Substitution of lysine with glutamic acid at position 193 in bovine CYP11A1 significantly affects protein oligomerization and solubility but not enzymatic activity.

CYP11A1, a mitochondrial cytochrome P450, catalyzes the conversion from cholesterol to pregnenolone, the crucial step in the steroid hormone biosynthesis of mammals. It was shown in prior investigations, that the putative F-G loop of this enzyme is involved in membrane attachment. We produced different bovine CYP11A1 variants by rational protein design and could show that a deletion of 20 amino acids comprising parts of the F-G loop results in an enzyme with a three-fold increased solubility, the highest solubility of a CYP11A1 variant obtained so far. Furthermore, a single amino acid mutation, K193E, could be identified which leads not only to a higher solubility of CYP11A1 as well as a 4-fold improved expression rate, but also lowers the oligomerization of the protein while its activity is only slightly decreased. Therefore, this mutant has many advantages for the biotechnological application of CYP11A1 and is an important step towards crystallization of this mitochondrial P450.

A new application of the yeast two-hybrid system in protein engineering.

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.

Cytochrome P450 systems--biological variations of electron transport chains.

Cytochromes P450 (P450) are hemoproteins encoded by a superfamily of genes nearly ubiquitously distributed in different organisms from all biological kingdoms. The reactions carried out by P450s are extremely diverse and contribute to the biotransformation of drugs, the bioconversion of xenobiotics, the bioactivation of chemical carcinogens, the biosynthesis of physiologically important compounds such as steroids, fatty acids, eicosanoids, fat-soluble vitamins and bile acids, the conversion of alkanes, terpenes and aromatic compounds as well as the degradation of herbicides and insecticides. Cytochromes P450 belong to the group of external monooxygenases and thus receive the necessary electrons for oxygen cleavage and substrate hydroxylation from different redox partners. The classical as well as the recently discovered P450 redox systems are compiled in this paper and classified according to their composition.

The "Bringer" strategy: a very fast and highly efficient method for construction of mutant libraries by error-prone polymerase chain reaction of ring-closed plasmids.

Random mutagenesis constitutes a keystone in many strategies of directed evolution of biocatalysts and is often done by error-prone polymerase chain reaction (epPCR). Traditionally, the epPCR-generated DNA fragments are then subcloned into an expression vector to obtain a mutant library, which in turn is transformed into a suited host and screened for mutants that display the desired property. However, the vast majority of epPCR-generated fragments generally are lost during the subcloning step, making it the bottleneck in the mutant library construction procedure. Here we report a rapid and convenient strategy based on the epPCR amplification of a ring-closed expression plasmid that contains the gene of interest; after a DpnI digest the product of the epPCR reaction constitutes the mutant library and can be used directly for screening procedures. Primers binding to the beta-lactamase gene were chosen to allow application of the strategy to as broad a range of target plasmids as possible. The functionality of this approach was demonstrated by mutating the alpha-peptide coding region of the lacZ gene.