title: Directed evolution of aniline dioxygenase for enhanced bioremediation of aromatic amines.
abstract: The objective of this study was to enhance the activity of aniline dioxygenase (AtdA), a multi-component Rieske non-heme iron dioxygenase enzyme isolated from Acinetobacter sp. strain YAA, so as to create an enhanced biocatalyst for the bioremediation of aromatic amines. Previously, the mutation V205A was found to widen the substrate specificity of AtdA to accept 2-isopropylaniline (2IPA) for which the wild-type enzyme has no activity (Ang EL, Obbard JP, Zhao HM, FEBS J, 274:928-939, 2007). Using mutant V205A as the parent and applying one round of saturation mutagenesis followed by a round of random mutagenesis, the activity of the final mutant, 3-R21, was increased by 8.9-, 98.0-, and 2.0-fold for aniline, 2,4-dimethylaniline (24DMA), and 2-isopropylaniline (2IPA), respectively, over the mutant V205A. In particular, the activity of the mutant 3-R21 for 24DMA, which is a carcinogenic aromatic amine pollutant, was increased by 3.5-fold over the wild-type AtdA, while the AN activity was restored to the wild-type level, thus yielding a mutant aniline dioxygenase with enhanced activity and capable of hydroxylating a wider range of aromatic amines than the wild type.
authors.affiliation: Department of Chemical Engineering and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
authors.forename: Ee Lui
authors.initials: EL
authors.lastname: Ang
authors.forename: Jeffrey P
authors.initials: JP
authors.lastname: Obbard
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
mesh_terms: D000150:Acinetobacter / Q000201:enzymology*; D000588:Amines / Q000378:metabolism*; D000814:Aniline Compounds / Q000378:metabolism; D001673:Biodegradation, Environmental; D004252:DNA Mutational Analysis; D019020:Directed Molecular Evolution; D008958:Models, Molecular; D016296:Mutagenesis; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D010105:Oxygenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: C519021:2-isopropylaniline; D000588:Amines; D000814:Aniline Compounds; D050505:Mutant Proteins; C009694:2,4-xylidine; D010105:Oxygenases; C046396:aniline oxygenase; C023650:aniline