Parasites of the phylum Apicomplexa cause substantial morbidity, mortality and economic losses, and new medicines to treat them are needed urgently. The shikimate pathway is an attractive target for herbicides and antimicrobial agents because it is essential in algae, higher plants, bacteria and fungi, but absent from mammals. Here we present biochemical, genetic and chemotherapeutic evidence for the presence of enzymes of the shikimate pathway in apicomplexan parasites. In vitro growth of Toxoplasma gondii, Plasmodium falciparum (malaria) and Cryptosporidium parvum was inhibited by the herbicide glyphosate, a well-characterized inhibitor of the shikimate pathway enzyme 5-enolpyruvyl shikimate 3-phosphate synthase. This effect on T. gondii and P. falciparum was reversed by treatment with p-aminobenzoate, which suggests that the shikimate pathway supplies folate precursors for their growth. Glyphosate in combination with pyrimethamine limited T. gondii infection in mice. Four shikimate pathway enzymes were detected in extracts of T. gondii and glyphosate inhibited 5-enolpyruvyl shikimate 3-phosphate synthase activity. Genes encoding chorismate synthase, the final shikimate pathway enzyme, were cloned from T. gondii and P. falciparum. This discovery of a functional shikimate pathway in apicomplexan parasites provides several targets for the development of new antiparasite agents.

The herbicide glyphosate (N‐phosphonomethy glycine) is a potent reversible inhibitor of the 5‐enolpyruvylshikimate‐3‐phosphate (EPSP) synthase activity of the purified arom multienzyme complex from Neurospora crassa. Inhibition of the EPSP synthase reaction by glyphosate is competitive with respect to phosphoenolpyruvate, with K i 1.1 μM, and uncompetitive with respect to shikimate‐3‐phosphate. The kinetic patterns are consistent with a compulsory order sequential mechanism in which either PEP or glyphosate can bind to an enzyme: shikimate‐3‐phosphate complex.

Rapid events in the processes of electron transfer and substrate binding to cytochrome P-450 BM3 from Bacillus megaterium and its constituent haem-containing and flavin-containing domains have been investigated using stopped-flow spectrophotometry. The formation of a blue semiquinone flavin form occurs during the NADPH-dependent reduction of the flavin domain and a species with a similar absorption maximum is also seen during reduction of the holoenzyme by NADPH. EPR spectroscopy confirms the formation of the flavin semiquinone. The formation of this semiquinone is transient during fatty acid monooxygenation by the holoenzyme, but in the presence of excess NADPH the species reforms once fatty acid is exhausted. Electron transfers through the reductase domain are too rapid to limit the fatty acid monooxygenation reaction. The substrate-binding-induced haem iron spin-state shift also occurs much faster than the k,,, at 25°C. The rate of first electron transfer to the haem domain is also rapid; but it is of the order of 5-10-times larger than the k,,, for the enzyme (dependent on the fatty acid used).Given that two successive electron transfers to haem iron are required for the oxygenation reaction, these rates are likely to exert some control over the rate of fatty acid oxygenation reactions. The presence of large amounts of NADPH also results in decreased rates of electron transfer from flavin to haem iron. In the difference spectrum of the active fatty acid hydroxylase, features indicative of a high-spin iron haem accumulate. These are in accordance with the presence of large amounts of an Fe'+-product bound enzyme during turnover and indicate that product release may also contribute to rate limitation. Taken together, these data suggest that the catalytic rate is not determined by the accumulation of a single intermediate in the reaction scheme, but rather that it is controlled in a series of steps.Keywords: cytochrome P-450; stopped-flow kinetics ; EPR; electron transfer.The cytochrome P-450 monooxygenases (P-450) are a ubiquitous superfamily of haem enzymes which catalyse insertion of oxygen into an enormous variety of both physiological and nonphysiological organic substrates [l -31. P-450 generally fall into one of two broad classes. Class I P-450 (bacterial/mitochondrial) are three component systems comprised of an NAD(P)H-binding flavoprotein reductase, a small iron-sulfur protein and the P-450, which is membrane bound in eukaryotic forms [4]. Class I1 P-450 (microsomal) are two component systems comprising an FAD-containing and FMN-containing NADPH-cytochrome P-450 reductase and the P-450. This class is found almost exclusively in eukaryotes, where both components are membrane bound [4].There are many prokaryotic class I P-450, the best characterised being the P-450cum camphor hydroxylase from Pseudomonus putida. P-450cum (CUP 101) was the first P-450 for which an atomic structure was determined [5]. More recently, a unique prokaryotic class I1 flavocytochrome P-450 from Bacillus megaterium has been char...

The structure of the type II DHQase from Streptomyces coelicolor has been solved and refined to high resolution in complexes with a number of ligands, including dehydroshikimate and a rationally designed transition state analogue, 2,3-anhydro-quinic acid. These structures define the active site of the enzyme and the role of key amino acid residues and provide snap shots of the catalytic cycle. The resolution of the flexible lid domain (residues 21-31) shows that the invariant residues Arg23 and Tyr28 close over the active site cleft. The tyrosine acts as the base in the initial proton abstraction, and evidence is provided that the reaction proceeds via an enol intermediate. The active site of the structure of DHQase in complex with the transition state analog also includes molecules of tartrate and glycerol, which provide a basis for further inhibitor design.

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