Actinobacterial Glutathione S-transferases: Catalysts for racemic kinetic resolution of Epoxides?

Epoxides are important platform chemicals in industry but also present in nature. Particularly chiral epoxides hold significant industrial interest due to their application in agrochemical or drug synthesis. A major challenge in organic chemistry is achieving high-yield production while maintaining regio- and enantioselectivity. Enzyme-catalyzed epoxidation offers these features but is limited by their selectivity, restricting its universal applicability. Some enzymes such as epoxide hydrolases (EHs), CoM-transferases (CoMTs) or glutathione S-transferases (GSTs) have shown epoxide conversion with certain selectivity. As the selectivity of GSTs is not well studied, these enzymes became our focus.

Lately, the bacterial GST GrStyI was found in the styrene degradation pathway of Gordonia rubripertincta CWB2. Unlike other styrene degraders, CWB2 can metabolize various styrene-related compounds, leading to the co-metabolic formation of ibuprofen for example. The presence of GSTs in bacterial styrene degradation is unusual but appears to broaden the substrate spectrum of this pathway. Similar gene clusters were found in styrene and isoprene-degrading bacteria, encoding GSTs styI and isoI. Consequently, we heterologously produced 5 GSTs in E. coli for characterization of substrate selectivity and enantiomeric preference. Herein, GrStyI appeared as the most promising enzyme for enzymatic kinetic resolution.

To explore GrStyIs potential for enantioselective epoxide opening, chiral GC-FID analysis was conducted on various aromatic and aliphatic epoxides. For styrene oxide derivatives, GrStyI exhibited high selectivity for (S)-enantiomers, while the enantiomeric preference of more diverse structures like aliphatic epoxides showed reduced enantioselectivity. Given its high enantioselectivity towards (S)-epoxides, we performed enzymatic kinetic resolution. To proof the feasibility, the reaction volume was subsequently increased to 150 ml for large-scale production, yielding approx. 14 mg (R)-styrene oxide, validated by NMR.

Summarizing, we identified GrStyI as highly enantioselective for (S)-styrene oxide and structural analogous. Our study highlights GrStyIs potential for large-scale production of enantiopure epoxides like styrene oxide. Moreover, GSTs from isoprene degradation appear as promising candidates for the production of enantiopure aliphatic epoxides.