Site-2 protease-like protein 2 mediates phenol tolerance in Rhodococcus ruber through transcriptomic and functional analyses

2. Materials and methods2.1. Strains and CulturesR. ruber SD3 or s2plp2-overexpressing glycerol strains were streaked on Luria-Bertani (LB) agar plates and activated by incubation at 35℃ for 48 h. Then, a single colony was inoculated into 5 mL LB medium and cultured at 35℃ with shaking at 200 rpm until the OD595 nm reached 1.0 for subsequent transfer and experimentation.2.2. Bioinformatics analysis of the S2PLP2 protein in R. ruber SD3To investigate the functional characteristics of the s2plp2 gene and its encoded protein, multiple bioinformatics tools were employed. The complete sequence of the s2plp2 gene and its protein product was obtained from the KEGG database (https://www.genome.jp/kegg/). Homologous sequences of the S2PLP2 protein were downloaded from the NCBI database, and multiple sequence alignment was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) on the EBI platform. A phylogenetic tree was then constructed using MEGA 11.0.13 software. Conserved domains were identified using the KEGG platform (https://www.kegg.jp/ssdb-bin/ssdb_motif?kid=rrz:CS378_21075). Physicochemical properties were analyzed using ProtParam (https://web.expasy.org/protparam/), and hydrophilicity/hydrophobicity profiles were generated with ProtScale (https://web.expasy.org/protscale/). Signal peptide prediction was performed using SignalP 5.0 (https://services.healthtech.dtu.dk/services/SignalP-5.0/). Transmembrane domain analysis was conducted using DeepTMHMM (https://services.healthtech.dtu.dk/services/DeepTMHMM-1.0/) [30]. Secondary structure was predicted using PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/), and tertiary structure was obtained from the AlphaFold DB (https://alphafold.ebi.ac.uk/).2.3. Subcellular localization analysis of S2PLP2To investigate the subcellular localization of S2PLP2, the coding sequence of the s2plp2 gene was cloned into the pNV18-GFP vector, generating the recombinant plasmid pNV18-GFP-s2plp2, which expresses an GFP-S2PLP2 fusion protein. The empty pNV18-GFP vector, expressing GFP alone, was used as a control. Both plasmids were introduced into R. ruber SD3 competent cells via electroporation. Following electroporation, the cells were plated on kanamycin selective medium and incubated to allow for plasmid maintenance and protein expression. For localization analysis, exponentially growing cells harboring either pNV18-GFP-s2plp2 or the empty pNV18-GFP control were harvested and washed by saline. The subcellular distribution of the GFP fluorescence was then examined using confocal laser scanning microscopy in 488 nm. The localization pattern of the GFP-S2PLP2 fusion protein was compared to that of the free GFP control.2.4. Gene cloning of S2PLP2The s2plp2 gene sequence was codon-optimized based on the codon usage bias of Escherichia coli (E. coli). The optimized gene was synthesized by General Biosystems (AnHui) and subsequently ligated into the pGEX-4T-1 vector with Glutathione S-Transferase (GST) tag. The ligation product was transformed into E. coli Top10 competent cells. The recombinant E. coli Top10 strain harboring pGEX-4T-1-s2plp2 was cultured in LB medium at 37℃ with shaking at 200 rpm for 16 h. Plasmid DNA was extracted using the SanPrep Column Plasmid Mini-Preps Kit (Sangon Biotech, ShangHai) according to the manufacturer's instructions. The plasmid was verified by double restriction enzyme digestion with BamH I and EcoR I. Confirmed plasmids were sent to Sangon Biotech (ShangHai) for DNA sequencing. Positive recombinant plasmid clones were identified, and 2 μL of the recombinant plasmid pGEX-4T-1-s2plp2 was transformed into 50 μL of competent E. coli BL21(DE3) pLysS cells for protein expression.2.5. Optimization of the induced expression conditions for S2PLP2 proteinThe E. coli BL21(DE3) pLysS strain harboring pGEX-4T-1-s2plp2 was cultured in LB medium containing 100 μg/mL ampicillin at 37℃ with shaking at 200 rpm for 16 h. The culture was then transferred into