Mihai Duduta

Bard College (MIT starting fall 2007)

Lippard Laboratory
Mentor: Mike McCormick

Summer 2007

Investigating Hydroxylase-Regulatory Protein Interaction in Phenol Hydroxylase by Site Directed Mutagenesis of Asn 204

Phenol hydroxylase (PH) belongs to a family of bacterial multicomponent monooxygenases (BMMs) with carboxylate-bridged diiron active sites. A characteristic of the BMM family, which includes soluble methane (sMMO) monooxygenase, is the necessity of the hydroxylase to bind to the regulatory protein component in order to become catalytically active. Of likely functional significance is a crystallographically observed hydrogen bond between an asparagine residue (N204) in the hydroxylase (PHH) and a serine residue (S72) in the regulatory protein (PHM), both of which are conserved throughout the BMM family. Structural characterization of the ToMO and MMO hydroxylases in various oxidation states reveals a rotamer shift in the conserved Asn residue upon reduction of the diiron center. To investigate this binding interaction, the analogous Asn unit in the wild type PH hydroxylase will be to be changed to Ala (PHH N204A) by site directed mutagenesis. Upon isolation, the wild type and mutant hydroxylases will be initially characterized using a colorimetric assay in which phenol oxidation to catechol is monitored via coupled oxidation of catechol to 2-hydroxymuconate semialdehyde by catechol 2,3-dioxygenase (C23O). Subsequently, the regulatory protein binding affinities of the wild type and mutant hydroxylases will be examined by isothermal titration calorimetry. Toward these goals, PHH was sequenced and the site of mutagenesis identified. The wild type PHH and PHM proteins as well as C23O were expressed, and also purified in the case of PHH and C23O. The preparation of the mutant hydroxylase is in progress. Results from these experiments will provide further insight into the hydroxylase-regulatory protein interaction in B MMs.