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J. Biol. Chem., Vol. 280, Issue 35, 31249-31256, September 2, 2005

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Crystal Structure of the Actinomadura R39 DD-peptidase Reveals New Domains in Penicillin-binding Proteins^*

Eric Sauvage, Raphaël Herman, Stephanie Petrella, Colette Duez¶, Fabrice Bouillenne, Jean-Marie Frère, and Paulette Charlier

From the Centre d'Ingénierie des Protéines, Université de Liège, Institut de Physique B5, B-4000 Liège, Belgium

Received for publication, March 24, 2005 , and in revised form, June 10, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Actinomadura sp. R39 produces an exocellular DD-peptidase/penicillin-binding protein (PBP) whose primary structure is similar to that of Escherichia coli PBP4. It is characterized by a high -lactam-binding activity (second order rate constant for the acylation of the active site serine by benzylpenicillin: k/K = 300 mM^-1s^-1). The crystal structure of the DD-peptidase from Actinomadura R39 was solved at a resolution of 1.8 Å by single anomalous dispersion at the cobalt resonance wavelength. The structure is composed of three domains: a penicillin-binding domain similar to the penicillin-binding domain of E. coli PBP5 and two domains of unknown function. In most multimodular PBPs, additional domains are generally located at the C or N termini of the penicillin-binding domain. In R39, the other two domains are inserted in the penicillin-binding domain, between the SXXK and SXN motifs, in a manner similar to "Matryoshka dolls." One of these domains is composed of a five-stranded -sheet with two helices on one side, and the other domain is a double three-stranded -sheet inserted in the previous domain. Additionally, the 2.4-Å structure of the acyl-enzyme complex of R39 with nitrocefin reveals the absence of active site conformational change upon binding the -lactams.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell wall assembly is catalyzed by a series of active-site serine DD-peptidases, known as penicillin-binding proteins (PBPs).¹ These enzymes catalyze the final steps in the synthesis of cell wall peptidoglycan, a highly cross-linked mesh that surrounds the bacteria and protects them from osmotic shock and lysis. They constitute the lethal targets of penicillins and more generally of -lactam antibiotics, which exhibit a rough structural analogy with the PBP peptide substrates, i.e. the D-alanyl-D-alanine C termini of peptidoglycan stem peptides. -Lactams acylate the active site serine of the penicillin-binding domain of PBPs and form a rather stable acyl-enzyme devoid of transpeptidase activity (1).

Multiple PBPs, from three in Neisseria meningiditis to twelve in Escherichia coli, are bound to the cytoplasmic membrane of all bacteria. These PBPs can be divided into two groups: the high molecular mass PBPs, which are multimodular enzymes containing a DD-transpeptidase domain associated either with a transglycosylase domain or with a domain of unknown function and the low molecular mass PBPs, which act mainly as DD-transpeptidases or DD-carboxypeptidases (2, 3). Low molecular mass PBPs are further divided into classes A, B, and C according to their sequences (1, 4). Structural information on class A and class B low molecular mass PBPs has been made available through crystal structures determination of E. coli PBP5 (5) and the Streptomyces K15 DD-peptidase (K15) (6), two representatives of the class A, and of the Streptomyces R61 DD-transpeptidase-carboxypeptidase (R61) (7), a well studied enzyme representative of class B. The overall fold of PBP5 is that of class A -lactamases with a C-terminal extension domain, whereas R61 is similar to class C -lactamases. Despite the absence of crystal structure of class C low molecular mass PBPs, similarities with class A -lactamases have been highlighted. Compared with the class A -lactamase structures, these proteins exhibit an additional domain of about 200 residues inserted between the SXXK and the SXN conserved motifs characteristic of the penicillin-binding domain (8, 9).

The Actinomadura sp. strain R39 DD-peptidase (R39) is a class C low molecular mass PBP that is related to E. coli PBP4 (8), Bacillus subtilis PBP4a (10, 11), and Neisseria gonorrhoeae PBP3 (12). The members of this class of PBPs exhibit medium to high sensitivity to -lactams, DD-carboxypeptidase, and transpeptidase activity. They also exhibit a so-called endopeptidase activity, i.e. they can release a C-terminal residue bearing a large and complex side chain, which is in fact a facet of their carboxypeptidase activity. Moreover, in contrast to most other PBPs, which are strongly anchored in the membrane, class C low molecular mass PBPs are soluble or loosely bound to the membrane via an amphiphilic C-terminal helix (10, 13). Under laboratory growth conditions E. coli PBP4 appears to be dispensable, and similar results were found for B. subtilis PBP4a and N. gonorrhoeae PBP3.

