TY - JOUR
T1 - Directed Evolution of a Cytochrome P450 Monooxygenase for Alkane Oxidation
AU - Farinas, Edgardo T
AU - Schwaneberg, Ulrich
AU - Glieder, Anton
AU - Arnold, Frances H
N1 - Publisher Copyright:
Copyright © 2001 WILEY-VCH Verlag GmbH, Weinheim, Fed. Rep. of Germany.
PY - 2001/8
Y1 - 2001/8
N2 - Cytochrome P450 monooxygenase BM-3 (EC 1.14.14.1) hydroxylates fatty acids with chain lengths between C12and C18. It is also known to oxidize the corresponding alcohols and amides. However, it is not known to oxidize alkanes. Here we report that P450 BM-3 oxidizes octane, which is four carbons shorter and lacks the carboxylate functionality of the shortest fatty acid P450 BM-3 is known to accept, to 4-octanol, 3-octanol, 2-octanol, 4-octanone, and 3-octanone. The rate is much lower than for oxidation of the preferred fatty acid substrates. In an effort to explore the plasticity and mechanisms of substrate recognition in this powerful biocatalyst, we are using directed evolution − random mutagenesis, recombination, and screening − to improve its activity towards saturated hydrocarbons. A spectrophotometric assay has been validated for high throughput screening, and two generations of laboratory evolution have yielded variants displaying up to five times the specific activity of wild-type P450 BM-3.
AB - Cytochrome P450 monooxygenase BM-3 (EC 1.14.14.1) hydroxylates fatty acids with chain lengths between C12and C18. It is also known to oxidize the corresponding alcohols and amides. However, it is not known to oxidize alkanes. Here we report that P450 BM-3 oxidizes octane, which is four carbons shorter and lacks the carboxylate functionality of the shortest fatty acid P450 BM-3 is known to accept, to 4-octanol, 3-octanol, 2-octanol, 4-octanone, and 3-octanone. The rate is much lower than for oxidation of the preferred fatty acid substrates. In an effort to explore the plasticity and mechanisms of substrate recognition in this powerful biocatalyst, we are using directed evolution − random mutagenesis, recombination, and screening − to improve its activity towards saturated hydrocarbons. A spectrophotometric assay has been validated for high throughput screening, and two generations of laboratory evolution have yielded variants displaying up to five times the specific activity of wild-type P450 BM-3.
KW - alkanes
KW - cytochrome P450 BM-3
KW - enzyme catalysis
KW - enzyme engineering
KW - in vitro evolution
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U2 - 10.1002/1615-4169(200108)343:6/7<601::AID-ADSC601>3.0.CO;2-9
DO - 10.1002/1615-4169(200108)343:6/7<601::AID-ADSC601>3.0.CO;2-9
M3 - Article
AN - SCOPUS:85165594655
SN - 1615-4150
VL - 343
SP - 601
EP - 606
JO - Advanced Synthesis and Catalysis
JF - Advanced Synthesis and Catalysis
IS - 6-7
ER -