Crystal Structure and Biochemical Characterization of

Nov 25, 2015 - domain (CTD). The mode of cofactor binding to CgDapD, elucidated by determining the structure in complex with succinyl-. CoA, reveals t...
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Crystal Structure and Biochemical Characterization of Tetrahydrodipicolinate N‑Succinyltransferase from Corynebacterium glutamicum Hye-Young Sagong and Kyung-Jin Kim* School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Korea ABSTRACT: Tetrahydrodipicolinate N-succinyltransferase (DapD) is an enzyme involved in the biosynthesis of L-lysine by converting tetrahydrodipicolinate into N-succinyl-L-2-amino-6-oxopimelate, using succinyl-CoA as a cofactor. We determined the crystal structure of DapD from Corynebacterium glutamicum (CgDapD). CgDapD functions as a trimer, and each monomer consists of three domains: an N-terminal helical domain (NTD), a left-handed β-helix (LβH) domain, and a β C-terminal domain (CTD). The mode of cofactor binding to CgDapD, elucidated by determining the structure in complex with succinylCoA, reveals that the position of the CTD changes slightly as the cofactor binds to the enzyme. The superposition of this structure with that of Mycobacterium tuberculosis shows differences in residues that make up cofactor-binding sites. Moreover, we determined the structure of CgDapD in complex with the substrate analogue 2-aminopimelate and revealed that the analogue was stabilized by conserved residues. The catalytic and substrate binding sites of CgDapD were confirmed by site-directed mutagenesis experiments. KEYWORDS: Corynebacterium glutamicum, tetrahydrodipicolinate N-succinyltransferase, L-lysine

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INTRODUCTION L-Lysine is an essential amino acid and an industrially important material used in animal feed, food and dietary supplements, and pharmaceuticals. Use in animal feed accounts for the majority of lysine consumption, and as the global demand for meat has grown in recent years, L-lysine use has correspondingly increased. By 2020, the global market for L-lysine is expected to reach U.S. $6.96 billion.1 Furthermore, meso-tetrahydrodipicolinate N-succinyltransferase (mDAP), the intermediate precursor of L-lysine, is a key component of the cell wall in some Gram-negative bacteria.2 Because the L-lysine/mDAP biosynthetic pathway is absent in mammals, it has become recognized as a target for antibacterial drugs.3 In industry, most L-lysine is manufactured by fermentation using Corynebacterium glutamicum. C. glutamicum (initially reported as Micrococcus glutamicus) is a small, nonpathogenic, Gram-positive bacterium known for its ability to produce various amino acids such as L-glutamate and L-lysine.4,5 Over the past few decades, various mutants of C. glutamicum that produce significant amounts of different L-amino acids have been isolated. In 2003, the complete genome of C. glutamicum (ATCC 13032) was sequenced, providing a large amount of information on metabolic pathways with products of industrial importance.6,7 In addition, during the past decade various genetic engineering techniques for this bacterium have been developed and used to increase its industrial productivity. In C. glutamicum, L-lysine is synthesized from aspartate.8−10 First, aspartate is converted into L-aspartate semialdehyde (ASA), a precursor for the biosynthesis of L-threonine, Lisoleucine, L-methionine, and L-lysine, by two consecutive enzymes. Then dihydrodipicolinate (DHDP) synthase converts ASA into DHDP and pyruvate. Next, DHDP reductase catalyzes the reduction of DHDP to produce tetrahydrodipi© XXXX American Chemical Society

