Differential Inhibition of Class I and Class II 5-Enolpyruvylshikimate-3-phosphate Synthases by Tetrahedral Reaction Intermediate Analogues †,‡ Todd Funke, §,| Martha L. Healy-Fried, §,| Huijong Han, § David G. Alberg, ⊥ Paul A. Bartlett, ⊥ and Ernst Scho ¨nbrunn* ,§,∇ Department of Medicinal Chemistry, UniVersity of Kansas, Lawrence, Kansas 66045, and Department of Chemistry, UniVersity of California, Berkeley, California 94720-1460 ReceiVed June 4, 2007; ReVised Manuscript ReceiVed September 9, 2007 ABSTRACT: The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase or EPSPS) is best known as the target of the herbicide glyphosate. EPSPS is also considered an attractive target for the development of novel antibiotics since the pathogenicity of many microorganisms depends on the functionality of the shikimate pathway. Here, we have investigated the inhibitory potency of stable fluorinated or phosphonate-based analogues of the tetrahedral reaction intermediate (TI) in a parallel study utilizing class I (glyphosate-sensitive) and class II (glyphosate-tolerant) EPSPS. The (R)-difluoromethyl and (R)-phosphonate analogues of the TI are the most potent inhibitors of EPSPS described to date. However, we found that class II EPSPS are up to 400 times less sensitive to inhibition by these TI analogues. X-ray crystallographic data revealed that the conformational changes of active site residues observed upon inhibitor binding to the representative class I EPSPS from Escherichia coli do not occur in the prototypical class II enzyme from Agrobacterium sp. strain CP4. It appears that because the active sites of class II EPSPS do not possess the flexibility to accommodate these TI analogues, the analogues themselves undergo conformational changes, resulting in less favorable inhibitory properties. Since pathogenic microorganisms such as Staphylococcus aureus utilize class II EPSPS, we conclude that the rational design of novel EPSPS inhibitors with potential as broad-spectrum antibiotics should be based on the active site structures of class II EPSP synthases. The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase or EPSPS; 1 EC 2.5.1.19) catalyzes the sixth step of the shikimate pathway. The product of the shikimate pathway, chorismate, serves as a starting material for the biosynthesis of essential compounds including the aromatic amino acids and cofactors such as ubiquinone, vitamin K, and folate in plants, fungi, and microorganisms (1-3), while mammals obtain necessary aromatic compounds from their diets. A variety of evidence suggests that EPSPS and other shikimate pathway enzymes represent enticing drug targets. The deletion of the gene encoding EPSPS has been shown to result in attenuated virulence in Staphylococcus aureus, Streptococcus pneumoniae, and Bordetella bronchiseptica (4-6). Many organisms, including Mycobacterium tuber- culosis, Pseudamonas aeruginosa, Vibrio cholerae, and Yersinia pestis, require the production of chorismate-derived siderophores for pathogenicity (7, 8). Further, glyphosate has been shown to inhibit the in vitro growth of apicomplexan parasites including Toxoplasma gondii, Plasmodium falci- parum, and Cryptosporidium parVum (9). As the molecular target of glyphosate (the active ingredient in Roundup herbicide), EPSPS has been the subject of extensive investigation (10, 11). While glyphosate has proven to be a potent inhibitor of EPSPS from plants and Escherichia coli, several glyphosate-tolerant forms of EPSPS have been identified (12-16). Glyphosate-tolerant EPSPS isolated from organisms including S. aureus, S. pneumoniae, Pseudomonas sp. strain PG2982, and Agrobacterium sp. strain CP4 are termed class II enzymes, while the plant and E. coli enzymes are considered class I enzymes (16-18). The molecular basis for glyphosate tolerance in the prototypical class II enzyme Agrobacterium sp. strain CP4 EPSPS has been elucidated recently (19). Although glyphosate restricts the growth of apicomplexan parasites, its antibiotic properties are poor, and pathogens such as S. pneumoniae and S. aureus have class II EPSPS which present unsuitable targets for glyphosate (16, 17). Thus, the effective targeting of microbial EPSPS requires new strategies toward the design of novel inhibitors. In † This work was supported by the National Institutes of Health (Grant 1R01 GM70633-02). ‡ The atomic coordinates and structure factors have been deposited in the Protein Data Bank (http://www.rcsb.org/), codes 2PQ9 for E. coli EPSPS liganded with 2F-TI, 2PQB for CP4 EPSPS liganded with 2F-TI, 2PQC for CP4 EPSPS liganded with RP-TI, and 2PQD for Ala100Gly CP4 EPSPS liganded with 2F-TI. * To whom correspondence should be addressed. Phone: (813) 745- 4703. Fax: (813) 745-6748. E-mail: ernst.schonbrunn@moffitt.org. § University of Kansas. | These authors contributed equally to this work. ⊥ University of California. ∇ Current address: Drug Discovery Program, H. Lee Moffitt Cancer Research Institute, Tampa, FL 33612. 1 Abbreviations: EPSP synthase or EPSPS, 5-enolpyruvylshikimate- 3-phosphate synthase; TI, tetrahedral reaction intermediate; S. aureus, Staphylococcus aureus; E. coli, Escherichia coli; CP4, Agrobacterium sp. strain CP4; PEP, phosphoenolpyruvate; S3P, shikimate 3-phosphate; RP-TI, (R)-phosphonate TI analogue; SP-TI, (S)-phosphonate TI analogue; 2F-TI, (R)-difluoromethyl TI analogue; TIAs, TI analogues. 13344 Biochemistry 2007, 46, 13344-13351 10.1021/bi701095u CCC: $37.00 © 2007 American Chemical Society Published on Web 10/25/2007