Laboratory evolution of toluene dioxygenase to accept 4-picoline as a substrate.
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 approximately 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 approximately 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.
Sakamoto
T
T
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Joern
J M
JM
Arisawa
A
A
Arnold
F H
FH
D000595:Amino Acid Sequence; D001483:Base Sequence; D001673:Biodegradation, Environmental; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D016296:Mutagenesis; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010847:Picolines / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D010847:Picolines; D010105:Oxygenases; C023172:toluene dioxygenase