colinate (THDP). Four different pathways from THDP to mDAP and L-lysine have so far been discovered:11,12 the succinylase pathway, the acetylase pathway, the mDAP dehydrogenase pathway, and the recently identified aminotransferase pathway.13 Although the majority of bacterial species use a single pathway to produce L-lysine, some bacterial species can use multiple pathways. C. glutamicum can synthesize L-lysine through both the succinylase and dehydrogenase pathways.14 The succinylase pathway generates a succinylated intermediate and is composed of four consecutive enzymecatalyzed reactions by THDP N-succinyltransferase, succinyldiaminopimelate (succinylDAP) aminotransferase, succinylDAP desuccinylase, and DAP epimerase. In the dehydrogenase pathway, THDP is converted into mDAP in a single step, catalyzed by DAP dehydrogenase. mDAP decarboxylase then catalyzes the decarboxylation of mDAP to yield L-lysine. Several studies have been done on the regulation of enzymes involved in L-lysine biosynthesis.15 Both branches are utilized at the rate of 30−70%, which is greatly affected by various conditions such as cultivation time and other parameters. For example, with decreasing extracellular ammonia concentration, dehydrogenase pathway activity also declined, indicating that the other succinylase pathway is used mainly for L-lysine production. 2,3,4,5-Tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase (DapD; EC 2.3.1.117) catalyzes the first step of the succinylase pathway (Figure 1A). It converts cyclic THDP into the acyclic compound N-succinyl-L-2-amino-6-oxopimelate, using succinyl-CoA as a cofactor. Biochemical and Received: October 1, 2015 Revised: November 20, 2015 Accepted: November 25, 2015

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DOI: 10.1021/acs.jafc.5b04785 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Overall shape of CgDapD. (A) Reaction catalyzed by CgDapD. (B) Alignment of amino acid sequences of DapD proteins. Secondary structure elements are shown and labeled on the basis of the structure of CgDapD. Identical and highly conserved residues are presented in red and blue characters, respectively. Residues involved in the succinyl-CoA and 2-aminopimelate binding are marked with blue and red triangles, respectively. Cg, Mt, Pa, and Mb represent Corynebaterium glutamicum, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Mycobacterium bovis, respectively. (C) Monomer structure of CgDapD. NTD, LβH domain, and CTD are distinguished with orange, light blue, and salmon colors, respectively. Succinyl-CoA and 2-aminopimelate molecules bound in the enzyme are shown as stick models and labeled. (D) Trimer structure of CgDapD presented as cartoon diagram. Mol I is presented in green and the other two molecules are shown in salmon and light blue colors. SuccinylCoA and 2-aminopimelate bound in the enzyme are shown as a sphere model with magenta and cyan colors, respectively. The right-side figure is a 90° rotation in the horizontal direction from the left-side figure. from C. glutamicum strain ATCC 13032 as a template. The PCR product was then subcloned into pET30a (Life Science Research), and the resulting expression vector pET30a:CgdapD was transformed into E. coli strain BL21(DE3)-T1R, which was grown in 1 L of LB medium containing kanamycin at 37 °C. After induction by the addition of 1 mM IPTG, the culture medium was maintained for a further 20 h at 18 °C. The culture was then harvested by centrifugation at 4000g for 20 min at 4 °C. The cell pellet was resuspended in buffer A (40 mM TrisHCl, pH 8.0) and then disrupted by ultrasonication. The cell debris was removed by centrifugation at 13500g for 25 min, and the lysate was applied to an Ni-NTA agarose column (Qiagen). After a washing with buffer A containing 30 mM imidazole, the bound proteins were eluted with 300 mM imidazole in buffer A. Finally, trace amounts of contaminants were removed by size exclusion chromatography by using a Superdex 200 prep-grade column (320 mL, GE Healthcare) equilibrated with buffer A. All purification experiments were

structural studies have been reported on DapDs from several microorganisms, such as Escherichia coli, Mycobacterium tuberculosis, and Mycobacterium bovis.16 However, the 3-D structure of DapD from C. glutamicum (CgDapD) has not yet been reported, despite the fact that this strain is responsible for a large proportion of L-lysine production. In the present study, we report a crystal structure of CgDapD in apo-form and in complex with its cofactor and substrate analogue. In addition, the biochemical properties of CgDapD were elucidated by kinetic analysis and site-directed mutagenesis experiments.

