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Biophysical and kinetic analysis of wild-type and site-directed mutants of the isolated and native dehydroquinate synthase domain of the AROM protein

¹ School of Cell and Molecular Biosciences, Catherine Cookson Building, University of Newcastle upon Tyne, NE2 4HH, UK
² Division of Structural Biology, The Henry Wellcome Building for Genomic Medicine, University of Oxford, Oxford OX3 7BN, UK
³ Department of Biological Sciences Centre for Molecular Microbiology and Infection, Imperial College London, London, SW7 2AZ, UK
⁴ Department of Chemistry, University of Glasgow, Glasgow, G12 8QQ, UK
⁵ Arrow Therapeutics, London, SE1 1DA, UK

(RECEIVED February 23, 2004; FINAL REVISION April 28, 2004; ACCEPTED April 28, 2004)

Dehydroquinate synthase (DHQS) is the N-terminal domain of the pentafunctional AROM protein that catalyses steps 2 to 7 in the shikimate pathway in microbial eukaryotes. DHQS converts 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) to dehydroquinate in a reaction that includes alcohol oxidation, phosphate -elimination, carbonyl reduction, ring opening, and intramolecular aldol condensation. Kinetic analysis of the isolated DHQS domains with the AROM protein showed that for the substrate DAHP the difference in K_m is less than a factor of 3, that the turnover numbers differed by 24%, and that the K_m for NAD⁺ differs by a factor of 3. Isothermal titration calorimetry revealed that a second (inhibitory) site for divalent metal binding has an approximately 4000-fold increase in K_D compared to the catalytic binding site. Inhibitor studies have suggested the enzyme could act as a simple oxidoreductase with several of the reactions occurring spontaneously, whereas structural studies have implied that DHQS participates in all steps of the reaction. Analysis of site-directed mutants experimentally test and support this latter hypothesis. Differential scanning calorimetry, circular dichroism spectroscopy, and molecular exclusion chromatography demonstrate that the mutant DHQS retain their secondary and quaternary structures and their ligand binding capacity. R130K has a 135-fold reduction in specific activity with DAHP and a greater than 1100-fold decrease in the k_cat/K_m ratio, whereas R130A is inactive.

Keywords: dehydroquinate synthase; AROM; biocalorimetry; site-directed mutagenesis

Reprint requests to: Alastair R. Hawkins, School of Cell and Molecular Biosciences, Catherine Cookson Building, University of Newcastle upon Tyne, Framlington Place, NE2 4HH, UK; e-mail: a.r.hawkins{at}ncl.ac.uk; fax: 44-191-2227424.

⁶ Present address: Vernalis (Cambridge) Ltd., Granta Park, Abington, Cambridge, Cambridgeshire, CB1 6GB, UK.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04705404.

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