Purification, properties, and crystallization of Saccharomyces cerevisiae dihydropterin pyrophosphokinase-dihydropteroate synthase.

The tri-functional enzyme of Saccharomyces cerevisiae dihydroneopterin aldolase (DHNA)-dihydropterin pyrophosphokinase (PPPK)-dihydropteroate synthase (DHPS) catalyzes three sequential steps in folate biosynthesis. A cDNA encoding the PPPK and DHPS domains of the tri-functional enzyme has been cloned. This bi-functional enzyme was expressed as a His(6) fusion protein in Escherichia coli and the protein was purified to apparent homogeneity. The purified protein possesses both PPPK and DHPS activities as measured by the incorporation of [(3)H]p-ABA into the appropriate substrate. The pH optimum of the DHPS activity was determined to be 8.5. Gel filtration measurement indicates that the protein exists as a dimer in solution. A robotic screening method was used to identify crystallization conditions. Bi-pyramidal crystals of the enzyme formed with the protein in the presence of a pterin substrate analog in phosphate buffer (pH 6.3) and these diffracted to 2.3A. Structural information from these crystals could be used to design novel drugs to inhibit folate biosynthesis.

The three-dimensional structure of the bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/dihydropteroate synthase of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae and other fungi, the enzymes dihydroneopterin aldolase, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) are encoded by a polycistronic gene that is translated into a single polypeptide having all three functions. These enzymatic functions are essential to both prokaryotes and lower eukaryotes, and catalyse sequential reactions in folate biosynthesis. Deletion or disruption of either function leads to cell death. These enzymes are absent from mammals and thus make ideal antimicrobial targets. DHPS is currently the target of antifolate therapy for a number of infectious diseases, and its activity is inhibited by sulfonamides and sulfones. These drugs are typically used as part of a synergistic cocktail with the 2,4-diaminopyrimidines that inhibit dihydrofolate reductase. A gene encoding the S.cerevisiae HPPK and DHPS enzymes has been cloned and expressed in Escherichia coli. A complex of the purified bifunctional polypeptide with a pterin monophosphate substrate analogue has been crystallized, and its structure solved by molecular replacement and refined to 2.3A resolution. The polypeptide consists of two structural domains, each of which closely resembles its respective monofunctional bacterial HPPK and DHPS counterpart. The mode of ligand binding is similar to that observed in the bacterial enzymes. The association between the domains within the polypeptide as well as the quaternary association of the polypeptide via its constituent DHPS domains provide insight into the assembly of the trifunctional enzyme in S.cerevisiae and probably other fungal species.

Analysis in Escherichia coli of Plasmodium falciparum dihydropteroate synthase (DHPS) alleles implicated in resistance to sulfadoxine.

Mutations in Plasmodium falciparum dihydropteroate synthase have been linked to resistance to the antimalarial drug, sulfadoxine, which competes with the dihydropteroate synthase substrate, p-aminobenzoate. In an effort to evaluate the role of these mutations in a simple model system, we have expressed six relevant alleles of the P. falciparum dihydropteroate synthase gene in Escherichia coli. When each construct was produced in a dihydropteroate synthase disrupted E. coli strain that required thymidine, the thymidine requirement was lost, indicating heterologous complementation had occurred. In the presence of sulfadoxine, the growth of the strain with the wild-type dihydropteroate synthase allele was inhibited while those containing each of the five mutant alleles grew, indicating that these mutations can confer sulfadoxine resistance in E. coli. When tested against twelve additional 'sulfa' drugs a variety of responses were obtained. All strains were resistant to sulfadiazine, but the wild-type allele conferred sensitivity to all other sulfa drugs. Three alleles conferred resistance to dapsone, a drug that is to be targetted for a new regime of malaria treatment in Africa. All mutant alleles remained sensitive to sulfachloropyridazine and sulfacetamide. These results suggest new drugs that could be tried for effective malaria treatment.

Promoter strength of folic acid synthesis genes affects sulfa drug resistance in Saccharomyces cerevisiae.

The enzyme dihydropteroate synthase (DHPS) is an important target for sulfa drugs in both prokaryotic and eukaryotic microbes. However, the understanding of DHPS function and the action of antifolates in eukaryotes has been limited due to technical difficulties and the complexity of DHPS being a part of a bifunctional or trifunctional protein that comprises the upstream enzymes involved in folic acid synthesis (FAS). Here, yeast strains have been constructed to study the effects of FOL1 expression on growth and sulfa drug resistance. A DHPS knockout yeast strain was complemented by yeast vectors expressing the FOL1 gene under the control of promoters of different strengths. An inverse relationship was observed between the growth rate of the strains and FOL1 expression levels. The use of stronger promoters to drive FOL1 expression led to increased sulfamethoxazole resistance when para-aminobenzoic acid (pABA) levels were elevated. However, high FOL1 expression levels resulted in increased susceptibility to sulfamethoxazole in pABA free media. These data suggest that up-regulation of FOL1 expression can lead to sulfa drug resistance in Saccharomyces cerevisiae.