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MATERIALS AND METHODS

Cloning, Expression, and Purification. The CgDapD gene was amplified by polymerase chain reaction (PCR) using genomic DNA B

DOI: 10.1021/acs.jafc.5b04785 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. Crystallographic Data and Refinement Statistics for CgDapDa CgDapD

a

CgDapD_SCoA

CgDapD_2-aminopimelate

PDB code

5E3P

5E3Q

5E3R

data collection wavelength (Å) unit cell (a, b, c; γ) (Å; deg) space group solvent content (%) protein chains in AU resolution range (Å) highest resolution shell (Å) unique reflections redundancy completeness (%) Rmerge (%) average I/σ(I)

0.97934 91.0, 91.0, 156.8; 120.0 P6322 61 1 50.00−2.01 2.03−2.00 24585 14.3 (7.7) 97.6 (94.9) 8.0 (27.1) 64.63 (8.1)

0.97934 92.4, 92.4, 282.3; 120.0 R32 69 1 50.00−1.80 1.83−1.80 41214 10.5 (10.5) 99.1 (100.0) 8.5 (32.3) 61.68 (15.0)

0.97934 92.4, 92.4, 279.6; 120.0 R32 68 1 50.00−1.85 1.88−1.85 36690 5.1 (3.9) 97.8 (97.8) 9.9 (36.8) 32.26 (4.15)

refinement R (%) Rfree (%) mean B valueb (Å2) B from Wilson plot (Å2) RMS deviation bond lengths (Å) RMS deviation bond angles (deg) no. of amino acid residues no. of water molecules

17.6 21.7 33.8 28.3 0.019 1.9 284 84

15.4 18.4 23.1 18.9 0.026 2.4 278 230

17.3 19.9 29.6 21.6 0.023 2.1 276 146

Ramachandran plot most favored regions (%) additional allowed regions (%)

97.9 1.4

98.9 0.7

98.2 1.4

Values in parentheses refer to the highest resolution shell. bMean B value is for both protein atoms and the solvent molecules.

performed at 4 °C. The degree of protein purification was confirmed by SDS-PAGE. The purified protein was concentrated to 85 mg/mL in 40 mM Tris-HCl, pH 8.0. Crystallization, Data Collection, snd Structure Determination. Crystallization of the purified protein was initially performed with commercially available sparse-matrix screens from Rigaku and Molecular Dimensions by using the hanging-drop vapor diffusion method at 20 °C. Each experiment consisted of mixing 1.0 μL of protein solution (85 mg/mL in 40 mM Tris-HCl, pH 8.0) with 1.0 μL reservoir solution and then equilibrating this against 500 mL of reservoir solution. CgDapD crystals of the best quality appeared in 50% polyethylene glycol 200, 0.1 M Tris-HCl, pH 7.0, and 50 mM lithium sulfate. The crystals were transferred to cryoprotectant solution containing 50% PEK 200, 0.1 M Tris-HCl, pH 7.0, 50 mM lithium sulfate, and 30% (v/v) glycerol, fished out with a loop larger than the crystals, and flash-frozen by immersion in liquid nitrogen. Data were collected to a resolution of 2.0 Å at the 7A beamline of the Pohang Accelerator Laboratory (PAL, Pohang, Korea), using a Quantum 270 CCD detector (ADSC, USA). All data were indexed, integrated, and scaled together using the HKL-2000 software package.17 The CgDapD crystals belonged to the space group P6322 with unit cell parameters a = b = 91.001 Å, c = 156.82 Å, α = β = 90.0°, and γ = 120.0°. Assuming one molecule of CgDapD (31.1 kDa) per asymmetric unit, the crystal volume per unit of protein mass was 3.01 Å3 Da−1, meaning that the solvent content was approximately 59.21%.18 CgDapD crystals in complex with succinyl-CoA and with 2-aminopimelate were crystallized with the 42% PEG 200, 0.1 M sodium cacodylate, pH 7.0, and 200 mM magnesium chloride hexohydrate, supplemented with 10 mM each of succinyl-CoA and 2-aminopimelate. The crystals in complex with succinyl-CoA belonged to space group R32, with unit cell parameters of parameters a = 92.446 Å, b = 92.446 Å, and c = 279.6 Å and α = 90.0°, β = 90.0°, and γ =

