Biochemical and Structural Basis of Triclosan Resistance in a Novel Enoyl-Acyl Carrier Protein Reductase

Bacterial strains, plasmids, culture conditions, and general DNA manipulation.E. coli strains DH5α, EPI300, and BL21(DE3) were grown at 37°C in Luria-Bertani (LB) broth or on LB agar media containing appropriate antibiotics: TCL (1 to 600 μg/ml; Sigma-Aldrich Co., St. Louis, MO), chloramphenicol (50 μg/ml), ampicillin (100 μg/ml), or kanamycin (50 μg/ml). Recombinant DNA manipulation was performed as previously described (45). Oligonucleotide synthesis and DNA sequencing were conducted at the DNA sequencing facility of MacroGen (Seoul, Republic of Korea). Nucleotide and amino acid sequences were compared using the online version of BLAST and ORF finder, publicly available at the National Center for Biotechnology Information (NCBI) portal (http://blast.ncbi.nlm.nih.gov). Multiple sequence alignments were performed using BioEdit v7.2.5 and GeneDoc v2.7 software.

Phylogenetic analysis.Phylogenetic analysis was performed as previously described (20) for metagenomic FabL2 ENR using amino acid sequences of FabL2 and its homologs, prototypic FabL, FabI, FabV, FabK ENRs, and prototypic 7-AHSDH from Comamonas testosteroni and its homologs retrieved from the UniRef50 database (updated on 19 September 2017). Top 10 entries were selected from each homology search. All identified sequences compiled together with the closely related prototypic ENRs and metagenomic FabL2, and redundant sequences were removed using the online Decrease Redundancy program (46). Sequence alignment and phylogenetic tree construction were performed with MEGA 6 (47) using the MUSCLE algorithm (48). To analyze the alignment output in MEGA 6, the maximum likelihood method was used in combination with the nearest-neighbor-interchange strategy, resulting in the deletion of gaps present in less than 50% of the sequences and generating 500 bootstrapped replicates resampling data sets to evaluate the confidence.

Expression and purification of FabL2 ENR.A gene encoding FabL2 ENR was PCR amplified from pBF1-4 (20) using gene-specific forward primer (5′-ATTCAAGGATCCTAGAGACATGACAAATATGAAAGGCAA-3′) and reverse primer (5′-TTATCATCTTTTAACATATAATAGATGGTCGACTTTCAA-3′) containing BamHI and SalI restriction sites (underlined), respectively. The amplified PCR product was digested with BamHI and SalI restriction endonucleases and cloned into pET-30b(+) expression vector to generate the recombinant vector pEBF1-4.