In addition to the low molecular mass PBPs, a few high molecular mass PBPs have been crystallized: Streptococcus pneumoniae PBP2x (14), Staphylococcus aureus PBP2a (15), and Enterococcus faecium PBP5 (PBP5fm) (16), three class B high molecular mass PBPs involved in determining the level of resistance to -lactam encountered in these bacteria. The crystal structure of R39 is the first structure of a class C low molecular mass PBP and reveals special structural features that characterize this class of PBPs.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Purification and Crystallization—R39 has been expressed and purified as described previously (8). Crystals were grown at 20 °C by hanging drop vapor diffusion. Native crystals were obtained by mixing 2.5 µl of 25 mg ml^-1 protein solution (containing 5 mM MgCl₂ and 0.1 M Tris) and 2.5 µl of well solution per drop (containing 1.5 M ammonium sulfate and 0.1 M Tris, pH 8). The crystallization conditions were optimized by a search around these conditions and by testing several additives, including different chloride salts. Crystals of the Co²⁺ derivative were prepared by mixing 2.5 µl of 25 mg ml^-1 protein solution (containing 5 mM MgCl₂ and 0.02 M Tris), 2 µl of well solution (containing 2.0 M ammonium sulfate and 0.1 M MES, pH 6), and 0.5 µl of 0.1 M CoCl₂ solution. Nitrocefin-soaked crystal were prepared using the same conditions and then transferred to 2 µl of well solution containing nitrocefin (1 mM) for 14 h. Before data collection, crystals were cryoprotected with 100% glycerol.

Data Collection, Phase Determination, and Structure Refinement—Native data were collected at 100 K on an ADSC Quantum 4R charge-coupled device detector at beamline ID29 at the European Synchrotron Radiation Facility (ESRF, Grenoble, France). SAD data and nitrocefin-soaked crystal data were collected at 100 K on a MAR charge-coupled device detector at beamline BM30a at ESRF. All data were processed and scaled using Mosflm (17) and CCP4 (18). The structure was solved by a SAD experiment at the peak wavelength of Co²⁺ K-edge (1.605 Å). The eighteen Co²⁺ sites were found with Solve (19). Phasing with Resolve (20) produced a clearly interpretable electron density map, from which a complete model could be built using Turbo-Frodo (21). This monomer was used as a search model for molecular replacement, with native data. The four monomers in the asymmetric unit were found using AMoRe (22). Subsequent refinement was carried out using CNS (23), and manual model fitting was done with Turbo-Frodo. The four monomers were refined individually. The figures were prepared with Molscript (24), Bobscript (25), and Raster3D (26).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Structure Determination—R39 is a soluble 538-amino acid protein, as deduced from DNA sequence analysis, including a 49-amino acid signal peptide. The tracing of the resulting 489-amino acid R39 molecule in the electron density maps showed that the 23-amino acid C-terminal peptide (467-489) is missing. Subsequent mass spectrometry analyses indicated that the purified enzyme contained 466 residues prior to crystallization.

Because a production system of R39 in E. coli was not available and selenomethionine-substituted protein could not be obtained, a search for the presence of anomalous scattering ions, firstly added to enhance crystal growth, was monitored by using a fluorescence detector available at the BM30a beamline at ESRF. A strong fluorescence peak was observed with a crystal grown in presence of Co²⁺. Thus the use of Co²⁺ ions in the crystallization conditions facilitated the structure determination by single anomalous diffraction at the cobalt resonance wavelength (Table I). R39 crystallizes in space group P2₁ (a = 104.56 Å; b = 94.37 Å; c = 106.96 Å; = 94.05°) with four molecules and eighteen Co²⁺ ions in the asymmetric unit (Fig. 1A). The crystallographic environment is similar for molecules A and D and molecules B and C but is different for both pairs. Five Co²⁺ ions are found in molecules A and D, whereas only four are found in molecules B and C. Each Co²⁺ ion is ligated by at least one histidine and one aspartic or glutamic acid residues. In molecule A, a Co²⁺ ion is located between His²⁸² and Glu⁴⁶⁶ of a crystallographic symmetry-related molecule of molecule A. The same situation arises for molecule D. Because the crystallographic environment is different for molecules B and C, the absence of cobalt near residue 466 results in a disordered 464-466 segment with elevated temperature factors for residues 464 and 465 and loss of interpretable density for residue 466. The root mean square deviation between any two molecules of the asymmetric unit is between 0.35 and 0.40 Å. The final structure has been refined at 1.8 Å against data collected from a crystal grown in slightly different conditions in the absence of Co²⁺. In this refined structure, four Mg²⁺ ions are present in the asymmetric unit in sites of four of the 18 Co²⁺ ions. Two of these Mg²⁺ ions are found in molecule A and two in molecule D.

