tailieunhanh - Báo cáo khoa học: Probing the molecular determinants of aniline dioxygenase substrate specificity by saturation mutagenesis

Aniline dioxygenase is a multicomponent Rieske nonheme-iron dioxygenase enzyme isolated from Acinetobactersp. strain YAA. Saturation mutagen-esis of the substrate-binding pocket residues, which were identified using a homology model of theasubunit of the terminal dioxygenase (AtdA3), was used to probe the molecular determinants of AtdA substrate specificity. | ễFEBS Journal Probing the molecular determinants of aniline dioxygenase substrate specificity by saturation mutagenesis Ee L. Ang1 2 Jeffrey P. Obbard3 and Huimin Zhao1 4 5 1 Department of Chemicaland Biomolecular Engineering University of Illinois at Urbana-Champaign Urbana IL USA 2 Department of Chemicaland Biomolecular Engineering NationalUniversity of Singapore Singapore 3 Division of EnvironmentalScience and Engineering NationalUniversity of Singapore Singapore 4 Center for Biophysics and ComputationalBiology University of Illinois at Urbana-Champaign Urbana IL USA 5 Department of Chemistry University of Illinois at Urbana-Champaign Urbana IL USA Keywords aniline dioxygenase homology modeling saturation mutagenesis substrate specificity Correspondence H. Zhao Department of Chemical and Biomolecular Engineering University of Illinois at Urbana-Champaign 600 South Mathews Avenue Urbana IL 61801 USA Fax 1 217 333 5052 Tel 1 217 333 2631 E-mail zhao5@ Received 28 October 2006 revised 5 December 2006 accepted 8 December 2006 doi Aniline dioxygenase is a multicomponent Rieske nonheme-iron dioxygenase enzyme isolated from Acinetobacter sp. strain YAA. Saturation mutagenesis of the substrate-binding pocket residues which were identified using a homology model of the a subunit of the terminal dioxygenase AtdA3 was used to probe the molecular determinants of AtdA substrate specificity. The V205A mutation widened the substrate specificity of aniline dioxygenase to include 2-isopropylaniline for which the wild-type enzyme has no activity. The V205A mutation also made 2-isopropylaniline a better substrate for the enzyme than 2 4-dimethylaniline a native substrate of the wild-type enzyme. The I248L mutation improved the activity of aniline dioxygenase against aniline and 2 4-dimethylaniline approximately and respectively. Thus it is shown that the a subunit of the terminal dioxygenase indeed plays a part in the .