The Enzymic Synthesis of Dihydropteroate and Dihydrofolate by Plasmodium berghei

Abstract

SYNOPSIS. Cell‐free extracts of the rodent malaria parasite Plasmodium berghei synthesized dihydropteroate (H2pteroate) and dihydrofolate (H2folate) from 2‐amino‐4‐hydroxy‐6‐hydroxymethyl‐7,8‐dihydropteridine (hydroxymethyldihydropteridine) and p‐aminobenzoate (pAB) or p‐aminobenzoylglutamate (pABG). The reaction was demonstrated also in extracts of Plasmodium gallinaceum, Plasmodium lophurae and Plasmodium knowlesi, by the use of a microbiologic assay method and pABG as cosubstrate. Some of the properties of the enzymes involved were investigated in P. berghei preparations, utilizing a radioactive assay which measures the conversion of [7‐14C]pAB to [14C]H2pteroate. Apparent Km values of 0.28 μM for [7‐14C]pAB, 0.037 mM for pABG and 0.8 μM for hydroxymethyldihydropteridine were obtained. The reaction had absolute requirements for ATP and Mg++, and was stimulated by dithiothreitol. The enzymes required for the reaction were eluted together from Sephadex G‐200 columns in a molecular weight range of 200,000–250,000. In bacteria hydroxymethyldihydropteridine is converted 1st by a pyrophosphokinase to pyrophosphorylmethyldihydropteridine, and this compound is then condensed with pAB to form H2pteroate by H2pteroate synthetase. Both enzymic activities were demonstrated in P. berghei preparations and separated by DEAE‐Sephadex chromatography. The enzymic synthesis of H2pteroate by P. berghei is inhibited by several sulfonamides and diaminodiphenylsulfone (DDS). The latter compound is shown to be competitive with pAB, with a Ki value of 0.38 μM; pABG is also a competitive inhibitor. These data establish an enzymic basis of support for the evidence obtained in vivo which indicate that malaria synthesize their folate cofactors de novo. It is suggested that the antimalarial action of sulfonamides and DDS is due to their inhibition of plasmodial H2pteroate synthetase.

Interaction of sulfonamide and sulfone compounds with Toxoplasma gondii dihydropteroate synthase.

“…In investigating the Michaelis-Menten constants for the DHPS enzyme with respect to both substrates, we found the affinity constant for pABA to be similar to those reported for enzymes isolated from both P. berghei (0.28 uM) and E. coli (0.21 uM) by Ferone and co-workers (16,28), but somewhat tighter than that reported by others (1.1-2.8 MM) (29,30). These differences may relate to the dependence of affinity on the purity of the enzyme preparation.…”

“…Purification of DHPS from potential inhibitors present in the crude preparations represents an additional possibility that has not been excluded. Instability of the DHPS enzymatic activity isolated from other organisms has not previously been noted and may be due to differences in the purity or source of the preparations used in other studies (16,(27)(28)(29)(30). The DHPS activity in crude cytosolic preparations of T. gondii was stable and became unstable only with purification > 100-200-fold.…”

“…We found that the Km for pABA differed by a factor of 10 between crude and pure enzyme, while the Km for the pterine substrate showed little dependence on the purity of the enzyme. The affinity of T. gondii DHPS for the pterin pyrophosphate substrate is slightly weaker (-5 uM) than that reported for either P. berghei or E. coli (0.5-1.4 uM) (16,28).…”

“…Each point represents the mean of three to six independent experiments. (16,30,31). DHPS protein purified through the green Sepharose affinity column did not appear to be homogeneous, as at least one additional protein (74,000 mol wt) was consistently noted to copurify.…”

Sequence Variation of the Hydroxymethyldihydropterin Pyrophosphokinase: Dihydropteroate Synthase Gene in Lines of the Human Malaria Parasite, Plasmodium falciparum, with Differing Resistance to Sulfadoxine

“…This suggests, on the basis of the M, predicted from our sequence (83 398), that P. falciparum CH,OH-H,pterinPP kinase: H,Pte synthase is dimeric in vivo, as found for bacterial H,Pte synthase molecules [40]. However, the covalent association of CH,OH-H,pterinPP kinase and H,Pte synthase may not be general among all Plasmodium species, as the activities from another rodent parasite, Plasmodium berghei, are reported to be separable [41]. Relative to the other known CH,OH-H,pterinPP kinase sequences, the P. falciparum domain is much larger, containing two long insertions of 95 and 92 amino acid residues, the latter having an unusual, highly Asn-rich (25 %) sequence.…”