Purification and Characterization of the Flavoprotein Tryptophan 2-Monooxygenase Expressed at High Levels in Escherichia coli

• Published in: Archives of biochemistry and biophysics Vol. 316; no. 1; pp. 241 - 248
• Main Authors: Emanuele, J.J., Heasley, C.J., Fitzpatrick, P.F.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10-01-1995
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; AGENT PATHOGENE; Amino Acids - metabolism; BACTERIA; Binding Sites; ESCHERICHIA COLI; Escherichia coli - genetics; EXPRESION GENICA; EXPRESSION DES GENES; Flavoproteins - biosynthesis; Flavoproteins - genetics; Flavoproteins - metabolism; Glycine - analogs & derivatives; Glycine - pharmacology; Indoleacetic Acids - metabolism; Mixed Function Oxygenases - antagonists & inhibitors; Mixed Function Oxygenases - biosynthesis; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Models, Chemical; ORGANISMOS PATOGENOS; OXIDORREDUCTASAS; OXYDOREDUCTASE; PSEUDOMONAS; Pseudomonas - enzymology; PURIFICACION; PURIFICATION; Recombinant Proteins - biosynthesis; Recombinant Proteins - metabolism; Spectrophotometry; Substrate Specificity; TRANSFERENCIA DE GENES; TRANSFERT DE GENE; TRIPTOFANO; Tryptophan - metabolism; TRYPTOPHANE
• DOI: 10.1006/abbi.1995.1034
• EISSN: 1096-0384
• ISSN: 0003-9861

Tryptophan 2-monooxygenase from Pseudomonas savastanoi is a flavoprotein which catalyzes the formation of indoleacetamide from tryptophan. This is the first step in a two-step pathway for the formation of indoleacetic acid during infection of plants and subsequent gall formation by this and other bacteria. The enzyme has been expressed in Escherichia coli at high levels, and a purification procedure has been developed which generates micromolar amounts of protein. The purified enzyme contains tightly bound indoleacetamide; a method involving dialysis against 20% methanol has been developed for removing the indoleacetamide without significant loss of enzyme activity. Amino acids with large hydrophobic side chains are the best substrates. N-substituted phenylalanines will also act as substrates. N-ethylmaleimide, methyl methanethiol-sulfonate, and diethylpyrocarbonate act as active site-directed reagents, consistent with a histidine and a cysteine at or near the enzyme active site. Vinylglycine partially inactivates the enzyme, while propargylglycine has no effect.