title: A new application of the yeast two-hybrid system in protein engineering.
abstract: Cytochromes P450 are involved in the biosynthesis of steroid hormones in mitochondria of the adrenal gland. The electrons required for these reactions are provided via a redox chain consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx). A prerequisite for a fast and efficient electron transfer as well as high catalytic activity is the formation of functional complexes between the different redox partners. To improve the protein-protein interactions by directed evolution, we developed a new in vivo selection system. This high-throughput screening method is based on the yeast two-hybrid system. It enables a background-free screening for increased protein-protein interactions between stable and functional species including cofactor-containing proteins (FAD, [2Fe-2S], heme). The method was successfully applied for the directed evolution of Adx and selected variants were analyzed biochemically and biophysically. All analyzed proteins exhibit typical characteristics of [2Fe-2S]-cluster-type ferredoxins. Adx-dependent substrate conversion assays with different cytochromes demonstrated that the improved ability of the mutants to form complexes results in an enhanced catalytic efficiency of the cytochrome P450 system.
authors.affiliation: FR 8.3--Biochemie, Universität des Saarlandes, D-66041 Saarbrücken, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Bichet
authors.forename: Frank
authors.initials: F
authors.lastname: Hannemann
authors.forename: Matthias
authors.initials: M
authors.lastname: Rekowski
authors.forename: Rita
authors.initials: R
authors.lastname: Bernhardt
mesh_terms: D000325:Adrenodoxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D019020:Directed Molecular Evolution; D015202:Protein Engineering; D012441:Saccharomyces cerevisiae; D020798:Two-Hybrid System Techniques
chemical_list: D003067:Coenzymes; D000325:Adrenodoxin