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SHIKIMATE ENZYMOLOGY

The shikimate pathway is the biosynthetic pathway by which the aromatic amino acids [C Abell book chapter in "Comprehensive Natural Products Chemistry - Vol. 1", ed. U Sankawa, 1999, Elsevier, Amsterdam, pp 573-607, ISBN: 0080431534 (v.1)], phenylalanine, tyrosine and tryptophan are assembled. The pathway is present in plants, microorganisms and fungi but not mammals. It is the target for Glyphosate, a very important herbicide.

Shikimate Pathway

The Shikimate Pathway

Our interests were to understand the enzymology of the pathway. We are using detailed knowledge of the mechanism and structure of specific enzymes to design novel enzyme inhibitors. Such compounds could potentially be herbicides, fungicides, antibiotics or antiparasitic agents. To get information about the mechanism of an enzyme we study its interaction with isotopically labelled substrates [Tetrahedron Lett., 1991, J. Am. Chem. Soc., 1990, Biochemistry, 1995, Biochem. J., 1995], or substrate analogues [J. Chem. Soc., Perkin Trans. I, 1996]. We have a particular interest in fluorinated analogues of pathway intermediates [J. Am. Chem. Soc., 1992, Bioorg. & Med. Chem. Lett., 1995, J. Chem. Soc. Perkin Trans. I, 1997, J. Org. Chem., 1997, J. Org. Chem., 1998, Bioorg. Med. Chem. Letts., 2000, BioMed. Chem. Letts., 2000].

Our studies were focused on two areas:
(1) The mechanism and structure of chorismate-utilising enzymes
Formation of p-aminobenzoate
We have previously shown that (6R)- and (6S)-6-fluoroEPSPs are potent inhibitors of chorismate synthase [J. Am. Chem. Soc., 1991]. Incubation of (6R)-6-fluoroEPSP with chorismate synthase provided the first evidence for the novel radical mechanism used by this enzyme [J. Am. Chem. Soc., 1992 and Proceedings of the Eleventh International Symposium on Flavins and Flavoproteins, Walter de Gruyter & Co., Berlin 1994], while (6S)-6-fluoroEPSP is slowly converted into 2-fluorochorismate [J. Biol. Chem., 1995]. We subsequently showed that 2-fluorochorismate irreversibly inhibited PabB by modifying the active site residue Lys274 [J. Am. Chem. Soc., 2004]. This was a key piece of evidence in the discovery of the mechanism of this enzyme. We were the first group to detect an unprecedented covalent intermediate [ChemBioChem, 2005].
Formation of salicylate
We have characterised the first salicylate synthase, Irp9 from Yersinia [J. Bact., 2005]. We have solved the crystal structure with and without product bound [J. Mol. Biol., 2006] and are now studying relation to the structurally homologous enzyme TrpE. Based on their close structural similarity we have used site directed mutagenesis to convert Irp9 into an anthranilate synthase [ChemMedChem, 2007].

Active site of Irp9

Active site of Irp9 with salicyclate and pyruvate bound

(2) Developing inhibitors of type II dehydroquinase
Dehydroquinase catalyses the dehydration of dehydroquinate to form dehydroshikimate. The type I enzyme involves an imine intermediate [J. Am. Chem. Soc., 1991, Biochem. Soc. Trans., 1998] and catalyses a syn elimination with loss of the equatorial C-2 hydrogen. The mechanism of the type II enzyme proceeds through an enolate [Biochem. J., 1996] and involves loss of the axial hydrogen at C-2 in an anti elimination [BioMed. Chem. Lett., 1993, J. Chem. Soc., Chem. Commun., 1993]. On the basis of these mechanistic differences we designed first generation inhibitors that are specific to type I [BioMed. Chem. Letts., 2000] and type II enzymes [J. Org. Chem., 1999, Chem. Commun., 2002]. Insights into the structure of type II dehydroquinase [Structures, 2002] have led to a new strategy for developing potent inhibitors of the enzyme [Org. Biol. Chem., 2003, Org. Biomol. Chem., 2005, ChemMedChem, 2007].

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