fresh LB at a 1:50 ratio and grown at 37℃ with shaking until the OD595 nm reached 0.4-0.6. Protein expression was induced with 0, 0.1, 0.2, 0.5, or 1.0 mM IPTG at 16℃ or 37℃ for 10 h. Following induction, cells were harvested by centrifugation at 10,000 ×g for 1 min.The pellet obtained from 300 μL of culture was resuspended in 30 μL of PBS (pH 7.4), mixed with 30 μL of 2× SDS-PAGE protein loading buffer, vortexed, and boiled for 15 min. The optimal expression conditions were determined by evaluating the intensity of the target band.2.6. Expression and purification of S2PLP2 proteinS2PLP2 was expressed under optimized conditions in a scaled-up culture volume of 300 mL. Cells were harvested by centrifugation at 5,000 ×g for 10 min. Pellets were resuspended in 30 mL PBS (pH 7.4), lysed by sonication (650 W × 30%, 4 s on/6 s off, 30 min, on ice), and fractionated into total, soluble, and insoluble proteins by centrifugation at 14,000 ×g for 5 min at 4℃. According to previous reports, buffer systems containing Triton X-100, NaCl, and Tris-HCl have been successfully used for the extraction of active membrane proteins[31]. Since GST-S2PLP2 localized to the insoluble fraction, pellets were solubilized in 6 mL inclusion body buffer (1 mol/L NaCl and 0.5 mol/L Tris-HCl, 1% Triton X-100, pH 8.0) at 4℃ for 1 h with shaking, followed by mild sonication (650 W × 10%, 2 s on/3 s off, 10 min, on ice). After centrifugation at 14,000 ×g for 5 min at 4℃, the solubilized supernatant was collected, and the process was repeated once. Solubilization efficiency was verified by SDS-PAGE. GST-S2PLP2 was purified from the combined supernatants using ProteinIso® GST Resin (TransGen Biotech, DP201) according to manufacturer's protocol. 2.7. Preparation of polyclonal antibodiesFor primary immunization, 0.5 mL of 1 mg/mL S2PLP protein was emulsified with 0.5 mL complete freund's adjuvant (Merck, F5881) by repeated passage through a 1 mL syringe until a stable water-in-oil emulsion formed. Each of 10 Kunming mice received 200 μL of the emulsion via intraperitoneal and subcutaneous injections. One week later, a secondary immunization was performe using antigen emulsified 1:1 (v/v) with incomplete freund's adjuvant (Merck, F5506). Booster immunizations were administered weekly for a total of three doses. Seven days after the final booster, three mice were randomly selected, anesthetized, and blood was collected from the tail vein. Blood was allowed to clot at room temperature for 2 h, followed by centrifugation at 12,000 ×g for 10 min at 4℃. The supernatant was analyzed by Western blotting to assess antibody production. For mice confirmed to have produced antibodies, blood was collected via the retro-orbital venous plexus. Serum was stored at –80°C for subsequent use.2.8. Western blotting analysisA single colony was cultured in 5 mL LB medium at 35℃ with shaking at 200 rpm until OD595 nm reached 1.0. One milliliter of culture was pelleted by centrifuged at 10,000 ×g for 1 min, resuspended in 100 μL PBS (pH 7.4), and sonicated on ice (650W ×30%, 2 s on/3 s off, 3 min total). Lysates were centrifuged at 14,000 ×g for 5 min at 4℃, and the supernatant was collected. Then, 1 mg/mL cell lysate supernatant or 0.5 mg/mL purified GST-S2PLP2 recombinant protein was mixed with an equal volume of 2 ×SDS-PAGE loading buffer and denatured by boiling at 100℃ for 10 min. Samples were resolved on 12% SDS-PAGE gels at constant 200 V for 50 min. Proteins were followed transferred to a nitrocellulose (NC) membrane at constant 80 mA for 60 min. The NC membrane was blocked overnight at 4℃ in PBST (pH 7.4) containing 5% (m/v) skim milk. Primary antibody was diluted 1:100 in blocking buffer and incubated with the membrane at room temperature for 3 h with shaking. After five washes with PBST, HRP-conjugated goat anti-mouse IgG secondary antibody was diluted 1:2,000 in blocking buffer and incubated with the membrane for 45 min at room temperature. Following five additional washes, target proteins were visualized using the ECL Chemiluminescence Kit (Beyotime Biotechnology, GS009). Band