Characterization of the dehydrogenase StyH and its role in a glutathione-dependent styrene degradation pathway

Styrene is a precursor for thermoplastic polymers which are of industrial importance. A major issue is the anthropogenic release which burdens the environment. The toxic properties of styrene effectuated some soil-dwelling bacteria to develop degradation pathways detoxifying styrene or utilizing it as a carbon source. Consequently, these organisms became attractive targets for bioremediation. Recently, the unique degradation pathway of Gordonia rubripertincta CWB2 was described. A strain with further biotechnological relevance as CWB2 can degrade a styrene analog to produce ibuprofen in a co‑metabolic process.

Herein, we present a newly characterized dehydrogenase (StyH) with the aim to elucidate its function and role in the pathway. Thus, improving the overall understanding of the CWB2 styrene degradation pathway and increasing its value as a potential candidate for bioremediation or biotechnological applications.

StyH was heterologously produced in E. coli BL21 (DE3), purified by IMAC, and characterized using bioinformatics as well as biochemical methods. The biochemical characterization of StyH was conducted spectrophotometrically to investigate optimal reaction conditions, kinetics and substrate range.

A phylogenetic analysis showed that StyH is a member of the SDR family. Protocols for enzymatic substrate synthesis and purification were established. The process yielded about 72 % (S)-GSH conjugate validated by NMR. Enzyme production was successful. StyH forms a homodimer (confirmed by bioinformatics and SEC). It displayed higher activity at basic pH values. Higher Vmax-values were observed with the (S)-GSH conjugate compared to the (R)-enantiomer. StyH acts specifically on glutathione-conjugated substrates and catalyzes two subsequent reaction steps. The product was identified via LC-MS.

Overall, this study provides new information on the function of StyH in the glutathione-dependent styrene degradation pathway. The enzyme is enantioselective, first catalyzing the (S)-enantiomer in a racemic mixture. StyH demonstrated a two-step catalysis by oxidation of (S)-GSH conjugate the corresponding phenylacetaldehyde, further to (S)‑(1‑phenyl‑2‑acetic acid) glutathione. Only short-chain alcohols were previously found to be catalyzed via two-step reactions.