Abstract
The identification of novel substrates of cytochrome P450 enzymes by high-throughput screening assays is of utmost importance to further increase the scope of these enzymes for future applications. Most screens are either confined to individual substrate analogues or hampered by low throughput due to elaborate analysis techniques. Here we describe a general high-throughput screening assay that interrogates the activity of P450 enzymes with the aid of catalase as a reporter enzyme.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Jung ST, Lauchli R, Arnold FH (2011) Cytochrome P450: taming a wild type enzyme. Curr Opin Biotechnol. (6):809–817. doi: 10.1016/j.copbio.2011.02.008. Epub 2011 Mar 14
Guengerich FP (2002) Cytochrome P450 enzymes in the generation of commercial products. Nat Rev Drug Discov 1:359–366
Urlacher V, Schmid RD (2002) Biotransformations using prokaryotic P450 monooxygenases. Curr Opin Biotechnol 13:557–564
Kelly SL, Lamb DC, Kelly DE (2006) Cytochrome P450 biodiversity and biotechnology. Biochem Soc Trans 34:1159–1160
Rabe KS, Gandubert VJ, Spengler M, Erkelenz M, Niemeyer CM (2008) Engineering and assaying of cytochrome P450 biocatalysts. Anal Bioanal Chem 392(6):1059–1073. doi: 10.1007/s00216-008-2248-9. Epub 2008 Jul 13
Delaporte E, Rodrigues AD (2001) Cytochrome P450 assays. Curr Protoc Pharmacol Chapter 3: Unit 3.9 doi: 10.1002/0471141755.ph0309s15.
Peters MW, Meinhold P, Glieder A, Arnold FH (2003) Regio- and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3. J Am Chem Soc 125:13442–13450
Tsotsou GE, Cass AE, Gilardi G (2002) High throughput assay for cytochrome P450 BM3 for screening libraries of substrates and combinatorial mutants. Biosens Bioelectron 17:119–131
Rabe KS, Spengler M, Erkelenz M, Muller J, Gandubert VJ, Hayen H, Niemeyer CM (2009) Screening for cytochrome p450 reactivity by harnessing catalase as reporter enzyme. Chembiochem 10:751–757
Xu F, Bell SG, Rao Z, Wong LL (2007) Structure-activity correlations in pentachlorobenzene oxidation by engineered cytochrome P450cam. Protein Eng Des Sel 20:473–480
Acknowledgments
We are most grateful to Deutsche Forschungsgemeinschaft (DFG), Bundesministerium für Bildung und Forschung (BMBF), Alexander-von-Humboldt Stiftung, the European Union, and the Max-Planck Society for financial support of our work. KSR thanks the Deutscher Akademischer Austauschdienst (DAAD) for a postdoctoral fellowship.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Rabe, K.S., Niemeyer, C.M. (2013). Screening for Cytochrome P450 Reactivity with a Reporter Enzyme. In: Phillips, I., Shephard, E., Ortiz de Montellano, P. (eds) Cytochrome P450 Protocols. Methods in Molecular Biology, vol 987. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-321-3_13
Download citation
DOI: https://doi.org/10.1007/978-1-62703-321-3_13
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-320-6
Online ISBN: 978-1-62703-321-3
eBook Packages: Springer Protocols