intensities were quantified using ImageJ 1.51j. 2.9. Measurement of S2PLP2 expression in phenol-grown R. ruber SD3The R. ruber SD3 was transferred to fresh LB medium containing 0.02% or 0.04% (m/v) phenol at a 1:50 ratio, with no phenol as the control. After 24 h of incubation at 35℃ with shaking at 200 rpm, cells were harvested by centrifugation at 4,000 ×g for 10 min, washed twice with 0.85% NaCl, and pellets from 1 mL culture aliquots were resuspended in 100 μL PBS (pH 7.4). Bacterial lysis was performed via sonication on ice (650 W × 30%, 2 s on/3 s off, 3 min, on ice), followed by centrifugation at 14,000 ×g for 5 min at 4℃. Membrane proteins of R. ruber SD3 with or without phenol were extracted using a bacterial membrane protein extraction kit (solarbio, EX1940). Protein concentration was quantified using the BCA Kit (Beyotime Biotechnology, P0010), adjusted to 0.5 mg/mL, and resolved by SDS-PAGE. Based on the prestained marker, the 35-40 kDa region was clipped for western blotting, and the remaining gel bands was stained with coomassie blue as a loading control. Band intensities quantified using ImageJ 1.51j. All samples were analyzed in three biological replicates.2.10. Genomic DNA extraction from R. ruber SD3Genomic DNA extraction from R. ruber SD3 was performed refers to previous reports and is briefly described as follows: Each 3 mL of log-phase culture were collected with centrifugated at 12,000 ×g for 1 min[32, 33]. The pellet was resuspended in 500 μL sterile ddH₂O and centrifuged again, repeat three times. Then, the pellet resuspended in 300 μL sterile ddH₂O, boiled for 10 min, immediately frozen at -20℃ for 10 min, thawed, and centrifuged at 12,000 ×g for 1 min. The supernatant containing genomic DNA was collected and stored at -20℃.2.11. Construction of shuttle recombinant plasmids containing the s2plp2 geneTo construct the s2plp2 overexpressing strain (s2plp2E), the s2plp2 gene was recombined into the shuttle plasmid pNV18. The pNV18 vector was linearized by double digestion with EcoR I and Hind III. Gene-specific primers containing homologous arms for recombination were designed (forward: 5’- tatgaccatgattacgaattcATGCTGCGCGGATCGGTC-3’, reverse: 5’-acgacggccagtgcca-agcttCTACTCCGGCCGGGGCTG-3’). PCR amplification was performed using 2.5 μL of genomic DNA, 2.5 μL (10 μM) each primer, 12 μL of 2×P6 High-Fidelity Premix (Tolo Biotech, China, 21805), sterilized ddH2O added to 50 μL. PCR conditions were: denature the DNA at 95℃ for 5 min; 34 cycles of 95℃ for 1 min, 64℃for 30 s, and 72℃ for 55 s; and a final extension at 72℃ for 5 min and preservation at 16℃. PCR products were verified by 1% agarose gel electrophoresis and purified using Omega's Gel Extraction Kit. The linearized pNV18 and s2plp2 insert were recombined via homologous recombination (ToloBio, 24305) at 37℃ for 15 min. The product was transformed into E. coli Top 10 competent cells. Positive clones were cultured in LB with 50 μg/mL kanamycin at 37℃ and 200 rpm for 12-16 h, and plasmid DNA was extracted using the SanPrep Plasmid Mini-Prep Kit (Sangon Biotech, B518191).2.12. Preparation of competent cells of R. ruber SD3R. ruber SD3 was streaked onto LB plates and activated at 35℃ for 2 d. A single colony was inoculated into 50 mL antibiotic-free LB broth and cultured at 35℃ with shaking at 200 rpm until OD 595 nm≈0.7. Cells from 100 mL culture were harvested by centrifugation at 8,000 ×g for 20 min at 4℃. The pellet was resuspended in 20 mL ice-cold sterile 300 mM sucrose containing 10% (v/v) glycerol, and the centrifugation/resuspension cycle was repeated three times. The final pellet was gently resuspended in 2 mL sucrose-glycerol solution and aliquoted into 100 μL portions in pre-chilled sterile tubes. All steps were performed at 4°C.2.13. Construction an s2plp2-overexpressing strain in R. ruber SD3To construct the s2plp2-overexpressing strain in R. ruber SD3, 10 μL of shuttle recombinant plasmid was added to 100 μL of freshly prepared R. ruber SD3 competent cells. The mixture was gently flicked and incubated on ice for 100 min. Electroporation