Roles of two glutathione S-transferases in the final step of the β-aryl ether cleavage pathway in Sphingobium sp. strain SYK-6

Sphingobium sp. strain SYK-6 is an alphaproteobacterial degrader of lignin-derived aromatic compounds, which can degrade all the stereoisomers of β-aryl ether-type compounds. SYK-6 cells convert four stereoisomers of guaiacylglycerol-β-guaiacyl ether (GGE) into two enantiomers of α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV) through GGE α-carbon atom oxidation by stereoselective Cα-dehydrogenases encoded by ligD, ligL, and ligN. The ether linkages of the resulting MPHPV enantiomers are cleaved by stereoselective glutathione (GSH) S-transferases (GSTs) encoded by ligF, ligE, and ligP, generating (βR/βS)-α-glutathionyl-β-hydroxypropiovanillone (GS-HPV) and guaiacol. To date, it has been shown that the gene products of ligG and SLG_04120 (ligQ), both encoding GST, catalyze GSH removal from (βR/βS)-GS-HPV, forming achiral β-hydroxypropiovanillone. In this study, we verified the enzyme properties of LigG and LigQ and elucidated their roles in β-aryl ether catabolism. Purified LigG showed an approximately 300-fold higher specific activity for (βR)-GS-HPV than that for (βS)-GS-HPV, whereas purified LigQ showed an approximately six-fold higher specific activity for (βS)-GS-HPV than that for (βR)-GS-HPV. Analyses of mutants of ligG, ligQ, and both genes revealed that SYK-6 converted (βR)-GS-HPV using both LigG and LigQ, whereas only LigQ was involved in converting (βS)-GS-HPV. Furthermore, the disruption of both ligG and ligQ was observed to lead to the loss of the capability of SYK-6 to convert MPHPV. This suggests that GSH removal from GS-HPV catalyzed by LigG and LigQ, is essential for cellular GSH recycling during β-aryl ether catabolism.

High-performance liquid chromatography (HPLC) analysis of the reaction mixtures showed that MPHPV was converted into GS-HPV by purified LigF and LigE at 60 min ( Fig. S2A-C). The conversion of MPHPV by LigF and LigE stopped at approximately 50%, and approximately equimolar GS-HPV to the converted MPHPV was generated ( Fig. S2D and E). Chiral HPLC analysis was conducted to confirm whether LigF and LigE enantioselectively convert MPHPV. The MPHPV preparation that was used as the substrate was an equimolar mixture of (βR)-MPHPV and (βS)-MPHPV (53:47) (Fig. S3A). LigF and LigE completely converted (βS)-MPHPV and (βR)-MPHPV, respectively, indicating (βR)-GS-HPV and (βS)-GS-HPV generation in each reaction ( Fig. S3B and C). We attempted to remove the remaining MPHPV and guaiacol from the reaction mixtures by ethyl acetate extraction; however, we found that the prepared GS-HPV isomers racemized after incubation in buffer A at 30 °C for 24 h (data not shown). Hence, we prepared each GS-HPV isomer by incubating 400 µM MPHPV with purified LigF or LigE and 5 mM GSH in buffer A before usage.

Identification of the GST genes involved in GS-HPV isomer conversion.
Given these results, we designated SLG_04120 as ligQ and investigated the role of ligG and ligQ in converting GS-HPV isomers in SYK-6, as well as the enzymatic properties of these gene products.

Disruption of both ligG and ligQ affects the MPHPV catabolism. The resting cells of each mutant
were reacted with 200 µM racemic MPHPV in buffer A to investigate whether the disruption of ligG and ligQ in SYK-6 affects MPHPV conversion. Here ΔligG and ΔligQ cells converted MPHPV similar to the wild type, whereas ΔligG ligQ cells lost the ability to convert MPHPV (Fig. 5A). By contrast, a cell extract of ΔligG ligQ was able to convert MPHPV in the presence of 5 mM GSH in buffer A at comparable levels with that of the wild type (Fig. S10). The introduction of pQFligG or pQFligQ into ΔligG ligQ cells restored their MPHPV-converting ability, indicating that the disruption of both ligG and ligQ led to the loss of the ability to convert MPHPV in vivo (Fig. 5B).

