Directed Evolution of Toluene ortho -Monooxygenase for Enhanced 1-Naphthol Synthesis and Chlorinated Ethene Degradation

Abstract

Trichloroethylene (TCE) is the most frequently detected groundwater contaminant, and 1-naphthol is an important chemical manufacturing intermediate. Directed evolution was used to increase the activity of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 for both chlorinated ethenes and naphthalene oxidation. When expressed in Escherichia coli, the variant TOM-Green degraded TCE (2.5 ؎ 0.3 versus 1.39 ؎ 0.05 nmol/min/mg of protein), 1,1-dichloroethylene, and trans-dichloroethylene more rapidly. Whole cells expressing TOM-Green synthesized 1-naphthol at a rate that was six times faster than that mediated by the wild-type enzyme at a concentration of 0.1 mM (0.19 ؎ 0.03 versus 0.029 ؎ 0.004 nmol/min/mg of protein), whereas at 5 mM, the mutant enzyme was active (0.07 ؎ 0.03 nmol/min/mg of protein) in contrast to the wild-type enzyme, which had no detectable activity. The regiospecificity of TOM-Green was unchanged, with greater than 97% 1-naphthol formed. The beneficial mutation of TOM-Green is the substitution of valine to alanine in position 106 of the ␣-subunit of the hydroxylase, which appears to act as a smaller "gate" to the diiron active center. This hypothesis was supported by the ability of E. coli expressing TOM-Green to oxidize the three-ring compounds, phenanthrene, fluorene, and anthracene faster than the wild-type enzyme. These results show clearly that random, in vitro protein engineering can be used to improve a large multisubunit protein for multiple functions, including environmental restoration and green chemistry.

Crystal Structure of the Toluene/o-Xylene Monooxygenase Hydroxylase from Pseudomonas stutzeri OX1

“…The I100Q and I100C mutants indicate that the residue in this position need not be hydrophobic for TMOs to be efficient catalysts and suggest that a small side chain is not beneficial, perhaps due to limited gating ability and greater exposure to solvent. A Val to Ala mutation of the analogous gate residue in T2MO from Burkholderia capacia G4, a member of the three-component PH family, resulted in enhanced ability to degrade naphthalene and threering fused aromatics including phenanthrene, fluorene, and anthracene (48). In the light of the present structures, these findings strongly suggest that members of the PH subclass have a similar substrate channel.…”

Insight into the mechanism of aromatic hydroxylation by toluene 4-monooxygenase by use of specifically deuterated toluene and p- xylene

“…First, the x-ray structure of the methane monooxygenase hydroxylase (MmoH) indicates that a short access path from solvent to the diiron center may pass through a ''Leu gate'' (27). Second, directedevolution studies of toluene 2-monooxygenase revealed that a V106A mutation in this ''gate'' region led to an increased reactivity with a larger substrate, naphthalene (28). Third, the position of G103L used in these studies is predicted to lie along this entry portal, in close proximity to the (Fe) 2 center.…”

Using multi-objective computational design to extend protein promiscuity

“…Proteins with several activities (multipurpose enzymes) may have a wide range of biotechnological applications related (but not limited) to industrial organic synthesis and metabolic engineering [12][13][14][15][16][17]. However, natural protein promiscuity (as described in the two preceding paragraphs) may perhaps be of limited use in the development of these multipurpose catalysts.…”