To express the FabL2 protein, pEBF1-4 was transformed into E. coli BL21(DE3) cells, and recombinant cells were selected on LB agar medium containing kanamycin. E. coli cells carrying pEBF1-4 were grown in 200 ml of LB supplemented with kanamycin at 37°C until reaching an optical density of 0.5 at 600 nm (OD₆₀₀). To induce protein expression, isopropyl β-d-1-thiogalactopyranoside (IPTG) (1 mM) was added to the bacterial culture during the late exponential phase. For protein purification, E. coli cells were harvested, resuspended in 5 ml of binding buffer (20 mM Tris-Cl, 0.5 M NaCl, 40 mM imidazole [pH 8.0]), and subjected to sonication (sonic dismembrator model 500; Fisher Scientific) for 2 min (pulse ON, 5 s; pulse OFF, 10 s). This mixture was then centrifuged at 3,500 × g for 6 min at 25°C. The supernatant was collected and recentrifuged at 17,000 × g for 10 min at 25°C and filtered using a 0.45-μm membrane filter. The fusion protein was purified using AKTA prime liquid chromatography system (GE Healthcare, Buckinghamshire, UK) with a His Trap HP affinity column (1-ml bed volume; GE Healthcare). The identity of the purified fusion protein was confirmed by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Analysis of FabL2 enzymatic activity.While the deduced amino acid sequence of FabL2 showed high sequence similarity to that of 7-AHSDH, fabL2 has previously been shown to complement ENR mutant E. coli (20). To test if FabL2 possesses dual enzymatic activities, enzyme assays were performed using the purified fusion protein. Biochemical characterization of the purified fusion protein was performed to determine the optimum reaction conditions and Michaelis-Menten kinetics for ENR activity. All enzyme assays were performed as previously described (33), with slight modifications. Briefly, ENR activity was measured in a 100-μl volume containing NADH/NADPH cofactors (250 μM), crotonyl-coenzyme A (crotonyl-CoA) substrate (200 μM), FabL2 protein (450 nM), and sodium phosphate buffer (100 mM; pH 7.0) at 25°C. Crotonyl-CoA, NADH, and NADPH were purchased from Sigma-Aldrich. Enzymatic reactions were monitored using a UV-visible (UV-Vis) spectrophotometer (DU730 Life Science; Beckman Coulter Inc., Fullerton, CA) at 340 nm and 30-s intervals for a total of 3 min. Because the protein did not exhibit ENR activity with NADH and preferred NADPH as a cofactor, the latter was used in all subsequent enzyme assays. To determine the value of the Michaelis constant (K_m) of the protein, 100 nM purified protein was added to 100 μl of the reaction mixture containing 200 μM NADPH and various concentrations of crotonyl-CoA (3, 6, 12, 24, 36, and 48 μM). To determine the K_m value of NADPH, 100 nM protein was added to 60 μM crotonyl-CoA and various concentrations of NADPH (5, 10, 15, 20, 30, 50, and 75 μM). The oxidation of NADPH cofactor was spectrophotometrically measured at 340 nm. The reaction mixtures were incubated at 25°C for 10 min. To determine the optimal buffer composition and pH for enzymatic activity of the protein, reactions were conducted using different buffers over a wide pH range: 100 mM sodium citrate buffer (pH 3.2, 4.2, 5.2, and 6.2) and 100 mM sodium phosphate buffer (pH 6.5, 7, 7.5, and 8.0). To determine whether FabL2 possessed 7-AHSDH activity, enzyme assays were performed using cholic acid (Sigma-Aldrich) as the substrate and NADH and NADPH as cofactors, as previously described (49). All kinetic reactions were performed in triplicates. The initial velocity of the reaction was calculated from the linear phase of the progress curves. To calculate the K_m values both for the substrate and cofactor, data were fitted to the standard Michaelis-Menten equation.

Site-directed mutagenesis.Sequence comparison, homology modeling, and docking analysis revealed that FabL2-type ENR carries an extrapolated highly flexible loop comprising six amino acid residues (Y96 to V101); this was specific to FabL2 ENR only, as other known ENRs lack this loop. To test if this extrapolated flexible loop was involved in TCL resistance and enzymatic activity, overlap extension PCR was used to delete the 18-bp loop (Fig. S1, region b). Briefly, two PCRs were performed to amplify the overlapping fragments A and C of the FabL2 gene (Fig. S1) from pBF1-4 using the primer pairs 7A-1/A-2 (7A-1, 5′-GCCAAAGCGTTGTCAGGTG-3′, and 7A-2, 5′-AATCATCGCATTGCTTACGAAG-3′) and 7A-3/A-4 (7A-3, 5′-TCGTAAGCAATGCGATGATTGGCGGATACGGTAAATTTAT-3′, and 7A-4, 5′-CCCGTCATATTACTCGTTCCCA-3′), respectively. The underlined sequences of 7A-2 and 7A-3 are complementary to each other to anneal for overlap extension. The PCR conditions were as follows: initial denaturation at 95°C for 3 min, 30 cycles of denaturation at 95°C for 30 s, annealing at variable temperatures (55°C or 63°C for the amplifying fragment A or C, respectively) for 30 s, and extension at 72°C for 30 s, followed by a final extension at 72°C for 5 min. The amplified PCR products A and C were gel purified. This was followed by a fusion PCR using fragments A and C as templates in equimolar concentrations without any primers and the following conditions: initial denaturation at 95°C for 3 min, 10 cycles of denaturation at 95°C for 1 min, annealing at 50°C for 30 s, and extension at 72°C for 1 min 35 s, followed by a final extension at 72°C for 5 min. The fusion product was subsequently amplified using primers 7A-1 and 7A-4 (Fig. S1) and the following conditions: initial denaturation at 95°C for 3 min, 20 cycles of denaturation at 95°C for 1 min, annealing at 63°C for 30 s, and extension at 72°C for 1 min 35 s, followed by a final extension at 72°C for 5 min. The purified fusion product was cloned into the pGEM-T Easy vector and transformed into E. coli DH5α to confirm the deletion of the loop and test for TCL resistance as previously described (20). The mutated version of FabL2 was designated mFabL2.

