PURIFICATION AND CHARACTERIZATION OF THE FLAVOPROTEIN TRYPTOPHAN 2-MONOOXYGENASE EXPRESSED AT HIGH-LEVELS IN ESCHERICHIA-COLI

Tryptophan 2-monooxygenase from Pseudomonas savastanoi is a flavoprote in which catalyzes the formation of indoleacetamide from tryptophan. T his is the first step in a two-step pathway for the formation of indol eacetic acid during infection of plants and subsequent gall formation by this and other bacteria. The enzyme has been expressed in Escherich ia coli at high levels, and a purification procedure has been develope d which generates micromolar amounts of protein, The purified enzyme c ontains tightly bound indoleacetamide; a method involving dialysis aga inst 20% methanol has been developed for removing the indoleacetamide without significant loss of enzyme activity, Amino acids with large hy drophobic side chains are the best substrates; N-substituted phenylala nines will also act as substrates, N-ethylmaleimide, methyl methanethi ol-sulfonate, and diethylpyrocarbonate act as active site-directed rea gents, consistent with a histidine and a cysteine at or near the enzym e active site, Vinylglycine partially inactivates the enzyme, while pr opargylglycine has no effect. (C) 1995 Academic Press,Inc.