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Rational Design of P450 Enzymes for Biotechnology

  • Chapter
Physics and Chemistry Basis of Biotechnology

Part of the book series: Focus on Biotechnology ((FOBI,volume 7))

Abstract

Nanobiotechnology is a novel field where bio-molecules are assembled on devices for exploitation in bio-analytical applications. The increased understanding of the structure-function relationship of redox proteins and enzymes combined with the progress made in protein engineering, molecular spectroscopy and structural biology allows today the possibility of creating genetically engineered proteins/enzymes to be used in arrays for high-through-put screening.

This paper reports on the use of small and well characterised electron transfer proteins/enzymes, suchasflavodoxin, cytochrome c 553 and cytochrome P450 as modules to design and construct covalently linked, artificial electron transfer chains. Functional characterisation of these molecular wires will increase our understanding on the structure-function relationships in electron transfer systems. This approach has been named “molecular lego”, and its application to cytochromes P450, an important class of enzymes responsible for the metabolism of a large number of drugs and xenobiotics, is particularly relevant to biotechnology.An efficient,artificialelectron transfer chain was obtained by fusing the flavodoxin from D. vulgaris and the soluble haem domain of cytochrome P450 from B. megaterium. Moving to a higher level of complexity, the scaffold of this soluble enzyme was also used to insert the key structural and functional elements of the human cytochrome P450 2E1. The chimeric protein containing the fused bacterial and human domains was successfully engineered. Finally, a method designed to identify active P450 mutants to be used for the assembly of arrays with different activity/specificity is presented.

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Authors and Affiliations

  1. Department of Biochemistry, Imperial College of Science Technology and Medicine, London, SW7 2AY, UK

    Sheila J. Sadeghi, Georgia E. Tsotsou, Michael Fairhead, Yergalem T. Meharenna & Gianfranco Gilardi

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  1. Sheila J. Sadeghi
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  2. Georgia E. Tsotsou
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  3. Michael Fairhead
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  4. Yergalem T. Meharenna
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  5. Gianfranco Gilardi
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  1. Interdisciplinaire Research Center, Katholieke Universiteit Leuven, Kortrijk, Belgium

    Marcel De Cuyper

  2. National Institutes of Health, Bethesda, MD, USA

    Jeff W. M. Bulte

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Sadeghi, S.J., Tsotsou, G.E., Fairhead, M., Meharenna, Y.T., Gilardi, G. (2001). Rational Design of P450 Enzymes for Biotechnology. In: De Cuyper, M., Bulte, J.W.M. (eds) Physics and Chemistry Basis of Biotechnology. Focus on Biotechnology, vol 7. Springer, Dordrecht. https://doi.org/10.1007/0-306-46891-3_3

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