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Laboratory Evolution of Toluene Dioxygenase To Accept 4-Picoline as a Substrate

Sakamoto, Takeshi and Joern, John M. and Arisawa, Akira and Arnold, Frances H. (2001) Laboratory Evolution of Toluene Dioxygenase To Accept 4-Picoline as a Substrate. Applied and Environmental Microbiology, 67 (9). pp. 3882-3887. ISSN 0099-2240. PMCID PMC93105.

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We are using directed evolution to extend the range of dioxygenase-catalyzed biotransformations to include substrates that are either poorly accepted or not accepted at all by the naturally occurring enzymes. Here we report on the oxidation of a heterocyclic substrate, 4-picoline, by toluene dioxygenase (TDO) and improvement of the enzyme's activity by laboratory evolution. The biotransformation of 4-picoline proceeds at only ~4.5% of the rate of the natural reaction on toluene. Random mutagenesis, saturation mutagenesis, and screening directly for product formation using a modified Gibbs assay generated mutant TDO 3-B38, in which the wild-type stop codon was replaced with a codon encoding threonine. Escherichia coli-expressed TDO 3-B38 exhibited 5.6 times higher activity toward 4-picoline and ~20% more activity towards toluene than wild-type TDO. The product of the biotransformation of 4-picoline is 3-hydroxy-4-picoline; no cis-diols of 4-picoline were observed

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Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2001, American Society for Microbiology. Received 31 January 2001/Accepted 31 May 2001 We thank R. W. T. Lee for technical assistance with NMR. We thank Maxygen Inc. for supporting this research.
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Issue or Number:9
PubMed Central ID:PMC93105
Record Number:CaltechAUTHORS:SAKaem01
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:1847
Deposited By: Archive Administrator
Deposited On:20 Feb 2006
Last Modified:02 Oct 2019 22:47

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