120.0°. Assuming one molecule of CgDapD in an asymmetric unit, the crystal volume per unit of protein mass was 3.73 Å3 Da −1, which means the solvent content was approximately 67.08%. Crystals in complex with 2-aminopimelate belonged to the same space as CgDapD-succinyl-CoA complex crystals with similar unit cell parameters. The structure of the apo-form of CgDapD was determined by molecular replacement with the CCP4 version of MOLREP19 using the structure of DapD from Mycobacterium tuberculosis (MtDapD, PDB code 3FSX) as a search model. Model building was performed manually using the program WinCoot,20 and refinement was performed with CCP4 refmac5.21 The structures of CgDapB in complex with succinyl-CoA and 2-aminopimleate were solved by molecular replacement using the crystal structure of the apo-form of CgDapD. The data statistics are summarized in Table 1. The refined model of apo-form of CgDapD and those in complex with succinylCoA and with 2-aminopimelate were deposited in the Protein Data Bank with PDB codes of 5E3P, 5E3Q and 5E3R, respectively. Site-Directed Mutagenesis and Activity Assay. Site-specific mutations were created with the QuikChange kit (Stratagene), and sequencing was performed to confirm correct incorporation of the mutations. Mutant proteins were purified in the same manner as for wild type. For DapD activity assays, the succinyl group from succinylCoA was transferred to the substrate analogue 2-aminopimelate, and the formation of free CoA was measured with 5,5′-dithiobis-2nitrobenzoic acid (DTNB, Ellman’s reagent) at 412 nm. Activity assays were performed at room temperature with a reaction mixture of 0.5 mL total volume. The reaction mixture contained 0.1 M Tris-HCl, pH 8.0, 0.2 mM succinyl-CoA, 0.5 mM DTNB, 0.1−30 mM 2aminopimelate, and 30 μg of wild-type or mutant CgDapD protein. The reaction was initiated by the addition of 0.2 mM succinyl-CoA. The kinetic statistics of the enzyme were calculated using SigmaPlot C

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Figure 2. Cofactor binding mode of CgDapD. (A) Electron density map of succinyl-CoA. The electron density map of the bound succinyl-CoA is shown as a gray mesh and contoured at 1.0 σ. (B) Electrostatic potential surface presentation of succinyl-CoA binding mode of CgDapD. The CgDapD structure is shown as an electrostatic potential surface presentation. The bound succinyl-CoA is presented as a stick model with magenta color. (C) Domain movement upon succinyl-CoA binding. CgDapD structure of an apo-form and in complex with succinyl-CoA are superimposed. In apo-form, the flexible loop is shown in red and CTD in orange. The cofactor bound form of enzyme is shown in gray. (D) Stereoview of succinylCoA binding in CgDapD. The bound succinyl-CoA is presented in a stick model with a magenta color and labeled appropriately. Each monomer is distinguished by blue and salmon colors. Residues involved in the succinyl-CoA stabilization are shown as stick models. Hydrogen bonds formed between succinyl-CoA and neighboring residues are shown with red dotted lines. MtDapD is superimposed with CgDapD and presented as a cartoon diagram in gray. The residues that contribute to the formation of compact succinyl-CoA binding pocket are shown as a stick model, and the corresponding residues in MtDapD are shown as a stick model in gray. version 13, from Systat Software, Inc., San Jose, CA, USA (www. sigmaplot.com).

