LICRED: A Versatile Drop-In Vector for Rapid Generation of Redox-Self-Sufficient Cytochrome P450s

TY - JOUR

T1 - LICRED

T2 - A Versatile Drop-In Vector for Rapid Generation of Redox-Self-Sufficient Cytochrome P450s

AU - Sabbadin, Federico

AU - Hyde, Ralph

AU - Robin, Aelig

AU - Hilgarth, Eva-Maria

AU - Delenne, Marie

AU - Flitsch, Sabine

AU - Turner, Nicholas

AU - Grogan, Gideon

AU - Bruce, Neil C.

PY - 2010/5/3

Y1 - 2010/5/3

N2 - Cytochromes P450 (P450s) are a family of haem-containing oxidases with considerable potential as tools for industrial biocatalysis. Organismal genomes are revealing thousands of gene sequences that encode P450s of as yet unknown function, the exploitation of which will require high-throughput tools for their isolation and characterisation. In this report, a ligation-independent cloning vector "LICRED" is described that enables the high-throughput generation of libraries of redox-self-sufficient P450s by fusing a range of P450 haem domains to the reductase of P450RhF (RhF-Red) in a robust and generically applicable way. Cloning and expression of fusions of RhF-Red with the haem domains of P450cam and P450-XplA resulted in soluble, active, redox-self-sufficient, chimeric enzymes. In vitro studies also revealed that electron transfer from NADPH to haem was primarily intramolecular. The general applicability of the LICRED platform was then demonstrated through the creation of a library of RhF-Red fusion constructs by using the diverse complement of P450 haem domains identified in the genome of Nocardia farcinica. The resultant fusion-protein library was then screened against a panel of substrates; this revealed chimeric enzymes competent for the hydroxylation of testosterone and methyltestosterone, and the dealkylation of 7-ethoxycoumarin.

AB - Cytochromes P450 (P450s) are a family of haem-containing oxidases with considerable potential as tools for industrial biocatalysis. Organismal genomes are revealing thousands of gene sequences that encode P450s of as yet unknown function, the exploitation of which will require high-throughput tools for their isolation and characterisation. In this report, a ligation-independent cloning vector "LICRED" is described that enables the high-throughput generation of libraries of redox-self-sufficient P450s by fusing a range of P450 haem domains to the reductase of P450RhF (RhF-Red) in a robust and generically applicable way. Cloning and expression of fusions of RhF-Red with the haem domains of P450cam and P450-XplA resulted in soluble, active, redox-self-sufficient, chimeric enzymes. In vitro studies also revealed that electron transfer from NADPH to haem was primarily intramolecular. The general applicability of the LICRED platform was then demonstrated through the creation of a library of RhF-Red fusion constructs by using the diverse complement of P450 haem domains identified in the genome of Nocardia farcinica. The resultant fusion-protein library was then screened against a panel of substrates; this revealed chimeric enzymes competent for the hydroxylation of testosterone and methyltestosterone, and the dealkylation of 7-ethoxycoumarin.

KW - biocatalysis

KW - cytochromes

KW - enzyme catalysis

KW - heme proteins

KW - high throughput

KW - ESCHERICHIA-COLI

KW - NADPH-P450 REDUCTASE

KW - FUSION PROTEIN

KW - RHODOCOCCUS SP

KW - ACTIVE-SITE

KW - FUNCTIONAL EXPRESSION

KW - ELECTRON-TRANSFER

KW - SYSTEM

KW - DOMAIN

KW - ENZYME

UR - http://www.scopus.com/inward/record.url?scp=77951723377&partnerID=8YFLogxK

U2 - 10.1002/cbic.201000104

DO - 10.1002/cbic.201000104

M3 - Article

SN - 1439-4227

VL - 11

SP - 987

EP - 994

JO - Chembiochem

JF - Chembiochem

IS - 7

ER -