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PEDS Advance Access originally published online on December 19, 2007
Protein Engineering Design and Selection 2008 21(1):29-35; doi:10.1093/protein/gzm074
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Laboratory evolution of P450 BM3 for mediated electron transfer yielding an activity-improved and reductase-independent variant

Jovana Nazor, Stephanie Dannenmann, Richard Obeng Adjei, Yaw Boafo Fordjour, Isaac Tyrone Ghampson, Milan Blanusa, Danilo Roccatano and Ulrich Schwaneberg1

Jacobs University, Campus Ring 8, 28759 Bremen, Germany

1To whom correspondence should be addressed. E-mail: u.schwaneberg{at}jacobs-university.de

One of the main obstacles in employing P450 monooxygenases for preparative chemical syntheses in cell-free systems is their requirement for cofactors such as NAD(P)H. In order to engineer P450 BM3 from Bacillus megaterium for cost-effective process conditions in vitro, a validated medium throughput screening system based on cheap Zn dust as an electron source and Cobalt(III)sepulchrate (Co(III)sep) as a mediator was reported. In the current study, the alternative cofactor system Zn/Co(III)sep was used in a directed evolution experiment to improve the Co(III)sep-mediated electron transfer to P450 BM3. A variant, carrying five mutations (R47F F87A V281G M354S D363H, Table  I), P450 BM3 M5 was identified and characterized with respect to its kinetic parameters. P450 BM3 M5 achieved for mediated electron transfer a 2-fold higher kcat value and a 3-fold higher catalytic efficiency compared with the starting point mutant P450 BM3 F87A (kcat: 62 min–1 compared with 28 min–1; kcat/Km: 62 µM–1min–1 compared to 19 µM–1min–1). For obtaining first insights on electron transfer contributions, three reductase-deficient variants were generated. The reductase-deficient mutant of P450 BMP M5 exhibited a catalytic efficiency of 69% and a kcat value of 89% of the values obtained for P450 BM3 M5.


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  		Table I. List of all P450 BM3 variants generated by site-directed mutagenesis and random mutagenesis

 

Keywords: cofactor/directed evolution/high-throughput screening/mediator/monooxygenase

Received August 26, 2007; revised October 30, 2007; accepted November 15, 2007.


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