Discussion
We confirmed that LigG is highly specific for (βR)-GS-HPV, and LigQ converts both GS-HPV isomers. Our study further elucidated that both ligG and ligQ participate in converting (βR)-GS-HPV in SYK-6 cells, whereas only ligQ is involved in converting (βS)-GS-HPV (Figs. 1, 4A,B). Considering that the rate of (βR)-GS-HPV  www.nature.com/scientificreports/ conversion by ΔligQ for 60 min was lower than that by ΔligG, ligQ appears to more involved in (βR)-GS-HPV conversion than ligG (Fig. 4A). However, the specific activity of LigQ for (βR)-GS-HPV was approximately twofold lower than that of LigG in the presence of 2.4 mM GSH. The reason why the disruption of ligQ had more influence on (βR)-GS-HPV conversion than the disruption of ligG may be the higher GSH affinity of LigQ than that of LigG (Table S1, Table S2, and Fig. S9). Alternatively, ligQ expression level in SYK-6 may be higher than that of ligG.
In N. aromaticivorans DSM 12444, NaGST Nu , which showed 58% amino acid sequence identity with LigQ, was reported to be the only enzyme essential for the conversion of both GS-HPV isomers. In Novosphingobium sp. strain MBES04, there are NmGST3 (MBENS4_2527) and NmGST6 (MBENS4_2530) that showed 29% identity with LigQ and 62% identity with LigG, respectively. NmGST3 had activities for both isomers, and NmGST6 was highly specific for (βR)-GS-HPV 22,23 ; however, the involvement of these genes in GS-HPV catabolism has not been investigated. We found another GST gene in the MBES04 genome, MBENS4_4395 (accession no. GAM07399), exhibiting 59% and 63% identity with LigQ and NaGST Nu , respectively. GAM07399 may be involved in GS-HPV isomer conversion besides the NmGST3 and NmGST6 genes. In Novosphingobium sp. strain PP1Y, NsLigG (PP1Y_AT11674), exhibiting 63% identity with LigG, was highly specific for (βR)-GS-HPV 26 . Furthermore, we found PP1Y_AT11650 (accession no. CCA92086) in the genome of this strain, which showed 80% identity with NmGST3; and PP1Y_AT20084 (accession no. CCA92884) that showed 57% and 66% identity with LigQ and NaGST Nu , respectively. These genes may also be involved in GS-HPV isomer conversion.
In the SYK-6 genome, the β-aryl ether catabolic genes, ligD, ligF, ligE, and ligG, constitute an operon, whereas ligQ locates at a different locus (Fig. S11) 14 . In the case of MBES04, the NmGST4-NmGST5-NmGST6 genes (MBENS4_2528-2530) corresponding to ligFEG are adjacent, and the NmGST3 gene (MBENS4_2527, a Nuclass GST gene) locates just upstream of MBENS4_2528 with a different transcription direction (Fig. S11) 22 . By contrast, another Nu-class GST gene MBENS4_4395 (accession no. GAM07399) locates at a different locus. Almost the same gene organization was also observed in the PP1Y genome (Fig. S11) 22 . Based on the conservation of these gene arrangements, the conversion of both GS-HPV isomers may become possible by acquiring a Nu-class GST gene in strains carrying ligFEG or by adding ligFEG to a Nu-class GST gene-carrying strain. Unlike the aforementioned strains, the genes corresponding to ligDFE are scattered through the DSM 12444 genome.
The K m values of LigG and LigQ for GSH were determined to be 1.19 ± 0.22 and 0.34 ± 0.10 mM, respectively (Table S1, Table S2, and Fig. S9). These values were considerably higher than K m of LigG for (βR)-GS-HPV (0.016 ± 0.001 mM) 21 and K m of LigQ for (βR)-GS-HPV (0.055 ± 0.007 mM) and (βS)-GS-HPV (0.011 ± 0.002 mM) 24 . The K m value of LigG for GSH is supported by the observation of Picart et al. that the specific activity of LigG was halved when reacted in the presence of 0.4 mM GSH compared to 1 mM GSH 26 . Based on the quantum mechanics/molecular mechanics simulations, Prates et al. suggested that HPV leaves the active site after GSof GS-HPV is transferred to Cys15 of LigG, giving enough space to accommodate a second GSH for a subsequent thiol-disulfide reaction, which forms GSH disulfide and restores the catalytic ability of LigG 18 . The lack of specific interactions to bind the GSH in the active site of LigG was suggested to be associated   28,29 .
The mechanism for the cleavage of the thioether bond in GS-HPV by NaGST Nu was proposed based on X-ray crystal structure, modeling of substrate binding, and mutant analysis. Thr51 and Asn53 of NaGST Nu provide hydrogen bonds that stabilize a reactive GSH thiolate anion, which attacks the GS − moiety of GS-HPV to form GS-SG disulfide. The rupture of the thioether bond is facilitated by the formation of a transient enolate intermediate. This intermediate is stabilized by the interactions between GS-HPV and Tyr166 and Tyr224 hydroxy groups. The capture of a solvent-derived proton by the carbanion collapses the enolate to form HPV 24 . All these residues are conserved in LigQ, GAM07399, and CCA92884, suggesting that these GSTs catalyze GSH removal from GS-HPV using the same reaction mechanism as that of NaGST Nu .
Finally, we observed that ΔligG ligQ lost the ability to convert MPHPV in vivo (Fig. 5A). In ΔligG ligQ cells, intracellular GSH was seemingly depleted due to GS-HPV accumulation during MPHPV catabolism. Consequently, the reactions that are catalyzed by β-etherases (LigF, LigE, and LigP) could not proceed (Fig. 1). Conversion of GS-HPV by LigG and LigQ produces HPV and GSSG; the latter of which must be reduced to GSH by NAD(P)H-dependent GSSG reductase (Fig. 1) 30 . Hence, it can be concluded that GS − removal from GS-HPV by LigG and LigQ is essential for maintaining intracellular GSH concentration during β-aryl ether catabolism.

