title: DNA-mediated assembly of cytochrome P450 BM3 subdomains.
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.
authors.affiliation: TU Dortmund, Fakultät Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Strasse 6, D-44227 Dortmund, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Erkelenz
authors.forename: Chi-Hsien
authors.initials: CH
authors.lastname: Kuo
authors.forename: Christof M
authors.initials: CM
authors.lastname: Niemeyer
mesh_terms: D000494:Allosteric Regulation; D001419:Bacteria / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D007848:Laurates / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007848:Laurates; D011993:Recombinant Fusion Proteins; C425970:p-nitrophenoxydodecanoic acid; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases