Laboratory Evolution of Toluene Dioxygenase To Accept 4-Picoline as a Substrate
Abstract
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
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.Attached Files
Published - SAKaem01.pdf
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Additional details
- PMCID
- PMC93105
- Eprint ID
- 1847
- Resolver ID
- CaltechAUTHORS:SAKaem01
- Maxygen, Inc.
- Created
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2006-02-20Created from EPrint's datestamp field
- Updated
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2023-06-01Created from EPrint's last_modified field