Characterization of the Glutathione S-Transferases Involved in Styrene Degradation in Gordonia rubripertincta CWB2

Abstract

The glutathione S-transferases carried on the plasmid for the styrene-specific degradation pathway in the Actinobacterium Gordonia rubripertincta CWB2 were heterologously expressed in Escherichia coli. Both enzymes were purified via affinity chromatography and subjected to activity investigations. StyI and StyJ displayed activity toward the commonly used glutathione S-transferase model substrate 1-chloro-2,4-dinitrobenzene (CDNB) with K_m values of 0.0682 ± 0.0074 and 2.0281 ± 0.1301 mM and V_max values of 0.0158 ± 0.0002 and 0.348 ± 0.008 U mg^-1 for StyI and StyJ, respectively. The conversion of the natural substrate styrene oxide to the intermediate (1-phenyl-2-hydroxyethyl)glutathione was detected for StyI with 48.3 ± 2.9 U mg^-1. This elucidates one more step in the not yet fully resolved styrene-specific degradation pathway of Gordonia rubripertincta CWB2. A characterization of both purified enzymes adds more insight into the scarce research field of actinobacterial glutathione S-transferases. Moreover, a sequence and phylogenetic analysis puts both enzymes into a physiological and evolutionary context. IMPORTANCE Styrene is a toxic compound that is used at a large scale by industry for plastic production. Bacterial degradation of styrene is a possibility for bioremediation and pollution prevention. Intermediates of styrene derivatives degraded in the styrene-specific pathways are precursors for valuable chemical compounds. The pathway in Gordonia rubripertincta CWB2 has proven to accept a broader substrate range than other bacterial styrene degraders. The enzymes characterized in this study, distinguish CWB2s pathway from other known styrene degradation routes and thus might be the main key for its ability to produce ibuprofen from the respective styrene derivative. A biotechnological utilization of this cascade could lead to efficient and sustainable production of drugs, flavors, and fragrances. Moreover, research on glutathione metabolism in Actinobacteria is rare. Here, a characterization of two glutathione S-transferases of actinobacterial origin is presented, and the utilization of glutathione in the metabolism of an Actinobacterium is proven.

Keywords: Actinobacteria; biotransformation; glutathione; glutathione S-transferases; microbial ibuprofen production; styrene metabolism; styrene oxide.

FIG 1

Phylogenetic trees of homologous sequences found via BLASTP in other organisms for the StyI (A) and StyJ (B) protein sequences. Both GSTs did not appear in the respective searches for another. The evolutionary history was inferred using the neighbor-joining method (52). The optimal tree is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches (47). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Jones-Taylor-Thornton (JTT) matrix-based method (48) and are in the units of the number of amino acid substitutions per site. This analysis involved 25 amino acid sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were 250 (A) or 252 (B) positions in the final data set. Evolutionary analyses were conducted in MEGA X (49). Tree generation using the maximum likelihood or minimum evolution methods resulted in similar trees as the ones displayed.

FIG 2

Protein staining pattern of SDS-PAGE (left) and immunodetection of a Western blot (right) of purified recombinant glutathione S-transferases StyI (estimated molecular weight from experiment was 35 kDa and theoretical molecular weight was 29.8 kDa) and StyJ (estimated molecular weight from experiment was 30 kDa and theoretical molecular weight was 30.1 kDa) from Gordonia rubripertincta CWB2.

FIG 3

Michaelis-Menten kinetics determined for 1.5 mM CDNB and various concentrations of GSH. (A) StyI resulted in a K_m value of 0.0682 ± 0.0074 mM and a V_max value of 0.0079 ± 0.0001 U (R² = 0.9474); 0.5 mg ml⁻¹ (16.8 μM) enzyme was applied. Enzyme-free background activity was 0.0031 ± 0 U with 6 mM GSH. (B) StyJ resulted in a K_m value of 2.0281 ± 0.1301 mM and a V_max value of 0.0348 ± 0.0008 U (R² = 0.9978); 0.1 mg ml⁻¹ (3.3 μM) enzyme was applied. Enzyme-free background activity was 0.0037 ± 0.0002 U with 11 mM GSH.

FIG 4

Degradation of styrene oxide by StyI and StyJ. (A) The reaction of styrene oxide with glutathione (GSH) catalyzed by a glutathione S-transferase (GST) as proposed for the styrene degradation pathway of Gordonia rubripertincta CWB2 by Heine et al. (7) would lead to the intermediate (1-phenyl-2-hydroxyethyl)glutathione (mass 427.14 g mol⁻¹). (B) Stacked HPLC chromatograms for samples drawn at respective time points (0 to 16 min) for the degradation of styrene oxide (retention time of 2.6 min) by StyI. Samples were drawn from reaction mixtures containing 1 mM styrene oxide and 5 mM GSH over 16 min (5 μg ml⁻¹ StyI) or 60 min (0.3 mg ml⁻¹ StyJ) and analyzed via HPLC. (C and D) Degradation of styrene oxide by StyI (C) and StyJ (D) was detected via HPLC. A decrease in the concentration of styrene oxide was calculated by applying a recorded standard curve. Shown are the reactions (▪) and controls excluding either GSH (●) or the GST (○). LCMS analysis of selected HPLC samples showed corresponding m/z values, as assigned accordingly (428 in positive mode [*], 426 in negative mode [*]).

FIG 5

Styrene-specific degradation pathway in Gordonia rubripertincta CWB2 as proposed by Heine et al. and Lienkamp et al. (7, 17). The styrene monooxygenase StyA/StyB oxidizes styrene to styrene oxide. The opening of the epoxide ring through the conjugation of glutathione is catalyzed through the glutathione S-transferase StyI and generates the intermediate (1-phenyl-2-hydroxyethyl)glutathione. Conversions were experimentally measured. Enclosed conversions of StyH and a deglutathionylation by StyJ lead to phenylacetic acid, which is the central metabolite in the styrene-specific degradation pathway and is then funneled into the central metabolism via multiple enzymatic steps. PaaK, phenylacetate coenzyme A ligase.