Studies on structure-function relationships of indolepyruvate decarboxylase from Enterobacter cloacae, a key enzyme of the indole acetic acid pathway

Eur J Biochem. 2003 May;270(10):2322-31. doi: 10.1046/j.1432-1033.2003.03602.x.

Abstract

Enterobacter cloacae, isolated from the rhizosphere of cucumbers, produces large amounts of indole-3-acetic acid. Indolepyruvate decarboxylase, the key enzyme in the biosynthetic pathway of indole-3-acetic acid, catalyses the formation of indole-3-acetaldehyde and carbon dioxide from indole-3-pyruvic acid. The enzyme requires the cofactors thiamine diphosphate and magnesium ions for catalytic activity. Recombinant indolepyruvate decarboxylase was purified from the host Escherichia coli strain JM109. Specificity of the enzyme for the substrates indole-3-pyruvic acid, pyruvic acid, benzoylformic acid, and seven benzoylformic acid analogues was investigated using a continuous optical assay. Stopped-flow kinetic data showed no indication for substrate activation in the decarboxylation reaction of indole-3-pyruvic acid, pyruvic acid or benzoylformic acid. Size exclusion chromatography and small angle X-ray solution scattering experiments suggested the tetramer as the catalytically active state and a pH-dependent subunit association equilibrium. Analysis of the kinetic constants of the benzoylformic acid analogues according to Hansch et al. [Hansch, C., Leo, A., Unger, S.H., Kim, K.H., Nikaitani, D & Lien, E.J. (1973) J. Med. Chem.16, 1207-1216] and comparison with indole-3-pyruvic acid conversion by pyruvate decarboxylases from Saccharomyces cerevisiae and Zymomonas mobilis provided some insight into the catalytic mechanism of indolepyruvate decarboxylase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Carboxy-Lyases / chemistry*
  • Catalysis
  • Chromatography
  • Crystallography, X-Ray
  • Dose-Response Relationship, Drug
  • Electrophoresis, Polyacrylamide Gel
  • Enterobacter / enzymology*
  • Escherichia coli / metabolism
  • Hydrogen-Ion Concentration
  • Indoleacetic Acids / chemistry*
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Recombinant Proteins / metabolism
  • Scattering, Radiation
  • Structure-Activity Relationship
  • Substrate Specificity
  • Thiamine Pyrophosphate / chemistry
  • Thiamine Pyrophosphate / pharmacology*
  • Time Factors
  • Tryptophan / chemistry
  • X-Rays

Substances

  • Indoleacetic Acids
  • Recombinant Proteins
  • indoleacetic acid
  • Tryptophan
  • indolepyruvate decarboxylase
  • Carboxy-Lyases
  • Thiamine Pyrophosphate