The role of cavity residue leucine 95 and channel residues glutamine 204, aspartic acid 211, and phenylalanine 269 on toluene o-xylene monooxygenase activity and regiospecificity

Conference Dates

September 15-19, 2019


The biocatalyst toluene-o-xylene monooxygenase (ToMO) of Pseudomonas sp. OX1 belongs to a remarkable family of bacterial multicomponent monooxygenases and has been shown to have a great potential for biotechnological and environmental applications. Structural analysis of ToMO hydroxylase revealed the presence of three hydrophobic cavities, a channel, and a pore leading from the protein surface to the active site. A structural study with the related enzyme toluene-4-monooxygenase (T4MO) hydroxylase with its respective regulatory protein confirmed that the channel undergoes extensive structural changes upon binding of the regulatory protein and transiently opens and closes during catalysis (Figure 1). Here, saturation mutagenesis was used to investigate the catalytic roles of alpha-subunit (TouA) second cavity residue L95 and TouA channel residues Q204, D211, and F269. By testing the substrates toluene, phenol, nitrobenzene, and/or naphthalene, these positions were found to influence the catalytic activity of ToMO. Several regiospecific variants were identified from TouA positions Q204, F269, and L95. For example, TouA variant Q204H had the regiospecificity of nitrobenzene changed significantly from 30 to 61 % p-nitrophenol. Interestingly, a combination of mutations at Q204H and A106V altered the regiospecificity of nitrobenzene back to 27 % p-nitrophenol. TouA variants F269Y, F269P, Q204E, and L95D improved the meta-hydroxylating capability of nitrobenzene by producing 87, 85, 82, and 77 % m-nitrophenol, respectively. Here, two additional TouA residues, S222 and A106, were also identified that may have important roles in catalysis. Most of the isolated variants from D211 remained active, whereas having a hydrophobic residue at this position appeared to diminish the catalytic activity toward naphthalene. The mutational effects on the ToMO regiospecificity described here suggest that it is possible to further fine tune and engineer the reactivity of multicomponent diiron monooxygenases toward different substrates at positions that are relatively distant from the active site.

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