A medium-throughput screening assay to determine catalytic activities of oxygen-consuming enzymes: a new tool for functional characterization of cytochrome P450 and other oxygenases

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.