We report the nucleotide sequences of iaaM and iaaH, the genetic determinants for, respectively, tryptophan 2-monooxygenase and indoleacetamide hydrolase, the enzymes that catalyze the conversion of L-tryptophan to indoleacetic acid in the tumor-forming bacterium Pseudomonas syringae pv. savastanoi. The sequence analysis indicates that the iaaM locus contains an open reading frame encoding 557 amino acids that would comprise a protein with a molecular weight of 61,783; the iaaH locus contains an open reading frame of 455 amino acids that would comprise a protein with a molecular weight of 48,515. Significant amino acid sequence homology was found between the predicted sequence of the tryptophan monooxygenase of P. savastanoi and the deduced product of the T-DNA tms-) gene of the octopine-type plasmid pTiA6NC from Agrobacteium tumefaciens. Strong homology was found in the 25 amino acid sequence in the putative FAD-binding region of tryptophan monooxygenase. Homology was also found in the amino acid sequences representing the central regions of the putative products of iaaH and tms-2 T-DNA. The results suggest a strong similarity in the pathways for indoleacetic acid synthesis encoded by genes in P. savastanoi and in A. tumefaciens T-DNA.The association of the tumor-forming bacterium Pseudomonas syringae pv. savastanoi (P. savastanoi) and its hosts, oleander and olive plants, provides a system for studying the molecular basis of virulence of a bacterium in plants. Tumor formation by these plants is a response to high concentrations of indoleacetic acid (IAA) introduced into infected tissue by the bacterium (1); thus, production of a tumor is used to assess virulence of the bacterium. The bacterium produces IAA from tryptophan, with indoleacetamide as the intermediate. The enzymes involved are tryptophan 2-monooxygenase [L-tryptophan:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.3], which catalyzes the conversion of L-tryptophan to indole-3-acetamide, and indoleacetamide hydrolase, which catalyzes the conversion of indoleacetamide to ammonia and IAA (2).The genes for the two enzymes, iaaM and iaaH, are part of an operon that is borne on a plasmid, pIAA, in oleander strains of the pathogen; in olive strains these genes are on the chromosome. Mutants cured of pIAA are weakly virulent on oleander; when transformed with pIAA,

Versions of the Y55F/Y88F modified form of Delta 5-3-ketosteroid isomerase in which the active-site tyrosine-14 is replaced by 2-fluorotyrosine, 3-fluorotyrosine, and 2,3-difluorotyrosine, amino acids having progressively greater acidity of their phenolic hydroxyls, have been expressed in an Escherichia coli host and purified to high homogeneity. The steady-state kinetic properties of Y55F/Y88F KSI and its fluorotyrosine modified forms have been determined. The mechanistic implications of the results are presented and discussed.

Tyrosine 14 of delta 5-3-ketosteroid isomerase plays an important role in the function of the enzyme, since its replacement by phenylalanine results in a decrease in kcat by a factor of 10(-4.7). This result and the fact that this residue resides in the enzyme's substrate binding site and is in close proximity to C-2 of the bound steroid suggests that it functions as an electrophile in the catalytic mechanism by protonation of or hydrogen bonding to the C-3 carbonyl oxygen of the substrate. In order to obtain more information about the role of tyrosine 14, we have prepared a modified form of the enzyme in which tyrosine 14 has been substantially replaced in vivo by exogenously supplied 3-fluorotyrosine, a tyrosine derivative in which the pKa' of the phenol hydroxyl should be decreased by about 1.5 log units. Site specificity of this modification has been ensured by mutation of the codons for the nonessential tyrosines 55 and 88 to phenylalanine. We find that replacement of tyrosine 14 by 3-fluorotyrosine in the Y55,88F modified form of the isomerase results in a 4-fold decrease in kcat. We interpret this result in terms of a mechanism in which the transition state for enolization is dienolate-like, characterized by relatively little proton transfer from tyrosine 14 in the transition state, and the intermediate in the overall reaction is dienol-like. An alternative mechanism in which the intermediate is stabilized by a short, strong hydrogen bond can also be consistent with the data.

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