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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

Lookup NU author(s): Dr Alison Park, Dr Heather Lamb, Professor Alastair Hawkins


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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 β-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 Km is less than a factor of 3, that the turnover numbers differed by 24%, and that the Km 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 KD compared to the catalytic binding site. Inhibitor studies have suggested the enzyme could act as a simple oxido-reductase 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 kcat/Km ratio, whereas R130A is inactive.

Publication metadata

Author(s): Park A, Lamb HK, Nichols C, Moore JD, Brown KA, Cooper A, Charles IG, Stammers DK, Hawkins AR

Publication type: Article

Publication status: Published

Journal: Protein Science

Year: 2004

Volume: 13

Issue: 8

Pages: 2108-2119

ISSN (print): 0961-8368

ISSN (electronic): 1469-896X

Publisher: Wiley-Blackwell


DOI: 10.1110/ps.04705404

PubMed id: 15273308


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Funder referenceFunder name
B20089Biotechnology and Biological Sciences Research Council