chromatography experiments (data not shown). Although all three domains contribute to trimer formation, the NTD and LβH domain contribute the most to intermolecular polar contact (Figure 1D). Cofactor Binding Mode of CgDapD. CgDapD catalyzes the conversion of THDP into N-succinyl-L-2-amino-6-oxopimelate by using succinyl-CoA as a cofactor. To investigate the cofactor-binding mode of CgDapD, we determined the structure of CgDapD in complex with succinyl-CoA at 1.79 Å resolution (Figure 2A). The succinyl-CoA molecule is bound in the narrow cleft formed between two adjacent subunits (Figure 2B). This succinyl-CoA binding pocket is made up of two loops (β9−β10 and β12−β13) and one β-strand from one subunit and two loops (β9−β10 and β11−α6) from the neighboring subunit. Interestingly, the loop (β9−β10) in the LβH domain that occupies the succinyl-CoA binding pocket in the apo-form moves away from the pocket and becomes flexible upon succinyl-CoA binding. Moreover, the CTD moves toward the pocket by about 4.63 Å, contributing to the binding of succinylCoA (Figure 2C). The 3-phosphoadenosinediphosphate is bound to the enzyme mainly through hydrogen bonds. The adenine ring interacts with the main chain of Arg272 and the side chain of Glu239. Ribose 3-phosphate is recognized by direct hydrogen bonds with Lys249 and Lys258. The residue Arg271 also contributes to the stabilization of succinyl-CoA. Pantotheine arm and β-mercaptoethylamine moieties interact with the main chains of Ala224 and Ala240, and the succinyl group is primarily stabilized by Arg183, Ser201, and Gly198 (Figure 2D). However, compared to the MtDapD, CgDapD contains a more compact succinyl-CoA binding pocket, where

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RESULTS AND DISCUSSION Overall Structure of CgDapD. To elucidate enzymatic properties of CgDapD, we determined its crystal structure at 2.0 Å resolution. The monomer consists of three distinct domains: an N-terminal helical domain (NTD, Met1−Ser140), a lefthanded β-helix (LβH) domain (Gly141−Ala246), and a β Cterminal domain (CTD, Gly247−Asn297) (Figure 1B,C). The NTD contains a four-stranded antiparallel β-sheet, flanked by two α-helices on one side and one α-helix on the opposite side of the sheet. The domain also contains an α-helix and an antiparallel β-hairpin that is involved in intersubunit interactions. In CgDapD, the NTD is relatively larger than those of other DapDs. The LβH domain constitutes the central domain of each subunit and is a common structural feature throughout this protein family. The LβH domain is involved not only in oligomerization but also in substrate and cofactor binding. One lithium ion binds to the hole of the trimer and is stabilized by interaction with two acidic residues, Asp146 and Asp148, in each trimer. In the middle of the domain, there is a flexible loop (Met203−His212) that might undergo open/ closed conformational change upon succinyl-CoA binding. The CTD consists of four β-strands and one α-helix. Two β-strands are antiparallel and are linked by a short looplike β-hairpin; the α-helix connects two β-hairpin-like structures. This domain is involved in cofactor binding and trimer formation. The asymmetric unit of the crystal contained one molecule, and a trimer can be generated by a crystallographic symmetry operation, which is consistent with our size exclusion D

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Journal of Agricultural and Food Chemistry Ile237 and Ile269 were positioned at the corresponding positions of Val255 and Leu288 in MtDapD. Substrate Binding Mode of CgDapD. To reveal the substrate binding mode of CgDapD, we also determined the structure in complex with the substrate analogue 2-aminopimelate at 1.85 Å resolution. The binding site of 2aminopimelate is an extension of the cofactor binding site, also existing between two adjacent subunits (Figure 3A). The

Kinetic and Mutagenesis Studies. To characterize the properties of CgDapD, kinetic analysis was performed by measuring transferase activity by using 2-aminopimelate and succinyl-CoA. Reaction rates corresponding to various concentrations of 2-aminopimelate were plotted and determined to obey Michaelis−Menten kinetics. On the basis of this kinetic analysis, Km and kcat values of CgDapD were determined to be 1.34 mM and 0.434 min−1, respectively (Figure 4A).

Figure 3. Substrate binding mode of CgDapD. (A) Electrostatic potential surface presentation of 2-aminopimelate binding mode of CgDapD. The CgDapD structure is shown as an electrostatic potential surface presentation. The bound 2-aminopimelate is presented as a stick model in cyan. (B) Substrate binding mode of CgDapD. 2Aminopimelate is shown as a stick model in cyan and labeled. Each monomer is characterized with different colors, blue and salmon. Residues involved in 2-aminopimelate binding are presented in stick models. Hydrogen bonds formed between 2-aminopimelate and neighboring residues are shown as red dotted lines.