was performed at 1.5 kV with a 2.8 ms pulse using pre-chilled sterile cuvettes. After two consecutive pulses, cells were immediately transferred to 500 μL LB medium and recovered at 35℃ with shaking at 200 rpm for 6 h. The resuscitated cells were plated on LB agar containing 50 μg/mL kanamycin and incubated at 35℃ for 48 h. Single-clone was inoculated into 50 μg/mL kanamycin-supplemented 5 mL LB broth and incubated at 35℃ with 200 rpm for 48 h. A PCR system with 50 μL was used, the reaction reagents were as follows: 2.5 μL of genomic DNA, 2.5 μL (10 μM) of forward primer, 2.5 μL (10 μM) of reverse primer, 12 μL of 2×P6 High-Fidelity Premix (Tolo Biotech, China, 21805), sterilized ddH2O added to 50 μL.Primary screening positive strain employed PCR with kanamycin resistance gene primers (forward: 5’-ATGATTGAACAAGATGG-3’, reverse: 5’-TCAGAAGAACTCGTCAAGAA-3’) under: denature the DNA at 95℃ for 5 min, and then proceed with PCR program consisting of 34 cycles of 95℃ for 1 min, 52℃for 30 s, and 72℃ for 55 s, followed by a final extension at 72℃ for 5 min and preservation at 16℃. Putative positives were verified by 1% agarose gel electrophoresis. Confirmatory PCR used junction-specific primers for pNV18-s2plp2 (forward: 5’-TTAGGCACCCCAGGCTTTACAC-3’, reverse:5’-GGCAGCAGGTTGAACACGG-3’) with modified annealing at 56℃, followed by gel analysis. Positive clones were preserved for subsequent studies.2.14. Verification of the expression level of s2plp2-overexpressing strainExpression level of S2PLP2/s2plp2 in s2plp2E were assessed by western blotting and quantitative real time polymerase chain reaction (qRT-PCR). R. ruber SD3 (WT) and s2plp2E were streaked on LB plate and activated at 35℃ for 2 d. Single colon were inoculated into 50 mL LB medium and cultured at 35℃ with shaking at 200 rpm for 3 d. Cultures were transferred into 5 mL fresh LB medium at a 1:50 ratio and grown at 35℃ with shaking at 200 rpm for 48 h. Each 1 mL aliquots were pelleted and analyzed by western blotting as described above. For qRT-PCR, 3 mL aliquots at OD595 nm≈0.5 were pelleted, washed twice with 0.85% NaCl, flash-frozen in liquid nitrogen (10-15 min), and stored at -80℃. Total RNA was extracted using Bio-Tek's Bacterial RNA Kit (Omega, R6950), and cDNA was synthesized from 1,000 ng RNA using the PrimeScript FAST RT reagent Kit with gDNA Eraser (Takara, RR092S/A). Gene-specific qRT-PCR primers were designed for s2plp2 (forward: 5’-GCAGCAGGTTGAACACGG-3’, reverse: 5’-CTACGGAGTGGGGGTGGA-3’) and 16S rRNA as the internal control (forward: 5’-ACTGGGCGTAAAGAGYTCGT-3’, reverse: 5’-CGCATTTCACCGCTACAC-3’) synthesized by Sangon Biotech. qRT-PCR reactions were carried out in 96-well plates using the TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, RR820A) with a total volume of 10 μL, the reaction reagents were as follows: 2 μL of cDNA, 5 μL of TB Green Premix Ex Taq II Fast qPCR（2 ×）, 0.2 μL (10 μM) of forward primer, 0.2 μL (10 μM) of reverse primer, 0.2 μL of ROX Reference Dye or Dye II, sterilized ddH2O added to 10 μL.. The reaction mixtures were run on a LightCycler 480 system (Roche, Germany). Quantitative PCR was performed using TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, RR820A) under: initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing and fluorescence measurements at 60 °C for 60 s. Quantified mRNA expression was normalized to 16S rRNA, and relative expression (2−ΔΔCt) was calculated in Excel and visualized using GraphPad Prism 8.0.1[34]. 