus dihydrofolate reductase (BsDHFR) wildtype and mutant D27N

Tetrahydrofolate is a molecule essential for DNA synthesis. This molecule comes from a reaction dependent on dihydrofolate reductase or DHFR. Dihydrofolate (DHF) is converted into tetrahydrofolate (THF) by DHFR, with the help of cofactor NADPH. The 27th position of BsDHFR has been studied in other organisms in which it plays an important role, as part of the active site. To study the role of this residue in catalysis, the position in Bacillus Stearothermophilus DHFR was mutated into residue D27N, which lies in the active site of the enzyme. This mutant was expressed and purified. Once the purity and concentration were established, catalytic tests were performed. We determined the catalytic activity (kcat) for the D27N mutant and compared it to the value for the wildtype enzyme.

This is the first step in finding the catalytic efficiency (kcat/km). Once we know how efficient the enzyme is, we would like to test temperature dependency on wildtype DHFR since Bacillus Stearothermophilus is a thermophile. These studies should determine the overall importance of aspartic acid at the 27th position for catalytic activity, which we can predict to be extremely important since the same aspartic acid is present in E.Coli DHFR and has been established as necessary for catalysis.