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

Protein Sci. 2004 Aug;13(8):2108-19. doi: 10.1110/ps.04705404.

Abstract

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 beta-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 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 kcat/Km ratio, whereas R130A is inactive.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Oxidoreductases / chemistry*
  • Alcohol Oxidoreductases / genetics
  • Amino Acid Substitution / genetics*
  • Animals
  • Binding Sites / genetics
  • Biophysical Phenomena
  • Biophysics
  • Calorimetry, Differential Scanning
  • Humans
  • Hydro-Lyases / chemistry*
  • Hydro-Lyases / genetics
  • Kinetics
  • Lyases / chemistry*
  • Lyases / genetics
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / genetics
  • Mutagenesis, Site-Directed / genetics
  • Phosphorus-Oxygen Lyases / chemistry*
  • Phosphorus-Oxygen Lyases / genetics
  • Phosphotransferases (Alcohol Group Acceptor) / chemistry*
  • Phosphotransferases (Alcohol Group Acceptor) / genetics
  • Protein Structure, Tertiary / genetics
  • Quinic Acid / analogs & derivatives*
  • Quinic Acid / chemistry
  • Substrate Specificity / genetics
  • Sugar Phosphates / chemistry
  • Transferases / chemistry*
  • Transferases / genetics

Substances

  • 3-dehydroquinic acid
  • Multienzyme Complexes
  • Sugar Phosphates
  • arom enzyme
  • Quinic Acid
  • 3-deoxy-D-arabino-heptulosonate-7-phosphate
  • Alcohol Oxidoreductases
  • Transferases
  • Phosphotransferases (Alcohol Group Acceptor)
  • Lyases
  • Hydro-Lyases
  • 3-dehydroquinate synthetase
  • Phosphorus-Oxygen Lyases