Laboratory evolution of P450 BM3 for mediated electron transfer yielding an activity-improved and reductase-independent variant.
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 k(cat) value and a 3-fold higher catalytic efficiency compared with the starting point mutant P450 BM3 F87A (k(cat): 62 min(-1) compared with 28 min(-1); k(cat)/K(m): 62 microM(-1)min(-1) compared to 19 microM(-1)min(-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 k(cat) value of 89% of the values obtained for P450 BM3 M5.
Nazor
Jovana
J
Jacobs University, Campus Ring 8, 28759 Bremen, Germany.
Dannenmann
Stephanie
S
Adjei
Richard Obeng
RO
Fordjour
Yaw Boafo
YB
Ghampson
Isaac Tyrone
IT
Blanusa
Milan
M
Roccatano
Danilo
D
Schwaneberg
Ulrich
U
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods*; D004579:Electron Transport; D007700:Kinetics; D007848:Laurates / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016296:Mutagenesis / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010088:Oxidoreductases / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D015032:Zinc / Q000378:metabolism
D001426:Bacterial Proteins; D003067:Coenzymes; D007848:Laurates; C425970:p-nitrophenoxydodecanoic acid; D003035:Cobalt; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D015032:Zinc