Heterologous expression of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania major

Grumont, Raelene J.; Sirawaraporn, Worachart; Santi, Daniel V.

doi:10.1021/bi00410a039

Cited by 47 publications

(26 citation statements)

References 29 publications

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“…Enzyme-The clone of the wild-type bifunctional TS-DHFR enzyme from L. major was a generous gift from C.-C. Kan and D. Matthews, then at Agouron Pharmaceuticals. This clone, harboring the pO2CLSA-4 plasmid in an E. coli Rue 10 expression vector, was used to obtain protein of high purity (Ͼ99%) using methods described previously for purification (28). The wild-type protein has both TS and DHFR activities similar to those reported previously (11,28,29).…”

Section: Methodsmentioning

confidence: 82%

“…This clone, harboring the pO2CLSA-4 plasmid in an E. coli Rue 10 expression vector, was used to obtain protein of high purity (Ͼ99%) using methods described previously for purification (28). The wild-type protein has both TS and DHFR activities similar to those reported previously (11,28,29). Mutations were made using a QuikChange mutagenesis kit (Stratagene).…”

Section: Methodsmentioning

confidence: 99%

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

Atreya

Johnson

Williamson

et al. 2003

Journal of Biological Chemistry

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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...

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Section: Methodsmentioning

confidence: 82%

Section: Methodsmentioning

confidence: 99%

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

Atreya

Johnson

Williamson

et al. 2003

Journal of Biological Chemistry

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“…They found that the recombinant protein accumulated to very high levels within E. coli, particularly under control of the T7 promoter, but most of the recombinant protein was present as inactive inclusion bodies and usually <2% of the soluble protein was catalytically active TS-DHFR. In contrast, Grumont et al (41) achieved better success in expressing the leishmanial TS-DHFR in yeast cells.…”

Section: Discussionmentioning

confidence: 96%

“…In general, however, the best way to identify the most effective expression system for a specific protein is largely empiric. For example, Grumont et al (41) expressed the bifunctional protein thymidylate synthetase-dihydrofolate reductase (TS-DHFR) of a protozoan parasite, Leishmania major, using several different vectors including those driven by tac, PL, and T7 RNA polymerase promoters. They found that the recombinant protein accumulated to very high levels within E. coli, particularly under control of the T7 promoter, but most of the recombinant protein was present as inactive inclusion bodies and usually <2% of the soluble protein was catalytically active TS-DHFR.…”

Section: Discussionmentioning

confidence: 99%

High level expression in Escherichia coli of soluble, enzymatically active schistosomal hypoxanthine/guanine phosphoribosyltransferase and trypanosomal ornithine decarboxylase.

Craig

Yuan

Kuntz

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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The bacterial alkaline phosphatase (phoA) promoter and signal peptide have been used previously to control recombinant expression and secretion of eukaryotic proteins in Escherichia coli. Other reports have shown that this expression system can generate relatively modest levels of active hypoxanthine/guanine phosphoribosyltransferase (HPRT; hypoxanthine phosphoribosyltransferase; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), which carries part of the signal peptide but remains in the cytosol of the bacteria. Herein, the phoA promoter without its associated signal peptide is used to regulate expression of the HPRT of Schistosoma mansoni and the ornithine decarboxylase (ODC; L-ornithine carboxy-lyase, EC 4.1.1.17) of Trypanosoma brucei, two enzymes that have been identified as potential targets for antiparasitic chemotherapy. The levels of recombinant expression range from 20% to 60% of the total bacterial protein, and the majority of both recombinant enzymes was soluble. The specific activity for the recombinant trypanosomal ODC was one-third to two-thirds that of the authentic native enzyme and yields were predicted to be 15-30 mg of active enzyme per liter of bacterial culture. The specific activity for the recombinant schistosomal HPRT was equivalent to that for the native enzyme purified from schistosomes and up to 10 mg ofenzymatically active HPRT has been purified from a 0.5-liter culture of treated bacteria. These results represent a breakthrough in recombinant expression of HPRT and ODC.Schistosoma mansoni and Trypanosoma brucei are the etiologic agents for the important tropical diseases, schistosomiasis and African sleeping sickness, respectively. Within S. mansoni, the hypoxanthine/guanine phosphoribosyltransferase (HPRT; hypoxanthine phosphoribosyltransferase; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) is responsible for the salvage of purine bases that are needed for cellular metabolism (1, 2), and since schistosomes do not have de novo synthetic pathways for purine bases, the HPRT has been proposed as a potential target for antiparasitic chemotherapy (2, 3). In T. brucei the ornithine decarboxylase (ODC; L-ornithine carboxy-lyase, EC 4.1.1.17) catalyzes the first step in the metabolic pathway for the biosynthesis of polyamines. This enzyme has been shown previously to be inhibited by the antitrypanosomal drug, difluoromethyl ornithine (DFMO; and, therefore, the enzyme is a target for antiparasitic chemotherapy. In humans, the metabolic importance ofthe homologue to the schistosomal HPRT is evidenced by the fact that mutations within the gene encoding this enzyme can result in gout (7) or Lesch-Nyhan syndrome (8). Similarly, the ODC of mammals has been shown to be essential for putrescine, spermidine, and spermine synthesis (9), as well as for cell proliferation (10).X-ray crystallographic analysis of three-dimensional structures of these enzymes has the potential for providing information that would be critical for the design or improvement of inhibitors targeted to...

