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Directed evolution and rational design were used to generate active variants of toluene-4-monooxygenase (T4MO) on 2-phenylethanol (PEA), with the aim of producing hydroxytyrosol, a potent antioxidant. Due to the complexity of the enzymatic system-four proteins encoded by six genes-mutagenesis is labor-intensive and time-consuming. Therefore, the statistical model of Nov and Wein (J. Comput. Biol. 12:247-282) was used to reduce the number of variants produced and evaluated in a lab. From an initial data set of 24 variants, with mutations at nine positions, seven double or triple mutants were identified through statistical analysis. The average activity of these mutants was 4.6-fold higher than the average activity of the initial data set. In an attempt to further improve the enzyme activity to obtain PEA hydroxylation, a second round of statistical analysis was performed. Nine variants were considered, with 3, 4, and 5 point mutations. The average activity of the variants obtained in the second statistical round was 1.6-fold higher than in the first round and 7.3-fold higher than that of the initial data set. The best variant discovered, TmoA I100A E214G D285Q, exhibited an initial oxidation rate of 4.4 ؎ 0.3 nmol/min/mg protein, which is 190-fold higher than the rate obtained by the wild type. This rate was also 2.6-fold higher than the activity of the wild type on the natural substrate toluene. By considering only 16 preselected mutants (out of ϳ13,000 possible combinations), a highly active variant was discovered with minimum time and effort.

Computational modeling combined with mutational and activity assays were used to underline the substrate migration pathways in toluene 4-monooxygenase, a member of the important family of bacterial multicomponent monooxygenases (BMM). In all structurally defined BMM hydroxylases, several hydrophobic cavities in the α-subunit map a preserved path from the protein surface to the diiron active site. Our results confirm the presence of two pathways by which different aromatic molecules can enter/escape the active site. While the substrate is observed to enter from both channels, the more hydrophilic product is withdrawn mainly from the shorter channel ending at residues D285 and E214. The long channel ends in the vicinity of S395, whose variants have been seen to affect activity and specificity. These mutational effects are clearly reproduced and rationalized by the in silico studies. Furthermore, the combined computational and experimental results highlight the importance of residue F269, which is located at the intersection of the two channels.

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