Methods
Bacterial strains, plasmids, and culture conditions. Table S3 lists the strains and plasmids that were used in this study. Sphingobium sp. strain SYK-6 and its mutants were grown in lysogeny broth (LB), Wx-SEMP, and Wx-SEMP containing 5 mM GGE at 30 °C. When necessary, 50 mg kanamycin/liter, 100 mg streptomycin/ liter, or 12.5 mg tetracycline/liter were added to the cultures. E. coli strains were grown in LB at 37 °C. The media for E. coli transformants were supplemented with 100 mg ampicillin/liter, 25 mg kanamycin/liter, or 12.5 mg tetracycline/liter.
Enzyme assays for cell extracts of SYK-6 and its mutants. GS-HPV-transforming activities of the cell extracts of SYK-6 and its mutants were determined by measuring HPV production using HPLC. The MPHPV-transforming activities of the cell extracts of SYK-6 and its mutants were determined by measuring the decrease in MPHPV using HPLC. The reaction products were also detected using HPLC analysis. SYK-6 and its mutant cells (ΔligG, ΔligQ, and ΔligG ligQ) grown in LB were washed with Wx medium, resuspended in Wx-SEMP medium to an optical density at 600 nm (OD 600 ) of 0.2, and incubated for 16 h. The resultant cells were washed twice with buffer A. The cells that were resuspended in the same buffer were then broken by an ultrasonic disintegrator (QSonica Q125; WakenBtech Co., Ltd.), and the supernatants were obtained as cell extracts after centrifugation (19,000 × g for 15 min at 4 °C). Protein concentration was determined using the Bradford method, using bovine serum albumin as the standard (Bio-Rad Laboratories).
Cell extracts (5-1000 µg protein/ml) were incubated with 100 µM (βR)-GS-HPV, (βS)-GS-HPV, or 200 µM racemic MPHPV in the presence of 2.4 mM (for GS-HPV) or 5.0 mM GSH (for MPHPV) in buffer A at 30 °C. The reactions were stopped by adding acetonitrile (final concentration of 50%) after 3 min (measurement of the specific activities for GS-HPV), 5 min (identification of GS-HPV metabolites), 20 min (measurement of the specific activities for GS-HPV in mutant-complemented strains), or 60 min (for MPHPV). Precipitated proteins were removed by centrifugation at 19,000 × g for 15 min, and the resulting supernatants were diluted with water to a final acetonitrile concentration of 12.5%, filtered, and analyzed using HPLC. The specific activity of GS-HPV conversion was expressed in moles of HPV produced per min per milligram of protein. The cells of SYK-6 grown in LB were washed with Wx medium, resuspended in Wx-SEMP medium to an OD 600 of 0.2, and incubated at 30 °C to examine the effects of GGE on enzyme induction. When the cultures reached an OD 600 of 0.4-0.5, we added 5 mM GGE to them. After 12 h of further incubation, cell extracts were prepared and used for the enzyme assay. For complementing ΔligG ligQ, pQFligG and pQFligQ were constructed by In-Fusion cloning (Takara Bio) of ligG and ligQ fragments amplified by PCR using SYK-6 total DNA and their corresponding primer pairs (Table S4)  www.nature.com/scientificreports/ ΔligG ligQ cells through electroporation. The transformed cells were grown in Wx-SEMP containing 100 µM cumate and tetracycline for 16 h, and the cell extracts were prepared and used for the enzyme assay.
Sequence analysis. Nucleotide sequences were determined through Eurofins Genomics. Sequence analysis was conducted using the MacVector program (MacVector, Inc.). Sequence similarity searches, pairwise alignments, and multiple alignments were conducted using the BLASTP program 32 , the EMBOSS Needle program through the EMBL-EBI server 33 , and the Clustal omega program 34 , respectively. For phylogenetic analysis, multiple alignments were conducted using the Clustal omega program, and then, phylogenetic trees were generated using the neighbor-joining algorithm of the MEGA 7 software ver. 7.0.26 (https ://www.megas oftwa re.net 35 ), applying 1,000 bootstrap replicates.
Expression of SYK-6 GST genes in E. coli and enzyme purification. DNA fragments carrying SLG_00360, ligQ (SLG_04120), ligF (SLG_08650), ligE (SLG_08660), ligG (SLG_08670), and SLG_29340 were amplified through PCR using SYK-6 total DNA and the primer pairs listed in Table S4, and each amplified fragment was cloned into pET-16b by In-Fusion Cloning. The nucleotide sequences of the inserts were confirmed by sequencing. Each expression plasmid was introduced into E. coli BL21(DE3), and the transformed cells were grown in LB. Gene expression was induced for 4 h at 30 °C by adding 1 mM isopropyl-β-D-thiogalactopyranoside when the OD 600 of the cultures reached 0.5. The cells were then harvested by centrifugation at 5000×g for 5 min at 4 °C and washed with buffer A. The cells were then resuspended in the same buffer and broken using an ultrasonic disintegrator. The supernatants were obtained as cell extracts after centrifugation at 19,000×g for 15 min at 4 °C. For purification of LigF, LigE, LigG, and LigQ, the cell extracts of E. coli BL21(DE3) harboring pET08650, pET08660, pET08670, and pET04120, respectively (Table S3), were transferred onto a His SpinTrap column (GE Healthcare). The purified fractions were subjected to desalting and concentration using an Amicon Ultra centrifugal filter unit (30 kDa cutoff; Merck Millipore), and the enzyme preparations were stored at − 80 °C. Gene expressions and the purity of the preparations were examined using SDS-12% PAGE. The protein bands in gels were stained with Coomassie Brilliant Blue.

