Thursday, August 2, 2007 - 3:15 PM
S187
Controlling Regiospecific Oxidation for Green Chemistry and Biofilm Formation: Homage to Indole
Thomas K. Wood, Chemical Engineering, Texas A & M University, 220 Jack E. Brown Building, College Station, TX 77843-3122
A primary goal of protein engineering is to control catalytic activity. Here we show that through mutagenesis of three active-site residues, the catalytic activity of a multi-component monooxygenase is altered so that it hydroxylates all three positions of toluene as well as both positions of naphthalene. Hence, for the first time, an enzyme has been engineered so that its regiospecific oxidation of a substrate can be controlled. Through the A107G mutation in the alpha subunit of toluene para-monooxygenase (TpMO), a variant was formed that hydroxylates toluene primarily at the ortho position while converting naphthalene to 1-naphthol. Conversely, the A107T variant produces >98% p-cresol and p-nitrophenol from toluene and nitrobenzene, respectively, as well as produces 2-naphthol from naphthalene. The mutation I100S/G103S produced a TpMO variant that forms 75% m-cresol from toluene and 100% m-nitrophenol from nitrobenzene; thus; for the first time a true meta-hydroxylating toluene monooxygenase was created. If the substrate is indole rather than toluene, colored compounds may be formed, and so we report the first DNA shuffling to make all the colors of the rainbow. We will show that these indigoids are also important for controlling bacterial biofilms, those sessile communities with high cell density that are ubiquitous in natural, medical, and engineering environments.