Directed evolution of CYP11A1 to generate the first soluble mitochondrial cytochrome P450 for crystallisation and kinetic investigation of this system
Zusammenfassung der Projektergebnisse
The mitochondrial steroid hydroxylase CYP11A1, an Integral mitochondrial membrane protein, catalyzes the first and rate limiting step in steroid hormone biosynthesis. Although 3D structures of microsomal P450s and of the mitochondrial CYP24 (2.5 Å resolution) are already known, there is no structure of this important mitochondrial P450 available so far. In this project we applied a novel approach, a combination of directed evolution combined with rational protein design to generate a soluble variant of CYP11A1 to access the structure of this membrane protein. Forty three CYP11A1 mutants were created by rational protein design based on the computermodel created by Usanov using site-directed mutagenesis. In addition, a total number of 23,100 mutants (6 libraries) were produced by random mutagenesis using error-prone PCR. In order to select soluble forms, two screening-systems were established. The solubility assay has been optimized for our requirements and a newly developed method (batch-screening) that is based on hydrophobic interaction chromatography has been studied. Our approach was shown to be successful. Two mutants were found which displayed improved solubility (up to 3.6-fold compared with CYP11A1 wild type), whereby CYP11A1-FG/K193E displays the most soluble CYP11A1 form described so far. To isolate these mutants for subsequent crystallization studies, protein expression in E. coli and protein purification were optimized resulting in a 10-fold higher yield of highly purified CYP11A1 mutants. Since mutant FG/K193E turned out to be unstable, the mutant with the highest yield (K193E) displaying two-fold higher solubility than CYP11A1 wild type, was analyzed by HPLC, stopped-flow- as well as surface-plasmon-resonance-measurements and was used for crystallization experiments. Micro-crystals could be obtained which demonstrate a significant progress in the crystallization of this mitochondrial cytochrome P450. In addition, it has been investigated whether the high dipole moment of adrenodoxin (803 Debye), the electron shuttle between the FAD-containing reductase and CYP11A1, has an influence on the recognition of the redox partners. The dipole moment has been significantly changed (from about 803 to 200 Debye) using site directed mutagenesis and the resultant proteins have been thoroughly investigated. The results of our studies give the first experimental evidence that the dipole moment does not influence the recognition in an electron transfer chain. This result was very unexpected and surprising and could have an impact on the situation in similar systems (photosynthesis, respiratory chain). Finally, the first crystal structure of a yeast ferredoxin, Etp1fd-SKF from Schizosaccharomyces pombe, being involved in the assembly of mitochondrial iron-sulfur clusters and in the formation of heme A, has been resolved. The structure is similar to that of adrenodoxin providing a rational basis for the inter-exchangeability of both proteins during steroid homione production. Thermodynamic studies of Etp1fd-SKF revealed that it shows a 20°C higher melting temperature than adrenodoxin. Comparison of the 3D structures of both proteins gives a rational explanation (higher number of H-bonds and salt bridges) for this observation. The physiological significance of this observation is, however, not clear at the moment.
Projektbezogene Publikationen (Auswahl)
- The interaction of bovine adrenodoxin with CYP11A1 (cytothrome P450scc) and CYP11B1 (cytochrome P45011beta). Acceleration of reduction and substrate conversion by sitedirected mutagenesis of adrenodoxin. J. Biol. Chem., 2001. 276(39): p. 36225-36232
Schiffler, B., M. Kiefer, A. Wilken, F. Hannemann, H.W. Adolph, and R. Bernhardt
- A new electron transport mechanism in mitochondrial steroid hydroxylase systems based on structural changes upon the reduction of Adrenodoxin. Biochemistry, 2002. 41(25): p. 7969- 7978
Beilke, D., R. Weiß, F. Löhr, P. Pristovsek, F. Hannemann, R. Bernhardt, and H. Rüterjans
- Deletions in the loop surrounding the iron-sulfur duster of adrenodoxin severely affect the interactions with its native redox partners adrenodoxin reductase and cytochrome P450scc (CYP11A1). Journal of Inorganic Biochemistry, 2002. 91(4): p. 644-654
Zollner, A., F. Hannemann, M. Lisurek, and R. Bernhardt
- Unfolding and conformational studies on bovine adrenodoxin probed by engineered intrinsic tryptophan fluorescence. Biochemistry, 2002. 41(36): p. 11008-11016
Hannemann, F., A.K. Bera, B. Fischer, M. Lisurek, K. Teuchner, and R. Bernhardt
- Bacterial (CYP101) and mitochondrial P450 systems-how comparable are they? Biochemical and Biophysical Research Communications, 2003. 312(1): p. 223-228
Schiffler, B. and R. Bernhardt
- Insights into the design of a hybrid system between anabaena ferredoxin-NADP+-reductase and bovine adrenodoxin. Eur J Biochem, 2003. 270(4): p. 726-735
Faro, M., B. Schiffler, A. Heinz, I. Nogues, M. Medina, R. Bernhardt, and C. Gomez-Moreno
- Stripping Down the Mitochondrial Cholesterol Hydroxylase System, a Kinetics Study. J. Biol. Chem., 2004. 279(33): p. 34269-34276
Schiffler, B., A. Zollner, and R. Bernhardt
- The adrenodoxin-like ferredoxin of Schizosaccharomyces pombe mitochondria. Journal of Inorganic Biochemistry, 2004. 98(7): p. 1229-1237
Schiffler, B., M. Bureik, W. Reinle, E.-C. Müller, F. Hannemann, and R. Bernhardt
- Cytochrome P450 and Steroid Hormone Biosynthesis Met. Ions Life Sci. 3, 361-396, 2007
Bernhardt, R. and Waterman, M.R.
- Cytochrome P450 systems - biological variations of electron transport chains. Biochim Biophys Acta. 1770, 330-344, 2007
Hannemann, F., Bichet, A., Ewen, K.M. and Bernhardt R.
- Self-association of adrenodoxin studied by using analytical ultracentrifugation. Biophys Chem. 125(1). 159-65, 2007
Behlke, J., Ristau, O., Müller, E.C., Hannemann, F. and Bernhardt, R.
- The dipole moment of the electron carrier adrenodoxin is not critical for redox partner interaction and electron transfer. J. of Inorg. Chem.103,997-1004, 2009
Hannemann, F., Guyot, A., Zöllner A., Müller, J. J. , Heinemann U. and Bernhardt, R.
