(613b) Protein Engineering Of Toluene Monooxygenases For Synthesis Of Chiral Sulfoxides

Enantiopure sulfoxides are valuable asymmetric starting materials that are able to bring about asymmetric transformations and are important chiral auxiliaries in organic synthesis. In addition, chiral sulfoxides possess a wide range of biological activity from flavor and aroma precursors to antimicrobial properties. Asymmetric oxidation of sulfides to chiral sulfoxides using biocatalyts is an attractive route with economical industrial potential. Toluene monooxygenases (TMOs) are soluble, non-heme, diiron multicomponent enzymes which utilize oxygen to catalyze the initial hydroxylation step in metabolic pathways for the oxidation of hydrocarbon substrates. TMOs were shown to be versatile biocatalysts capable of oxidizing a large spectrum of substrates such as substituted aromatic and phenolic compounds, as well as chlorinated aliphatic compounds. Here we show for the first time, that TMOs are also capable of performing enantioselective oxidation reactions of aromatic sulfides. Furthermore, through mutagenesis of a key position in the alpha-hydroxylase subunit of toluene-ortho-monooxygenase (TOM) of Burkholderia cepacia G4, and toluene-4-monooxygenases (T4MO) of Pseudomonas mendocina KR1, the catalytic activity is altered so that the rate and enantioselectivity are dramatically improved. Escherichia coli TG1 cells expressing TOM variant TomA3 V106A were found to hydroxylate methyl-phenyl-sulfide to the corresponding sulfoxide at a rate of 2.7 nmol/min/mg protein compared with 1.6 for the wild-type enzyme. The enantiomeric excess (ee) increased from 51% (pro-S) for the wild-type to 91% for the mutant. Similar results were obtained with V106M and V106E variants suggesting that position 106 can occupy a small hydrophobic residue, but also a large and charged residue. The structure of the sulfide had a dramatic influence on the activity and selectivity of the wild-type enzymes and variants as well. For example, methyl-para-tolyl sulfide (addition of a methyl group in the para position to the sulfur atom), was converted to the (R)-sulfoxide by TomA3 V106M, rather than to the (S)-enantiomer. The (R)-methyl-para-tolyl-sulfoxide (41% ee) was also obtained by wild-type T4MO in very low conversion rates, whereas variant TmoA I100A showed a ten-fold improvement in activity accompanied by high enantioselectivity (77% ee) towards the opposite, (S)-sulfoxide. In silico homology modeling was used to elucidate structure-function relationships of the substrates and mutants. This work demonstrates that position V106 of TOM (and the analogous residue I100 of T4MO) influences not only substrate regiospecificity, but also enantiospecificity.