Regulation of 3-indoleacetic Acid Production in Pseudomonas Syringae Pv. Savastanoi. Purification and Properties of Tryptophan 2-monooxygenase

J Biol Chem. 1985 May 25;260(10):6281-7.

Abstract

The oxidative decarboxylation of L-tryptophan to yield 3-indoleacetamide, catalyzed by tryptophan 2-monooxygenase, represents a controlling reaction in the synthesis of indoleacetic acid by Pseudomonas savastanoi (Pseudomonas syringae pv. savastanoi), a gall-forming pathogen of olive (Olea europea L.) and oleander (Nerium oleander L.). Production of indoleacetic acid is essential for virulence of the bacterium in its hosts. Tryptophan 2-monooxygenase was characterized to determine its role in indoleacetic acid metabolism in the bacterium. The enzyme was purified to apparent homogeneity from Escherichia coli cells containing the genetic locus for this enzyme obtained from P. savastanoi. The preparation contained a single polypeptide with a mass of 62,000 that cross-reacted immunologically with a homologous protein in P. savastanoi. The holoenzyme contained one FAD moiety/subunit with properties consistent with a catalytic function. The enzyme preparation catalyzed an L-tryptophan-dependent O2 uptake and yielded 3-indoleacetamide as a product. Enzyme activity fit simple Michaelis Menten kinetics with a Km for L-tryptophan of 50 microM. 3-Indoleacetamide and 3-indoleacetic acid were identified as regulatory effectors. The apparent Ki for 3-indoleacetamide was 7 microM; that for indoleacetic acid was 225 microM. At Km concentrations of tryptophan, enzyme activity was inhibited 50% by 25 microM 3-indoleacetamide. In contrast, 230 microM indoleacetic acid was required to effect a similar inhibition. Phenylalanine and tyrosine were ineffective as regulatory metabolites. These results indicate that IAA synthesis in P. savastanoi is regulated by limiting tryptophan and by feedback inhibition from indoleacetamide and indoleacetic acid.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Feedback
  • Flavin-Adenine Dinucleotide / metabolism
  • Indoleacetic Acids / metabolism*
  • Kinetics
  • Molecular Weight
  • Pseudomonas / metabolism*
  • Tryptophan Hydroxylase / immunology
  • Tryptophan Hydroxylase / isolation & purification
  • Tryptophan Hydroxylase / metabolism*

Substances

  • Indoleacetic Acids
  • Flavin-Adenine Dinucleotide
  • indoleacetic acid
  • Tryptophan Hydroxylase