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 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.
Emanuele
J J
JJ
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128.
Heasley
C J
CJ
Fitzpatrick
P F
PF
D000596:Amino Acids / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D005420:Flavoproteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D007210:Indoleacetic Acids / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D014364:Tryptophan / Q000378:metabolism*
D000596:Amino Acids; D005420:Flavoproteins; D007210:Indoleacetic Acids; D011994:Recombinant Proteins; C006507:vinylglycine; C030737:indoleacetic acid; C015950:indoleacetamide; D014364:Tryptophan; D006899:Mixed Function Oxygenases; C023204:tryptophan 2-monooxygenase; D005998:Glycine