Preparative synthesis with P450 monooxygenases is hampered in cell-free systems by the requirement for cofactors such as NAD(P)H as reduction equivalents. A validated medium-throughput screening system was designed for improving P450 monooxygenases by mediated electron transfer with zinc/cobalt(III)sepulchrate (Zn/Co(III)sep) as an alternative and cost-effective cofactor system. The monooxygenase P450 BM-3 F87A was used as a model system for developing the screening system in a 96-well format. A coefficient of variation of less than 10% was achieved under optimized screening conditions. The mediator evolution screen was validated by comparing the activity of P450 BM-3 to P450 BM-3 F87A and by screening a saturation mutagenesis library at amino acid position R47. For mediated electron transfer, two double mutants P450 BM-3(F87A R47F) and P450 BM-3 (F87A R47Y) were identified with a two-threefold increased catalytic efficiency (up to 32 microM(-1) min(-1) for P450 BM-3(F87A R47F) and 34 microM(-1) min(-1) for P450 BM-3 (F87A R47Y)) compared to P450 BM-3 F87A. The kinetic constants of the double mutants are, in contrast to those of P450 BM-3 F87A, dependent on Co(III)sep concentration in the presence of NADPH. kcat increases from 145 min(-1) (0.25 mM Co(III)sep) to 197 min(-1) (0.5 mM Co(III)sep), and Km decreases simultaneously from 7.0 microM to 3.7 microM, for P450 BM-3 (F87A R47F). For P450 BM-3 (F87A R47Y), kcat increases from 138 min(-1) (0.25 mM Co(III)sep) up to 187 min(-1) (0.5 mM Co(III)sep), and Km decreases from 8.2 microM to 4.2 microM. Due to lower Km values, the catalytic efficiencies were improved six times for P450 BM-3 (F87A R47F) and three times for P450 BM-3 (F87A R47Y), when comparing catalytic efficiencies of the mediated electron-transfer system to the natural reduction equivalent NADPH.
"Some are based on automated and fast, sometimes multiplexed or combinatorial, LC–MS procedures (Ansede and Thakker, 2004; Peng et al., 2003), but procedures based on the detection of radioactive, chromophoric or fluorescent substrates or products are used more extensively for highthroughput approaches. These are used in particular for inhibition-based assays (Crespi et al., 2002; Moody et al., 1999; Stresser et al., 2000; Yamamoto et al., 2002) or molecular breeding (Alcalde et al., 2004; Hannemann et al., 2006; Lussenburg et al., 2005; Nazor and Schwaneberg, 2006). Fluorescent compounds have also been replaced successfully by luminogenic substrates, derived from D-luciferin (Cali et al., 2006). "
[Show abstract][Hide abstract] ABSTRACT: A challenge of the post-genomic era is to determine the functions of a plethora of orphan genes. This is a more acute problem when dealing with large gene families, such as the superfamily encoding cytochrome P450 enzymes in higher plants. We propose here a new, simple, medium-throughput methodology to screen for potential substrates of orphan P450 mono-oxygenases. The same technique can also be applied to screening for inhibitors of the oxygenases involved in the biosynthesis of compounds essential for plant development, such as growth regulators. The method is based on a commercially available microplate system, which detects the oxygen consumed by the catalytic reaction via an oxygen-sensing fluorophore. It is optimized using as a model CYP73A1, the cinnamic acid hydroxylase from Helianthus tuberosus, expressed in yeast. We show that the procedure is suitable not only for the detection and real-time monitoring, but also for the quantitative evaluation of enzyme activity. This new method has broad application for the identification of candidate substrates and inhibitors in chemical libraries, to support determination of physiological substrates, development of plant growth regulators, investigations on herbicide and pollutant metabolism, synthesis of valuable compounds and drug design. It also provides a fast-assay platform for determination of catalytic and inhibition parameters. The method applies to plant P450 enzymes, but also to cytochromes P450 from other organisms, and all types of oxygenases. The critical steps, calculation of oxygen consumption from fluorescence signal, and limits of the methods are discussed.
The Plant Journal 08/2007; 51(2):331-40. DOI:10.1111/j.1365-313X.2007.03140.x · 5.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Synthosomes are mechanically stable vesicles with a block copolymer membrane and an engineered transmembrane protein acting as selective gate. The polymer vesicles are nanometer-sized (50-1000 nm) and functionalized by loading them with enzymes for bioconversions or encapsulating charged macromolecules for selective compound recovery/release. The Synthosome system might become a novel technology platform for biocatalysis and selective product recovery. Progress in Synthosome research comprises employed block copolymers, transmembrane channel engineering, and functionalizations, which are discussed here in detail. The challenges in transmembrane protein engineering, as well as cost-effective production, in block copolymer design and the state of the art in Synthosome characterization comprising quantification of encapsulated protein, translocation efficiency, number of transmembrane channels per vesicle, and enzyme kinetics are also presented and discussed. An assessment of the Synthosome technology platform for prospective applications in industrial (white) biotechnology concludes this review.
[Show abstract][Hide abstract] ABSTRACT: The cytochrome P450 enzymes (P450s or CYPs) form a large family of heme proteins involved in drug metabolism and in the biosynthesis of steroids, lipids, vitamins and natural products. Their remarkable ability to catalyze the insertion of oxygen into non-activated C-H bonds has attracted the interest of chemists for several decades. Very few chemical methods exist that directly hydroxylate aliphatic or aromatic C-H bonds, and most of them are not selective or of limited scope. Biocatalysts such as P450s represent a promising alternative: however, their applications have been limited by substrate specificity, low activity, poor stability and the need for cofactors. This review covers the attempts to overcome these limitations using approaches such as mutagenesis, chemical modifications, conditions engineering and immobilization.
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