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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 ϳ4.5% 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 ϳ20% 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.Dioxygenase enzymes involved in the catabolism of aromatic hydrocarbons by soil microorganisms nicely illustrate nature's ability to adapt to different carbon sources through evolution of the substrate specificity of the biodegradation enzymes (35). The biodegradation pathway often begins with the dioxygenasecatalyzed regio-and enantio-specific introduction of molecular oxygen into aromatic compounds to form the corresponding arene cis-diols, with consumption of NADH (13, 36) ( Fig. 1).It has been noted that the chiral products of the dioxygenase reaction can be converted in a variety of synthetic reactions to advanced intermediates for natural-product synthesis (8, 15). Toluene dioxygenase (TDO) from Pseudomonas putida readily dihydroxylates aromatic carbocycles with one or two small hydrophobic substituents (6, 33). Activity towards much larger, fused-ring substrates or substrates with polar or bulky substituents is considerably reduced or nonexistent (33).We are attempting to extend the utility of dioxygenase-catalyzed biotransformations by engineering the catalysts to accept a wider range of substrates, including small heterocyclic compounds and polar-substituted carbocyclic aromatics. N-Heterocyclic compounds are useful for the synthesis of biologically active compounds; recent reports describe the regioand/or stereo-controlled biooxidation of N-heterocycles (20,26). Bicyclic compounds such as quinolines and benzofurans are accepted by TDO, with oxygen insertion occurring primarily in the carbocylic rings (3-5).To date, reactions on single-ring heterocycles have not been reported. We have found that TDO catalyzes the oxygenation of 4-picoline (4-methylpyridine), although at a much slower rate than on its preferred substrate toluene.A strategy of DNA shuffling to create libraries of hybrid genes and screening has been used with some success to extend the substrate range of dioxygenases that degrade environmental pollutants such as polychlorinated biphenyls (7, 21). An alternative approach is to fine tune a single gene by accumulating beneficial mutat...

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We have developed a solid-phase, high throughput (10,000 clones/day) screen for dioxygenase activity. The cis-dihydrodiol product of dioxygenase bioconversion is converted to a phenol by acidification or to a catechol by reaction with cis-dihydrodiol dehydrogenase. Gibbs reagent reacts quickly with these oxygenated aromatics to yield colored products that are quantifiable using a microplate reader or by digital imaging and image analysis. The method is reproducible and quantitative at product concentrations of only 30 microM, with essentially no background from media components. This method is an effective general screen for aromatic oxidation and should be a useful tool for the discovery and directed evolution of oxygenases.