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“…Roos. These clones were used to obtain protein of high purity by previously described methods for purification (7,17 (18).…”

mentioning

confidence: 99%

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

Atreya¹,

Anderson

2004

Journal of Biological Chemistry

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This study presents a kinetic characterization of the recently crystallized bifunctional thymidylate synthasedihydrofolate reductase (TS-DHFR) enzyme from the apicomplexa parasite, Cryptosporidium hominis. Our study focuses on determination of the C. hominis TS-DHFR kinetic mechanism, substrate channeling behavior, and domain-domain communication. Unexpectedly, the unique mechanistic features of C. hominis TS-DHFR involve the highly conserved TS domain. At 45 s ؊1 , C. hominis TS activity is 10 -40-fold faster than other TS enzymes studied and a new kinetic mechanism was required to simulate C. hominis TS behavior. A large accumulation of dihydrofolate produced at TS and a lag in product formation at DHFR were observed. These observations make C. hominis TS-DHFR the first bifunctional TS-DHFR enzyme studied for which there is clear evidence against dihydrofolate substrate channeling. Furthermore, whereas with Leishmania major TS-DHFR there are multiple lines of evidence for domain-domain communication (ligand binding at one active site affecting activity of the other enzyme), no such effects were observed with C. hominis TS-DHFR.Protozoal parasites such as Cryptosporidium hominis, Plasmodium falciparum, Toxoplasma gondii, and Leishmania major are unusual in that their thymidylate synthase (TS) 1 and dihydrofolate reductase (DHFR) enzymes exist on the same polypeptide as part of a single bifunctional TS-DHFR enzyme.2 TS and DHFR are critical enzymes and established drug targets. TS represents the only means of de novo synthesis of 2Ј-deoxythymidylate (dTMP) for DNA synthesis, via reductive methylation of 2-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 tetrahydrofolate (H 4 folate), used for one carbon unit transfer reactions in several biochemical processes, including thymidylate, purine, and amino acid biosynthesis.Almost a decade of research on bifunctional TS-DHFR from protozoal parasites has relied on the only available TS-DHFR crystal structure, that from the kinetoplastid protozoa L. major (1). The recent solution of the crystal structures of TS-DHFR enzymes from two clinically relevant apicomplexan protozoa, P. falciparum and C. hominis, however, has revealed unanticipated deviations from the kinetoplastid model (2, 3). An obvious next question is whether these significant structural differences translate as significant mechanistic differences between parasite classes. We sought to address this question through a detailed kinetic characterization of TS-DHFR from C. hominis, again yielding surprising results.A major conclusion of the recent solution of the apicomplexan TS-DHFR structures is that there exist two families of bifunctional TS-DHFR structures: a short linker family with an Nterminal tail, as in the kinetoplastids (a class that includes Leishmania and the trypanosomes); and a long linker family with a donated or crossover helix, as in the apicomplexan paras...

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Heterologous expression of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania major

Cited by 47 publications

References 29 publications

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

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

High level expression in Escherichia coli of soluble, enzymatically active schistosomal hypoxanthine/guanine phosphoribosyltransferase and trypanosomal ornithine decarboxylase.

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

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