Kinetic studies of wild-type and mutant Staphylococcus aureus dihydrofolate reductase (SaDHFR) for drug discovery.

Dihydrofolate reductase (DHFR) is an attractive enzyme as a drug target since
inhibition of bacterial DHFR function will impede microorganism growth and enable us to
overcome the infections. Here, we focus on the study of DHFR from Staphylococcus aureus,
a gram-positive bacterium that is a common cause of hospital-associated infections. Since
antibiotic-resistant strains of S. aureus are becoming more prevalent, most researchers try to
discover new compounds with greater potential to counteract the resistant strains. In order to
discover a new drug, a large number of natural product analogs usually need to be
synthesized. Instead of synthesis of new compounds, which is time-consuming and
expensive, we have developed a computational method to select the distance inside the
binding pocket of proteins that should be expanded or contracted in order to achieve higher
affinity of inhibitor binding. To validate the computational prediction using SaDHFR as the
model sytem, we replaced Leu5 with Val to expand the distance and replaced Asp27 with Glu
to contract the distance. Kinetic experiments were used to determine the K i values of both
wild-type and mutant SaDHFR for the bacterial-specific DHFR inhibitor trimethoprim
(TMP). By expanding the distance as a result of the L5V mutation, the K i value is decreased
when compare to that of wild-type enzyme, which is in accordance with the prediction.
However, the D27E mutant does not behave as expected. One plausible explanation for this
observation is that the altered distance from the E27 could place the carboxylate group at a
position that is not optimal for catalysis.