2.15. Determination of bacterial growth ratesWT and s2plp2E strains were transferred to fresh LB broth supplemented with 0, 0.02%, or 0.04% (m/v) phenol at a 1: 50 ratio. Cultures were incubated at 35°C with shaking at 200 rpm, and OD595 nm was measured at 0, 12, 24, 36, 48, 60, 72, 84, 108, 132, and 156 h. Growth rates relative to the 12 h baseline were calculated and analyzed using GraphPad Prism 8.0.1. Each group were carried out with 3 biological replicates.2.16. Comparative transcriptomic analysis of s2plp2-overexpressing and R. ruber SD3 The WT and s2plp2E strains were harvested, resuspended in sterile LB, and transferred in fresh LB medium at a 1:50 ratio, then cultured at 35°C with shaking at 200 rpm for 24 h. For each strain, 10 mL cultures at OD595 nm≈1.0 were centrifuged, washed twice with 0.85 NaCl, flash-frozen in liquid nitrogen for 10-15 min, and submitted to Majorbio Biopharm Technology (Shanghai) for RNA sequencing. Each group were carried out with 3 biological replicates.Total RNA was extracted from tissue using the CTAB method, followed by genomic DNA removal. High-quality RNA underwent ribosomal RNA depletion with the RiboCop rRNA Depletion Kit (Lexogen, 125). Purified mRNA was fragmented to ~200 nt, followed by double-stranded cDNA synthesis with random hexamer primers (Illumina) incorporating dUTP during second-strand synthesis. Libraries were prepared using Illumina® Stranded mRNA Prep with end-repair, phosphorylation, and A-tailing. Paired-end sequencing (150 bp) was performed on Illumina Novaseq 6000. The processing of original images to sequences, base-calling, and quality value calculations. The clean reads by removing low-quality sequences, reads with more than 10% of N bases (unknown bases) and reads containing adaptor sequences. Clean reads were aligned to the reference genome using Bowtie2. Transcript abundance was quantified via RSEM (default), Kallisto, or Salmon, reporting expression in FPKM and TPM to normalize for gene length and sequencing depth. Differentially expressed genes (DEGs) were identified using edgeR/DESeq2 (FDR-adjusted P <0.05, |log₂FC| ≥ 1. GO enrichment (GOatools) across Cellular Component, Molecular Function, and Biological Process domains. Significant terms were identified by Fisher’s exact test with Benjamini-Hochberg FDR correction (P-value < 0.05). For validation of transcriptomic data, five randomly DEGs were selected: DCN13_13450 (nadA, quinolinate synthase A), DCN13_16655 (MaoC-like dehydratase), DCN13_16660 (acyl-CoA dehydrogenase), DCN13_16665 (acetyl/propionyl/crotonyl-CoA carboxylase α subunit), and DCN13_18505 (porin). qRT-PCR primers (Supplementary materials Table S1) were designed using Primer 5. RNA extraction, cDNA synthesis, and qRT-PCR were performed as described above, with 16S rRNA as the endogenous control. Triplicate biological replicates were analyzed via the 2-ΔΔCt method.2.17. Expression and Purification of GST-S2PLP2(H68C) and GST TagThe codon-optimized s2plp2 gene with H68C mutation (histidine at position 68 was mutated to cysteine) was synthesized (General Biosystems, Anhui), cloned into pGEX-4T-1, and transformed into E. coli Top 10. Plasmids were extracted (SanPrep Kit, Sangon Biotech), verified by double digestion and sequencing, and then transformed into BL21(DE3). GST-S2PLP2(H68C) was expressed and purified using the same protocol established for GST-S2PLP2. The only difference lies in that 5 mmol/L EDTA was added to the inclusion body dissolution solution. Coomassie-stained bands of purified GST-S2PLP2(H68C) were submitted to Sangon Biotech (ShangHai) for mass spectrometry identification.For comparison, GST tag protein expression was induced in LB with 0、0.1、0.5 or 1.0 mM IPTG at 16°C or 37°C for 10 h. Optimal conditions were determined by SDS-PAGE. Cells were lysed by sonication (650 W × 30%, 4 s on/6 s off, 30 min, ice bath). Soluble GST tag was purified via GST affinity chromatograph. Purified protein was confirmed by coomassie staining and mass spectrometry (Sangon Biotech，ShangHai). 2.18. GST pull-down assayTo avoid interference from the putative S2P proteolytic activity, the catalytically inactive mutant GST-S2PLP2(H68C) was utilized. GST tag lysate served as a negative control. ProteinIso® GST Resin was equilibrated with ice-cold 1×PBS (pH 7.4) for 3–5 cycles. GST-S2PLP2(H68C) lysate with 5 mmol/L EDTA was incubated with 2 mL resin at 4°C overnight. Resin-bound complexes were loaded onto a pre-chilled column. Flow-through was reloaded twice and conducted the flow rate with about 6 s/drop. After 10 washes with 50 mM Tris-HCl (pH 8.0) by 2-column volumes per wash. Meantime, R. ruber SD3 was cultured to OD595 nm at 1.0. Each 50 mL cells