Search

Nitrobenzene dioxygenase (NBDO) from Comamonas sp. is shown here to perform enantioselective oxidation of aromatic sulfides. Several para-substituted alkyl aryl sulfides were examined and it was found that the activity of the enzyme is dependent on the size of the substrate. Saturation mutagenesis was performed on different residues in the active site in order to improve activity and selectivity. Mutagenesis at position 258 in the α-hydroxylase subunit of NBDO improved both activity and enantioselectivity. Substitutions in position 293 improved the activity on all substrates and had diverse influence on enantioselectivity. Mutagenesis in position 207 provided two interesting variants, V207I and V207A, with opposite enantioselectivities. Furthermore, combining two favorable mutations, N258A and F293H, provided an improved variant with both higher activity (5.20 ± 0.01, 2.12 ± 0.21, 2.64 ± 0.14 and 4.01 ± 0.34 nmol min(-1) mg protein(-1) on thioanisole, ptolyl, Cl-thioanisole and Br-thioanisole, respectively, which is 1.7, 4.6, 7.1 and 26.7-fold compared with wild type) and improved enantioselectivity (e.g. 67% enantiomeric excess for Cl-thioanisole vs. 5% for wild type). Molecular docking and active site volume calculations were used to correlate between the structure of the substrates and the function of the enzymes. The results from this work suggest that the location of pro-chiral sulfides in the active site is coordinated by hydrophobic interactions and by steric considerations, which in turn influences the activity and enantioselectivity of NBDO.