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.

Molecular Evolution of a Steroid Hydroxylating Cytochrome P450 Using a Versatile Steroid Detection System for Screening

“…This fact and the nature of the substitution from a non polar alanine to a polar threonine imply a function of the mutant A106T in the binding of adrenodoxin. The effect of changed redox partnercytochrome P450 binding on the activity of the P450 was previously also shown for the couple adrenodoxin/ CYP11A1 [41].…”

Semi‐rational engineering of cytochrome P450sca‐2 in a hybrid system for enhanced catalytic activity: Insights into the important role of electron transfer

“…Biochemical analyses on P450sca‐2 variants revealed a number of interesting features for this P450sca‐2/Pdx/Pdr coupling system. While our current analyses using the Michaelis–Menten equation to analyze the complex P450 cycle can only be regarded as an approximation (Bichet et al, ; Guengerich, ; Xu et al, ; Yang et al, ), nonetheless several conclusions can be drawn. First, as four of the five originally chosen sites in the substrate binding pocket (T85), in the substrate access channel (V194), and at the Pdx interaction interface (T119 and N363) yielded mutants with improved activity, this suggests the importance of these regions for the overall P450sca‐2/Pdx/Pdr function.…”

“…Consistent with this view, CD spectral analyses of the P450sca‐2 variants revealed no significant changes in the heme active site microenvironments (data not shown; Andersson and Peterson, ). Additionally, it is worth mentioning that we had carried out surface plasmon resonance (SPR) biosensor analysis for the interaction of P450sca‐2 variants with immobilized Pdx (Bichet et al, ; Önell and Andersson, ). It was found that this binding appeared not to correlate with the apparent K M (Pdx) or k cat /K M (Pdx) for P450sca‐2 variants.…”

ROS production by adrenodoxin does not cause apoptosis in fission yeast

“…We then created three fission yeast strains that express functionally activated mutants of Adx: CAD46 (expressing Adx-S112W), CAD47 (expressing Adx-D113Y), and CAD48 (expressing Adx 1-108 ), respectively. Adx 1-108 is a strongly activated mutant that lacks the carboxy terminal 20 amino acids and displays an significantly lower redox potential (-344 mV) than Adx-WT (-273 mV) [30]; Adx-S112W is another activated mutant that lacks the carboxy terminal 16 amino acids and features a terminal tryptophane, leading to a increased affinity for its redox partner CYP11A1 and also a lower redox potential (-334 mV) [31]; Adx-D113Y is a full length Adx mutant with enhanced AdR binding ability, diminished CYP11B1 binding ability and a slightly changed redox potential (-298 mV) [32]. For each of the Adx expressing strains, correct subcellular localization of Adx was confirmed by Western blot analysis of mitochondrial protein lysates (Fig.…”