DNA-Mediated Assembly of Cytochrome P450 BM3 Subdomains
- 15 September 2011
- journal article
- Published by American Chemical Society (ACS) in Journal of the American Chemical Society
- Vol. 133 (40), 16111-16118
- https://doi.org/10.1021/ja204993s
Abstract
Cytochrome P450 BM3 is a versatile enzyme, which holds great promise for applications in biocatalysis and biomedicine. We here report on the generation of a hybrid DNA–protein device based on the two subdomains of BM3, the reductase domain BMR and the porphyrin domain BMP. Both subdomains were fused genetically to the HaloTag protein, a self-labeling enzyme, allowing for the bioconjugation with chloroalkane-modified oligonucleotides. The subdomain-DNA-chimeras could be reassembled by complementary oligonucleotides, thus leading to reconstitution of the monooxygenase activity of BM3 holoenzyme, as demonstrated by conversion of the reporter substrate 12-pNCA. Arrangement of the two chimeras on a switchable DNA scaffold allowed one to control the distance between both subdomains, as indicated by the DNA-dependent activity of the holoenzyme. Furthermore, a switchable chimeric device was constructed, in which monooxygenase activity could be turned off by DNA strand displacement. This study demonstrates that P450 BM3 engineering and strategies of DNA nanotechnology can be merged to open up novel ways for the development of novel screening systems or responsive catalysts with potential applications in drug delivery.This publication has 44 references indexed in Scilit:
- Cytochrome P450: taming a wild type enzymeCurrent Opinion in Biotechnology, 2011
- Control over Enzymatic Activity by DNA‐Directed Split Enzyme ReassemblyChemBioChem, 2010
- Directed evolution of a magnetic resonance imaging contrast agent for noninvasive imaging of dopamineNature Biotechnology, 2010
- A Panel of Cytochrome P450 BM3 Variants to Produce Drug Metabolites and Diversify Lead CompoundsChemistry – A European Journal, 2009
- Designer DNA NanoarchitecturesBiochemistry, 2009
- DNA-directed assembly of artificial multienzyme complexesBiochemical and Biophysical Research Communications, 2008
- Chemo-enzymatic fluorination of unactivated organic compoundsNature Chemical Biology, 2008
- Design of Molecular Logic Devices Based on a Programmable DNA‐Regulated Semisynthetic EnzymeAngewandte Chemie, 2007
- DNA Hairpins: Fuel for Autonomous DNA DevicesBiophysical Journal, 2006
- Critical Role of the Residue Size at Position 87 in H2O2-Dependent Substrate Hydroxylation Activity and H2O2 Inactivation of Cytochrome P450BM-3Biochemical and Biophysical Research Communications, 2001