Complementation.To investigate the ENR activity of mFabL2, complementation studies were performed. The recombinant pGEM-T Easy plasmid carrying mFabL2 was transformed into a conditional temperature-sensitive fabI mutant of E. coli, JP1111, which is unable to grow at the high temperature of 42°C (50). E. coli JP1111 organisms containing the mFabL2 vector were grown in triplicates on LB agar medium supplemented with ampicillin (100 μg/ml) and IPTG at 30°C and 42°C. The growth of E. coli JP1111 at 42°C for 48 h indicated complementation of FabI ENR activity.

TCL resistance test.To determine and compare the growth and TCL resistance of E. coli DH5α expressing either metagenomic FabL2 or mFabL2, growth assays were performed in LB broth supplemented with ampicillin and various concentrations of TCL (0 to 600 μg/ml). E. coli DH5α expressing the Bacillus velezensis FabL homolog (WP_003155478.1) was used as a positive control at similar TCL concentrations, and E. coli DH5α carrying empty pGEM-T Easy vector was used as a negative control. Bacterial growth was monitored using a UV-Vis spectrophotometer (DU730 Life Science; Beckman Coulter Inc., Fullerton, CA) by measuring OD₆₀₀ over 96 h.

Homology modeling of FabL2.Homology modeling is the construction of an atomic model of the target protein utilizing experimentally determined structures of evolutionarily related proteins (51). First, the FabL2 protein sequence (target) was analyzed against the Protein Data Bank (PDB) using the BLASTP tool in NCBI to identify the suitable protein structure (template) (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch). Subsequently, the target-template alignment was subjected to MODELLER program implemented in Discovery Studio v4.5. Ten iterative models of FabL2 were generated, and the best model was selected based on the lowest probability density function (PDF) total energy and discrete optimized protein energy (DOPE) score for further analysis.

It is noteworthy that the homology model does not reflect the conformation or orientation of amino acids comprising side chains in their physiological state. To obtain the native conformation of FabL2, an unrestrained molecular dynamic (MD) simulation was performed with CHARMm36 force field in GROMACS v5.0.7 (52). Briefly, the system was solvated in an octahedral box of transferable intermolecular potential three position (TIP3P) water model. Counterions (Na⁺) were added to neutralize the system. The steepest descent minimization with a maximum tolerance of 10 kJ/mol/nm was employed to avoid any unfavorable interactions. The system was equilibrated in two phases. In the first phase, NVT (number of particles at constant volume and temperature) equilibration was conducted for 100 ps at 300 K. The temperature was maintained with a V-rescale thermostat. In the second phase, heavy atoms were restrained, and solvent molecules with counterions were allowed to move during the 100-ps simulation at 300 K and 10⁵ Pa pressure using the Parrinello-Rahman barostat. The final production step was conducted for 10 ns under periodic boundary conditions with NPT (number of particles at constant pressure and temperature) ensemble and bond constraint algorithm, linear constraint solver (LINCS). The representative structure of FabL2 was extracted from the last 6-ns trajectory using the clustering method. The stereochemical quality of the MD-refined model of FabL2 was verified using PROCHECK implemented in SAVES web server (http://services.mbi.ucla.edu/PROCHECK/). The MD-refined model of FabL2 was also validated by ProSA-web (https://prosa.services.came.sbg.ac.at/prosa.php) for its accuracy of potential errors. The Z-score of ProSA measures the deviation of the total energy of the structure with respect to an energy distribution derived from random conformations.

Molecular docking simulation of TCL into FabL2.Molecular docking is a computational technique used to predict the binding affinity and orientation of a ligand in the binding site of a protein. The two-dimensional (2D) structure of TCL was drawn in Accelrys Draw v4.2 and converted into three-dimensional (3D) structure in Discovery Studio v4.5. The MD-refined model of FabL2 and TCL was used as input data in the Genetic Optimization of Ligand Docking (GOLD) v5.2.2 program. The binding site of FabL2 was traced from its catalytic residues using Define and Edit Binding Site tools implemented in Discovery Studio v4.5. Docking results were analyzed with the GOLD fitness score, which includes hydrogen bond (H bond) energy, van der Waals energy, and ligand torsion strains. The best docking pose was selected based on the GOLD fitness score and H bonding with catalytic residues.

Accession number(s).The nucleotide sequence of pBF1 harboring the FabL2 gene has been deposited in the GenBank database under accession number KT982367. The FabL2 ENR protein sequence has been deposited in the NCBI protein database under accession number AOR51268.1.