Probing Electrostatic Channeling in Protozoal Bifunctional Thymidylate Synthase-Dihydrofolate Reductase Using Site-directed Mutagenesis

Abstract

In this study we used site-directed mutagenesis to test the hypothesis that substrate channeling in the bifunctional thymidylate synthase-dihydrofolate reductase enzyme from Leishmania major occurs via electrostatic interactions between the negatively charged dihydrofolate produced at thymidylate synthase and a series of lysine and arginine residues on the surface of the protein. Accordingly, 12 charge reversal or charge neutralization mutants were made, with up to 6 putative channel residues changed at once. The mutants were assessed for impaired channeling using two criteria: a lag in product formation at dihydrofolate reductase and an increase in dihydrofolate accumulation. Surprisingly, none of the mutations produced changes consistent with impaired channeling, so our findings do not support the electrostatic channeling hypothesis. Burst experiments confirmed that the mutants also did not interfere with intermediate formation at thymidylate synthase. One mutant, K282E/R283E, was found to be thymidylate synthase-dead because of an impaired ability to form the covalent enzyme-methylene tetrahydrofolate-deoxyuridate complex prerequisite for chemical catalysis.Electrostatic channeling is a mechanism proposed based on the crystal structure of bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) 1 from Leishmania major that would enable negatively charged dihydrofolate produced at the TS active site to be handed off along a series of solventexposed lysine and arginine residues to the DHFR active site, where it is converted to tetrahydrofolate (H 4 folate) (1).TS and DHFR are crucial enzymes, found in all species. TS represents the only means of de novo synthesis of 2Ј-deoxythymidylate (dTMP) for DNA synthesis, via reductive methylation of 2Ј-deoxyuridate (dUMP) with methylene tetrahydrofolate (CH 2 H 4 folate), producing dihydrofolate (H 2 folate) in the process. DHFR catalyzes the reduction of H 2 folate by NADPH to generate H 4 folate, used for one carbon unit transfer reactions in several biochemical processes, including thymidylate, purine, and amino acid biosynthesis. Only in protozoal parasites and some plants however, are TS and DHFR found on the same polypeptide chain, leading to the hypothesis that in these organisms, H 2 folate may be channeled from the TS active site to that of DHFR, never equilibrating with bulk solution (Fig. 1A). When the crystal structure of L. major TS-DHFR was solved (2.9 Å resolution), a 40 Å "electrostatic highway" across the surface of the protein was proposed as an explanation for how channeling may occur (1, 2). We sought to test the electrostatic channeling hypothesis with two parallel approaches. In this report we present results of mutagenesis of solvent-exposed basic residues comprising the electrostatic highway. In an earlier paper, we reported the findings from targeted molecular docking searches to identify small molecule inhibitors that bind in this region, as a means to block the putative channel (3).There are precedents for channeling among bifunction...

Kinetic Characterization of Bifunctional Thymidylate Synthase-Dihydrofolate Reductase (TS-DHFR) from Cryptosporidium hominis

“…H 2 folate and the TS intermediate are plotted together to simulate the experimental data, but are modeled separately in the inset (same axis labels implied). material (8). Similar behavior is observed with T. gondii TS-DHFR (5).…”

“…High Performance Liquid Chromatography (HPLC) Analysis-Tritiated products of the rapid quench experiments were quantified by HPLC in combination with a radioactivity flow detector as detailed previously (8). The HPLC separation was performed using a BDSHypersil C18 reverse phase column (250 ϫ 4.6 mm, Keystone Scientific, Bellefonte, PA) with a flow rate of 1 ml/min.…”

“…Steady-State Spectroscopic Channeling Assays-The steady-state TS-DHFR reaction was monitored spectroscopically for a lag in NADP ϩ formation at DHFR using a procedure detailed previously (8,9). The predicted change in absorbance is very small, therefore a 10-cm path length quartz observation cell was used to enhance sensitivity.…”

“…Indeed, there is evidence for substrate channeling with both L. major and T. gondii TS-DHFR. In the case of these bifunctional enzymes, unlike with the combination of monofunctional TS ϩ DHFR, H 2 folate produced at the TS active site does not substantially accumulate in solution and there is no lag in product formation at DHFR, suggesting that the TS product does not leave the protein and enter bulk solution before binding at DHFR (5,7,8). Steady-state spectroscopic analysis, used to detect the presence or absence of a lag in NADP ϩ production via the DHFR-catalyzed reduction of H 2 folate formed at TS, also supports substrate channeling by both L. major and T. gondii TS-DHFR (9,10).…”

Crystal Structure of Liganded Rat Peroxisomal Multifunctional Enzyme Type 1

“…The mechanism that allows for substrate channeling of such extensive polar molecules is poorly understood and has been addressed only in studies of few enzymes, for example dihydrofolate reductase-thymidylate synthase and the bacterial fatty acid oxidation ␣ 2 ␤ 2 complex. Electrostatic interactions have been proposed to be important for the channeling in the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (23,24), whereas for the bacterial fatty acid oxidation complex, a substrate-anchored diffusion mechanism has been proposed (25).…”