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Directed Evolution of a Cytochrome P450 Monooxygenase for Alkane Oxidation

Farinas, Edgardo T. and Schwaneberg, Ulrich and Glieder, Anton and Arnold, Frances H. (2001) Directed Evolution of a Cytochrome P450 Monooxygenase for Alkane Oxidation. Advanced Synthesis and Catalysis, 343 (6-7). pp. 601-606. ISSN 1615-4150. doi:10.1002/1615-4169(200108)343:6/7<601::AID-ADSC601>3.0.CO;2-9.

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Cytochrome P450 monooxygenase BM‐3 (EC hydroxylates fatty acids with chain lengths between C_(12) and C_(18). 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.

Item Type:Article
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URLURL TypeDescription<601::AID-ADSC601>3.0.CO;2-9DOIArticle
Schwaneberg, Ulrich0000-0003-4026-701X
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2001 WILEY‐VCH Verlag GmbH, Weinheim, Fed. Rep. of Germany. Received March 23, 2001; Accepted May 23, 2001. Special Issue: Biocatalysis. We thank Yaniv Dubowski, Dr. Peter Green and Patrick Cirino for their assistance with the GC/MS. This work is supported by U. S. National Science Foundation Grants DBI-9807460 (ETF) and BES-9981770 (FHA).
Funding AgencyGrant Number
Subject Keywords:alkanes; cytochrome P450 BM‐3; enzyme catalysis; enzyme engineering; in vitro evolution
Issue or Number:6-7
Record Number:CaltechAUTHORS:20180822-144646102
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Official Citation:Farinas, E. , Schwaneberg, U. , Glieder, A. and Arnold, F. (2001), Directed Evolution of a Cytochrome P450 Monooxygenase for Alkane Oxidation. Adv. Synth. Catal., 343: 601-606. doi:10.1002/1615-4169(200108)343:6/7<601::AID-ADSC601>3.0.CO;2-9
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89062
Deposited By: Tony Diaz
Deposited On:22 Aug 2018 21:53
Last Modified:16 Nov 2021 00:31

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