were pelleted at 5,000 ×g for 10 min, resuspended in 8 mL ice-cold 1×PBS (pH 7.4), and sonicated (150 W ×50%; 4 s on/6 s off, 20 min, ice bath). Supernatant was collected at 14,000 ×g with 5 min and 4°C, and then incubated with resin-bound GST-S2PLP2(H68C) at 4°C overnight. Resin-bound complexes were loaded onto a pre-chilled column. Flow-through was reloaded twice and conducted the flow rate with about 6 s/drop. After 10 washes with 50 mM Tris-HCl (pH 8.0) by 2-column volumes per wash. Bound proteins were eluted 8-10 times using ice-cold 1 mL 10 mM glutathione, with 3 min on-ice incubations before each elution. Eluates with clearest bands from GST-S2PLP2(H68C) and GST control groups were submitted to Sangon Biotech for interaction proteomics. 2.19. Molecular docking analysisSingle-pass transmembrane proteins in pull-down results (excluding GST controls) were prioritized using UniProt and NovoPro, referencing known S2P substrates are single-pass transmembrane proteins in E. coli RseA[35] and M. jannaschii CED-9[36]. The structures of S2PLP2 and candidate proteins were derived from AlphaFoldDB. Candidate proteins were docked to S2PLP2 via Z-dock (https://zdock.wenglab.org/)[37]. Binding interfaces were analyzed using PDBePISA, with top 10 complexes ranked by binding free energy selected for further evaluation. 2.20. Cloning of gene sbpThe SPFH/Band7/PHB domain-containing protein (UniProt accession number: A0A098BIA4, designated SBP), was cloned, expressed, and purified. The pET-28a(+) vector was linearized by double digestion with BamH I and Xho I, analyzed via 1% (m/v) agarose gel electrophoresis, and gel-purified (Omega DNA Gel Extraction Kit). The sbp gene was amplified by PCR using primers (forward:5’-cagcaaatgggtcgcggatccATGGAAGCGCTGATCGTGC-3’, reverse: 5’-gtggtggtggtgctcgagTCACTGGGGTTCGCCGTG-3’). PCR products were purified and assembled with linearized pET-28a(+) via homologous recombination. The product was transformed into E. coli Top10, and plated on LB with 50 μg/mL kanamycin. Positive clones were screened by BamH I/Xho I digestion and confirmed by sequencing (Sangon Biotech, Shanghai). The verified plasmid was transformed into E. coli BL21(DE3). 2.21. Heterologous expression and purification of candidate interacting protein His-SBPE. coli BL21(DE3) harboring pET-28a(+)-sbp plasmid was grown in 5 mL LB with 50 μg/mL kanamycin at 37°C with shaking at 200 rpm for 16 h. Cultures were transferred into fresh LB, grown to OD₅₉₅ₙₘ at 0.4–0.6, and induced with 0.1, 0.5, or 1.0 mM IPTG at 16°C or 37°C (100 rpm, 20 h). Optimal conditions were determined by SDS-PAGE. Large-scale expression under optimal conditions yielded pellets from 300 mL culture. Cells were lysed by sonication, and inclusion bodies were solubilized in inclusion body buffer with 5 mM EDTA as GST-S2PLP2(H68C). Solubilized proteins were analyzed by SDS-PAGE.2.22. Identification of GST-S2PLP2 Interaction with His-SBP ProteinsGST pull-down assay were conducted as fowling: ProteinIso® GST Resin was equilibrated with PBS (pH 7.4). Purified GST-S2PLP2(H68C) and His-SBP were added to the resin and incubated overnight at 4°C. After washing, bound proteins were eluted with 10 mM glutathione.Microscale thermophoresis (MST) was performed as following: Purified His-SBP (10 μM) was incubated with 30 μM NT-647-NHS fluorescent labeled dye at room temperature for 30 min in the dark. GST-S2PLP2(H68C) was serially diluted with PBS buffer and mixed with fluorescently labeled His-SBP and then incubated for 5 min at room temperature. The samples (10 μL) were loaded to capillaries and MST analysis was performed on a NanoTemper Monolith NT.115T device (Nano Temper Technologies GmbH). By plotting the change per milliliter of normalized fluorescence as a function of metabolites concentration [∆Fnorm (‰) = (Metabolite fluorescence - Control fluorescence) / Control fluorescence]. The GST tag protein was used as the control. Curve fit was performed using the GraphPad Prism software 8.0.1, and dissociation constant (Kd) values could be determined. Three independent samples were tested.