Enzyme assays for cell extracts of E. coli transformants and purified enzymes. GS-HPV-trans-
forming activities were determined by measuring HPV production using HPLC. The reaction products were also detected through HPLC analysis. The cell extracts (100 µg protein/ml) and purified enzymes (LigG, 1.25 and 250 µg protein/ml for the conversion of (βR)-and (βS)-GS-HPV, respectively; LigQ, 0.2 and 1.0 µg protein/ ml for the conversion of (βS)-and (βR)-GS-HPV, respectively) were incubated in the 100 µl reaction mixtures containing 100 µM (βR)-GS-HPV or (βS)-GS-HPV, 2.4 mM GSH, and buffer A at 30 °C. The reactions were stopped by adding acetonitrile or methanol to a final concentration of 50% after 30 min of incubation (for cell extracts) and 15 s (for purified enzymes). Protein precipitates were removed through centrifugation at 19,000×g for 15 min. The resulting supernatants were diluted with water to a final concentration of acetonitrile or methanol of 12.5%, filtered, and analyzed using HPLC. Specific activities for GS-HPV conversion of purified enzymes were calculated after 15 s incubation and expressed in moles of HPV produced per min per milligram of protein.
The K m values of LigG and LigQ for GSH were determined using 100 µM (βR)-GS-HPV under the following protein and GSH concentrations: LigG, 1.25 µg/ml protein and 0.25-4.9 mM GSH; LigQ, 5.0 µg/ml protein and 0.1-1.9 mM GSH. The K m values were calculated through non-linear regression analysis using the GraphPad Prism 7 software (GraphPad Software Inc.; https ://www.graph pad.com/scien tific -softw are/prism /) fitted to the Michaelis-Menten equation and expressed as means ± standard deviations of three independent experiments.

Construction of mutants.
To construct the ligG and ligQ mutants, the upstream and downstream regions of the genes were amplified through PCR from SYK-6 total DNA using the primer pairs listed in Table S4. The resulting fragments were cloned into pAK405 by In-Fusion Cloning. Each of the resulting plasmids was introduced into the SYK-6 cells by triparental mating, and the resulting mutants were selected as described previously 36 . Gene deletion was confirmed through colony PCR using the primer pairs listed in Table S4.

Data availability
All data supporting this study are available within the article and its Supplementary Information or are available from the corresponding author upon request.