Figure 4. Enzyme kinetics of CgDapD. (A) Michaelis−Menten equation-based plot of reaction velocity versus substrate concentrations. For the various substrate concentrations, 0.1, 0.2, 0.3, 0.5, 1, 2, 3, 5, 10, and 20 mM 2-aminopimelate were used. (B) Site-directed mutagenesis of CgDapD. Residues involved in substrate binding were replaced by alanine residues. The relative activities of recombinant mutant proteins were measured and compared with that of wild-type CgDapD. Each experiment was performed in triplicate.

α-carboxyl group is stabilized by direct interaction with Asn169 and a water-mediated hydrogen bond with Glu181. The carboxyl group in the side chain of 2-aminopimelate directly interacts with the guanidinium group of two arginine residues (Arg143 and Arg151) from a neighboring subunit. In addition, several hydrophobic residues, namely, Phe90, Met163, Phe170, and Met179, also contribute to the constitution of the 2aminopimelate binding pocket. Most of these residues are conserved throughout DapDs from other microorganisms (Figure 3B).

To confirm the residues involved in the enzyme catalysis and substrate-binding mode of CgDapD, we performed site-directed mutagenesis experiments based on our structural observations of the protein and compared the enzyme activity of the mutants with that of the wild-type protein. To confirm its importance as a catalytic residue, Glu181 was mutated to alanine, and the E181A mutant showed almost complete loss of activity compared to the wild-type protein. In addition, we mutated residues involved in the stabilization of the substrate to alanine (Arg143, Arg151, Met163, and Asn169). As expected, the E

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(12) Dogovski, C.; Atkinson, S. C.; Dommaraju, S. R.; Gerrard, J. A. Lysine biosynthesis in bacteria: an unchartered pathway for novel antibiotic design. Biotechnology; EOLSS; Vol. XI. (13) McCoy, A. J.; Adams, N. E.; Hudson, A. O.; Gilvarg, C.; Leustek, T.; Maurelli, A. T. L,L-Diaminopimelate aminotransferase, a trans-kingdom enzyme shared by Chlamydia and plants for synthesis of diaminopimelate/lysine. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 17909−17914. (14) Sonntag, K.; Eggeling, L.; Degraaf, A. A.; Sahm, H. Flux partitioning in the split pathway of lysine synthesis in Corynebacteriumglutamicum quantification by C-13-NMR and H-1-NMR spectroscopy. Eur. J. Biochem. 1993, 213, 1325−1331. (15) Cremer, J.; Treptow, C.; Eggeling, L.; Sahm, H. Regulation of enzymes of lysine biosynthesis in Corynebacterium glutamicum. Microbiology 1988, 134, 3221−9. (16) Schuldt, L.; Weyand, S.; Kefala, G.; Weiss, M. S. The threedimensional structure of a mycobacterial DapD provides insights into DapD diversity and reveals unexpected particulars about the enzymatic mechanism. J. Mol. Biol. 2009, 389, 863−879. (17) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997, 276, 307−326. (18) Matthews, B. W. Solvent content of protein crystals. J. Mol. Biol. 1968, 33, 491−497. (19) Vagin, A.; Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 22−25. (20) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126−2132. (21) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 240−255.

enzymatic activity of all of these mutants was lower than that of the wild-type enzyme (Figure 4B). These results indicate that 2-aminopimelate is stabilized by residues Arg143, Arg151, and Asn169 through a direct hydrogen bond network and that Met163 also contributes to the formation of the substratebinding pocket. In this study, we report the crystal structure of CgDapD both in apo-form and in complex with either succinyl-CoA or 2aminopimelate. Although CgDapD showed an overall fold similar to those of other reported DapDs, the protein exhibited somewhat distinctive structural features at the succynyl-CoA binding site. In conclusion, we are confident that our study provides valuable structural information that will be utilized for structure-based protein engineering to increase L-lysine productivity.

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AUTHOR INFORMATION

Corresponding Author

*(K.-J.K.) Phone: +82-53-950-5377. Fax: +82-53-955-5522. Email: [email protected]. Funding

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (2014R1A2A2A01005752 and 2014M1A2A2033626) and by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the MEST. Notes

The authors declare no competing financial interest.

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REFERENCES

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