title: Molecular Lego: design of molecular assemblies of P450 enzymes for nanobiotechnology.
abstract: This paper reports on the application of the molecular Lego approach to P450 enzymes. Protein domains are used as catalytic (P450 BM3 haem domain and human P450 2E1) or electron transfer (flavodoxin and P450 BM3 reductase) modules. The objectives are to build assemblies with improved electrochemical properties, to construct soluble human P450 enzymes, and to generate libraries of new P450 catalytic modules based on P450 BM3. A rationally designed, gene-fused assembly (BMP-FLD) was obtained from the soluble haem domain of cytochrome P450 BM3 from Bacillus megaterium (BMP) and flavodoxin from Desulfovibrio vulgaris (FLD). The assembly was expressed successfully and characterised in its active form, displaying improved electrochemical properties. Solubilisation of the human, membrane-bound P450 2E1 (2E1) was achieved by fusing key elements of the 2E1 enzyme with selected parts of P450 BM3. An assembly containing the first 54 residues of P450 BM3, the whole sequence of P450 2E1 from residue 81 and the reductase domain of P450 BM3 was constructed. The 2E1-BM3 assembly was successfully expressed in the cytosol of Escherichia coli. The soluble form of 2E1-BM3 was reduced in carbon monoxide atmosphere and displayed the typical absorption peak at 450 nm, characteristic of a folded and active P450 enzyme. Finally, the alkali method previously developed in this laboratory was used to screen for P450 activity within a library of random mutants of P450 BM3. A number of variants active towards non-physiological substrates, such as pesticides and polyaromatic hydrocarbons were identified, providing new P450 catalytic modules. The combination of these three areas of research provide interesting tools for exploitation in nanobiotechnology.
authors.affiliation: Department of Biological Sciences, Biochemistry building, Imperial College of Science, Technology and Medicine, London SW7 2AY, UK. g.gilardi@ic.ac.uk
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Gilardi
authors.forename: Yergalem T
authors.initials: YT
authors.lastname: Meharenna
authors.forename: Georgia E
authors.initials: GE
authors.lastname: Tsotsou
authors.forename: Sheila J
authors.initials: SJ
authors.lastname: Sadeghi
authors.forename: Michael
authors.initials: M
authors.lastname: Fairhead
authors.forename: Silva
authors.initials: S
authors.lastname: Giannini
mesh_terms: D001426:Bacterial Proteins; D001709:Biotechnology; D020650:Combinatorial Chemistry Techniques; D019392:Cytochrome P-450 CYP2E1 / Q000737:chemistry*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D006801:Humans; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D008958:Models, Molecular; D009251:NADPH-Ferrihemoprotein Reductase; D036103:Nanotechnology; D010084:Oxidation-Reduction; D019151:Peptide Library; D015202:Protein Engineering
chemical_list: D001426:Bacterial Proteins; D019151:Peptide Library; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019392:Cytochrome P-450 CYP2E1; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases