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

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 ba...

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Bibliographic Details
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
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Summary:	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.
Bibliography:	9545767 H20 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0003-9861 1096-0384
DOI:	10.1006/abbi.1995.1034