Directed evolution of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 previously created the hydroxylase ␣-subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity). In the present study, saturation mutagenesis was performed at position A106 with Escherichia coli TG1/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation. Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells (V max of 9.3 versus 4.5 nmol ⅐ min ؊1 ⅐ mg of protein ؊1 and unchanged K m ), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography. The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 ؎ 0.07 versus 1.30 ؎ 0.06 nmol ⅐ min ؊1 ⅐ mg of protein ؊1 [mean ؎ standard deviation]) at 91 M and has a substantial decrease in regiospecificity, since o-cresol (50%), m-cresol (25%), and p-cresol (25%) are formed, in contrast to the 98% o-cresol formed by wild-type TOM. The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography (V max of 2.61 versus 0.95 nmol ⅐ min ؊1 ⅐ mg of protein ؊1 and unchanged K m ) and chloride release (0.034 ؎ 0.002 versus 0.012 ؎ 0.001 nmol ⅐ min ؊1 ⅐ mg of protein ؊1 ). The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 ؎ 0.3 versus 1.30 ؎ 0.06 nmol ⅐ min ؊1 ⅐ mg of protein ؊1 ). A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with o-cresol (28%), m-cresol (18%), and p-cresol (54%) being formed. Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis.Toluene ortho-monooxygenase (TOM) is encoded by the Burkholderia cepacia G4 gene cluster tomA012345 (33) and belongs to an evolutionarily related family of nonheme diiron hydroxylases found in a wide range of bacteria (16). Examples of the family include soluble methane monooxygenase (sMMO) from methanotrophic bacteria (6, 37) as well as other toluene monooxygenases, such as toluene 4-monooxygenase from Pseudomonas mendocina KR1 (40), toluene 3-monooxygenase from Ralstonia pickettii PKO1 (23), and toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1 (1). TOM is a three-component complex consisting of a 211-kDa hydroxylase (tomA1A3A4) with two binuclear iron centers in the (␣␥) 2 quaternary structure, a 40-kDa NADH-oxidoreductase (tomA5), and a 10.4-kDa regulatory protein (tomA2) involved in the electron transfer between the hydroxy...