Expression and characterization of recombinant human-derived Pneumocystis carinii dihydrofolate reductase

Abstract

Dihydrofolate reductase (DHFR) is the target of trimethoprim (TMP), which has been widely used in combination with sulfa drugs for treatment and prophylaxis of Pneumocystis carinii pneumonia. While the rat-derived P. carinii DHFR has been well characterized, kinetic studies of human-derived P. carinii DHFR, which differs from rat-derived P. carinii DHFR by 38% in amino acid sequence, have not been reported to date. Here we report on the expression and kinetic characterization of the recombinant human-derived P. carinii DHFR. The 618-bp coding sequence of the human-derived P. carinii DHFR gene was expressed in Escherichia coli. As determined by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis, the purified enzyme had a molecular mass of 25 kDa, consistent with that predicted from the DNA sequence. Kinetic analysis showed that the K(m) values for dihydrofolate and NADPH were 2.7 +/- 0.3 and 14.0 +/- 4.3 microM, respectively, which are similar to those reported for rat-derived P. carinii DHFR. Inhibition studies revealed that both TMP and pyrimethamine were poor inhibitors of human-derived P. carinii DHFR, with K(i) values of 0.28 +/- 0.08 and 0.065 +/- 0.005 microM, respectively, while trimetrexate and methotrexate were potent inhibitors, with K(i) values of 0.23 +/- 0.03 and 0.016 +/- 0.004 nM, respectively. The availability of purified recombinant enzyme in large quantities should facilitate the identification of antifolate inhibitors with greater potency and higher selectivity for human-derived P. carinii DHFR.

FIG. 1

Coomassie blue-stained SDS-polyacrylamide gel of recombinant human-derived P. carinii DHFR. Lane 1, crude extract from cells containing vector pET28(+) alone; lane 2, crude extract from cells containing pET-DHFR; lane 3, soluble extract from cells containing pET-DHFR; lane 4, purified DHFR preparation (arrow). The migrations of protein size markers (kilodaltons) are indicated at the left.

FIG. 2

Determination of the K_m for dihydrofolate (DHF). Initial velocity measurements (micromoles per minute per milliliter) were performed at various concentrations of NADPH in the presence of different fixed concentrations of DHF, and the results were fitted to the Hanes equation (10) a/V = a/V_m + K_m/V_m, where a is the substrate concentration, V is the reaction velocity, and V_m is the maximal velocity. In the primary plot of [NADPH]/V versus [NADPH], each DHF concentration gives a straight line whose slope represents the reciprocal of the apparent maximal velocity (Vapp). In the secondary plot (inset), [DHF]/Vapp is plotted against [DHF], and the intercept on the [DHF] axis is the value for −K_m. This experiment was repeated five times, and representative results are shown.

FIG. 3

Determination of the K_i for the inhibitor TMP. Initial velocities (micromoles per minute per milliliter) were determined at various concentrations of dihydrofolate (DHF) in the presence of different fixed concentrations of TMP, and the results were fitted to the equation a/V = a/V_m + K_m(1 + i/K_i)/V_m, which is derived from the Dixon equation (8), where V is the reaction velocity, a is the dihydrofolate concentration, V_m is the maximal velocity, and i is the inhibitor concentration. Plots of [DHF]/V versus [DHF] at different concentrations of TMP give straight lines with y intercepts of K_m(1 + i/K_i)/V_m, which refer to Kapp/Vapp. These y intercepts were replotted against [TMP] (inset), and the intercept on the [TMP] axis is the value for −K_i. This experiment was repeated five times, and representative results are shown.

FIG. 4

Determination of the K_i for the tight-binding inhibitor trimetrexate (TMTX). The enzyme was preincubated with 75 μM NADPH and various concentrations of TMTX (between 4.0 and 64 nM), and then the reaction was started by adding 45 μM (Δ) or 90 μM (+) dihydrofolate (DHF). Steady-state velocities were measured and fitted to the Henderson equation (3, 12) I_t/(1 − V_i/V₀) = K_i(1 + A_t/K_a) V₀/V_i + E_t, where I_t is the total concentration of inhibitor, V_i is the velocity in the presence of inhibitor, V₀ is the velocity without inhibitor, K_i is the inhibition constant, A_t is the concentration of competing substrate DHF, K_a is the Michaelis constant for the DHF, and E_t is the enzyme concentration. Plots of I_t/(1 − V_i/V₀) against V₀/V_i give straight lines with slopes of K_i(1 + A_t/K_a); then K_i = slope/(1 + A_t/K_a). The crossing lines on the ordinate suggested competitive inhibition. This experiment was repeated three times, and representative results are shown.