Protein engineering of nirobenzene dioxygenase for enantioselective synthesis of chiral sulfoxides

Abstract

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.

Altering 2‐Hydroxybiphenyl 3‐Monooxygenase Regioselectivity by Protein Engineering for the Production of a New Antioxidant

“…The organic supernatant was taken for solvent evaporation with nitrogen and then resuspended (1:1, v / v ) in HPLC‐grade ethanol, due to the sensitivity of the chiral column to organic solvents. The enantiomeric excess (% ee ) of the products was determined by HPLC on a 1100‐series instrument (Agilent Technologies) equipped with LUX 5 μ m Cellulose‐2 chiral column (250×4.6 mm, Phenomenex) as described before . Detection was carried out with a UV/Vis detector at 225 nm.…”

“…Detection was carried out with a UV/Vis detector at 225 nm. The absolute configuration of all sulfoxides was determined as previously described …”

Improvement of Biocatalysts for Industrial and Environmental Purposes by Saturation Mutagenesis

“…An increasing number of recent papers proposes the application of saturation mutagenesis to biocatalysts of applicative interest, for “greener” industrial processes [67,68] improved bulk enzymes [41,69,70], biotechnology [71,72] bioremediation [73], fine chemical synthesis [74,75,76,77,78,79,80], biofuels production [55,81,82] and biomass exploitation [82,83,84,85]. …”

“…In order to enhance the performance of biocatalysts for fine chemistry, for example, for the synthesis of chiral sulfoxides and asymmetric ketone reduction, other redox enzymes such as nitrobenzene dioxygenase [77], alcohol dehydrogenase [78] and carbonyl reductase [79] were also recently optimized by saturation mutagenesis.…”

Polycyclic Ketone Monooxygenase (PockeMO): A Robust Biocatalyst for the Synthesis of Optically Active Sulfoxides

“…Biocatalytic procedures present some advantages, such as the use of mild and environmentally friendly reaction conditions and oxidants [7,8]. Several types of oxidative enzymes can be used for the synthesis of chiral sulfoxides, which include peroxidases [9][10][11], dioxygenases [12,13] and different types of monooxygenases [14,15]. BVMOs represent a class of flavin-containing monooxygenases that catalyze the Baeyer-Villiger oxidation of aldehydes and ketones, but they are also able to perform epoxidations and the oxygenation of sulfides and other heteroatom-containing compounds, often with high chemo-, regio-and/or enantioselectivity [16][17][18][19].…”