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RATIONAL DESIGN OF P450 ENZYMES FOR BIOTECHNOLOGY
SHEILA J. SADEGHI, GEORGIA E. TSOTSOU, MICHAEL FAIRHEAD, YERGALEM T. MEHARENNA AND GIANFRANCO GILARDI*
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, UK
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, such as flavodoxin, cytochrome c553 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, artificial electron 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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M. De Cuyper and J.W.M. Bulte (eds.), Physics and Chemistry Basis of Biotechnology, 71-104. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
Sheila J. Sadeghi et al
1. Introduction
Electron transfer (ET) is essential for life: not only it provides means for harnessing solar energy through photosynthesis but it is also of crucial importance for the generation of metabolic energy in living cells [1-3]. This process is extremely interesting from a technological point of view as many reactions of biotechnological interest such as degradation of pollutants and drug and food processing are based on redox systems. The biotechnological exploitation of this process together with the rapidly growing field of biosensors and bioelectronics leads to fascinating possibilities especially in the creation of amperometric biosensors.
However, the key factor for the successful exploitation of new and interesting redox enzymes in amperometric devices remains the efficient interaction with electrode surfaces. Much success has been achieved in the modification of electrode surfaces with various strategies aiming at rendering the electrode surface more compatible with the biological matrix, in its delicate balance of the folded and active state [4-7]. In some cases the rate of success is limited by the fact that the relevant redox centre is deeply buried in the biological matrix, that in this way can control the properties and reactivity of such centre, but with the detriment of the bio-electrochemical signal. A notable example of such case is the well-known and highly relevant family of cytochromes P450 [8-9].
Recent progress made in the area of protein engineering, molecular spectroscopy and structural biology allows today the possibility of rationally designing site-directed mutants of proteins and enzymes with properties tailored, for example, for the oriented immobilisation on electrode surfaces [10-12].
Despite the great amount of reactions carried out simultaneously by many enzymes on the numerous substrates in the cell, the living systems exhibit highly efficient redox chains, where electrons are tunnelled in specific directions to sustain life. This success is the result of the slow process of evolution that can today be speeded up and mimicked by the protein engineer towards targets for the benefit of bioelectrochemistry. In particular, novel protein engineering methods based on random mutagenesis and in vivo selection and/or in vitro screening allows the creation of combinatorial libraries of proteins and enzymes that can be evolved for specific biotechnological targets [13-14]. This approach to engineering proteins is complementary to the rational, site-directed approach (Figure 1), and it can extend the capabilities in the generation of new proteins, beyond their physiological functions.
This paper reports on the progress made on selected aspects of protein engineering applied to the amperometric biosensor area, with particular reference to P450 enzymes and its implications in the pharmaceutical and bioreinediation areas of application.
1.1. INTERPROTEIN ELECTRON TRANSFER
The fundamental importance of protein ET in biology and the potential biotechnological implications that it underpins have attracted much research effort over the last decade. This has mostly been directed towards elucidating the nature of the
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Rational design of P450 enzymes for biotechnology
interactions between hypothetical physiological ET partners demonstrating how interprotein ET is the result of a number of events that occur in several stages [15-16]. The initial event leading to the reaction is the diffusional encounter of the protein pairs stirred by electrostatic and steric forces communicated through the solvent electrolyte medium; among these stirring parameters, long-range electrostatics are often the overruling forces [15]. The net charges on the proteins not only enhance the rate of collision by diffusion, but also cause their dipole moments [17-19] to line up in a specific orientation. The second event leading to inter-protein ET is the formation of a complex between the redox pairs, where a specific interaction is required to produce a configuration that actually results in rapid ET. The complex formation is expected to depend upon the nature of the protein surface topology and the distribution of the charged residues. A good fit between the surfaces of the two proteins allows their redox centres to be brought into close proximity under the control of short range forces such as hydrogen bonds, van der Waals forces, electrostatic and hydrophobic interactions [15]. The balance between these forces can vary markedly in different ET complexes. Once an ET competent complex has been formed, the ET process occurs, leading to the third stage of the reaction. This in turn depends on a variety of factors including the geometric disposition of donor and acceptor sites in the complex, the difference in free energy of the oxidised and reduced states, the activation energy, reorganisation energy and the electronic coupling between the two redox centres. These parameters and their influence over the kinetics and thermodynamics of this process have been described by Marcus and Sutin [20].
Figure 1. Flow chart depicting the several experimental stages involved in rational design and directed evolution.
When the rates of ET are very/extremely fast, other processes may become rate limiting. For instance, when the pre-complexes have to undergo dynamic reorientation
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