Bacterial toluene monooxygenases (TMOs) are known for their ability to oxidize a wide range of aromatics, degrade a variety of organic pollutants, and produce industrially important compounds. Acetanilide, which was once a commonly used drug, is mostly metabolized into 4-acetamidophenol (4-AAP) in the body, which gives its analgesic and antipyretic properties. 4-AAP, also known as acetaminophen and sold under the name Tylenol, has replaced acetanilide in therapy and its derivative 2-AAP has been shown to have therapeutic potential. In this study, metabolism of acetanilide was examined with Escherichia coli strains expressing wild-type TMOs including toluene o-xylene monooxygenase (ToMO) of Pseudomonas sp. OX1, toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4, toluene para-monooxygenase (TpMO) of Ralstonia pickettii PKO1, and toluene 4-monooxygenase (T4MO-BTAi1) of Bradyrhizobium sp. BTAi1 for the very first time. Oxidation activity levels and regiospecificities were determined using reverse-phase high-performance liquid chromatography (HPLC) on a C18 column using a water/methanol/2-propanol solvent system. We found that ToMO successfully metabolized acetanilide and produced mixtures of 2-AAP, 3-AAP, and 4-AAP. TOM metabolized acetanilide into 2-AAP, whereas TpMO and T4MO-BTAi1 metabolized acetanilide primarily at the para position and formed &gt;70% 4-AAP. After these new reactions were discovered, selected engineered enzymes containing mutations in ToMO alpha hydroxylase (TouA) were examined in order to generate further improvements in ToMO regiospecificity. We found that the selectivity of 2-AAP was enhanced by 3.6-fold and the selectivity of 4-AAP was enhanced by 3.2-fold by TouA variants. The results of the present study show the capacity of TMOs and ToMO variants for regiospecific oxidation of acetanilide for the synthesis of acetaminophen and its derivatives using environmentally-benign biological methods rather than currently used traditional chemical processes. Furthermore, this study also shows the potential of non-human TMOs and ToMO variants in drug metabolism applications; hence, their applicability to selectively hydroxylate other drug molecules will be investigated in the future.

Characterization of toluene monooxygenases and engineered toluene o-xylene monooxygenases of Pseudomonas sp. OX1 for the metabolism of acetanilide

ACS Spring 2022

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