Laboratory evolution of toluene dioxygenase to accept 4-picoline as a substrate

We are using directed evolution to extend the range of dioxygenase-catalyze d biotransformations to include substrates that are either poorly accepted or not accepted at all by the naturally occurring enzymes. Here we report o n the oxidation of a heterocyclic substrate, 4-picoline, by toluene dioxyge nase (TDO) and improvement of the enzyme's activity by laboratory evolution . The biotransformation of 4-picoline proceeds at only similar to4.5% of th e rate of the natural reaction on toluene. Random mutagenesis, saturation m utagenesis, and screening directly for product formation using a modified G ibbs assay generated mutant TDO 3-B38, in which the wild-type stop codon wa s replaced with a codon encoding threonine. Escherichia coli-expressed TDO 3-B38 exhibited 5.6 times higher activity toward 4-picoline and similar to 20% more activity towards toluene than wild-type TDO. The product of the bi otransformation of 4-picoline is 3-hydroxy-4-picoline; no cis-diols of 4-pi coline were observed.