View larger version (38K): [in this window] [in a new window]   FIG. 1. Structure of R39. A, asymmetric unit cell representation. Co2+ ions are shown as small brown spheres. Crystallographic environment is similar for molecules A and D and molecules B and C but different for both pairs; B, overall structure of R39 shown as a ribbon diagram. The penicillin-binding domain is green, domain II is yellow and brown, and domain III is blue. The position of the active site serine is indicated by a red'S'; C, active site of R39. Secondary structure elements of the penicillin-binding domain have been labeled in accordance with the labeling scheme of class A -lactamases. Ser49 and Lys52 form the first motif SXXK characteristic of the penicillin-binding proteins active site. Ser298 and Asn300 form the second motif SXN, and the third motif, KTGT, consists of Lys410, Thr411, Gly412, and Thr413.

View larger version (35K): [in this window] [in a new window]   FIG. 2. Interaction between R39 and nitrocefin. A, structure of nitrocefin; B, nitrocefin-R39 acyl-enzyme structure. Stereo view of a Fobs-Fcalc electron density omit map calculated without the nitrocefin molecule, contoured at 2. Nitrocefin is magenta, the penicillin-binding domain is green, and domain II is yellow. The side chain of nitrocefin fills a pocket formed by Trp139, Asp142, Tyr147, Arg351, and Met414; C, comparison between the structures of the R39 apo-enzyme and the nitrocefin acyl-enzyme. Apo-enzyme is gray, and nitrocefin acyl-enzyme is colored as in B.

View larger version (32K): [in this window] [in a new window]   FIG. 3. Structure of R39 domain II. A, structural comparison of R39 domain II, d-Tyr-tRNA deacylase, and the N-terminal domain of MinC. Secondary structures of domain II are labeled; B, dipolar head of R39 domain II. Nitrogen atoms are colored blue; oxygen atoms are colored red. Connections with the penicillin-binding domain and domain III are represented by small red spheres.

Non Penicillin-binding Domains—Domain II (residues 66-165 and 237-284) is a five-stranded -sheet with two helices packed on one side (Fig. 3A). The sheet is composed of one antiparallel and four parallel strands in the order 5_II-1_II-2_II-3_II-4_II with 1_II anti-parallel. Strand 2_II is followed by helix 1_II, and strand 3_II is followed by helix 2_II. The topology as well as the three-dimensional arrangement of the subdomain formed by 2_II-1_II-3_II-2_II-4_II corresponds to one-half of a Rossmann fold. Using a three-dimensional DALI search (32) with residues 66-165 and 237-284, domain II was found to topologically resemble domains of E. coli d-Tyr-tRNA^Tyr deacylase (33) (PDB 1JKE [PDB] ) and barley 1,3-1,4--glucanase (34) (PDB 1AQ0 [PDB] ) with Z-scores of 5.5 and 4.7, respectively. Indeed, the five strands and the two helices can be superimposed with a root mean square value of 3.1 Å for 82 aligned C atoms for the d-Tyr-tRNA^Tyr deacylase and 3.5 Å for 74 aligned C atoms for the 1,3-1,4--glucanase. Domain II is also topologically similar to the N-terminal domain of MinC (35) and the 1A region of FtsA (36), two proteins interacting with FtsZ and involved in the regulation of the septum formation in cell division. However the resemblance between R39 and both MinC and FtsA remains limited. Moreover, R39 lacks sequence homology with all these topologically similar proteins and the loops connecting the secondary structures are completely different.

A hairpin connects strands 1_II and 2_II. Two arginines (Arg⁸¹ and Arg⁸²) constitute the head of the hairpin with their side chain protruding out of the core of domain II. Similarly, the side chains of Arg¹¹⁶ and Arg¹¹⁹, located in the loop connecting 1_II and 3_II, extend on the same side of domain II. These four arginines, together with Lys²⁶¹, form a positively charged surface on the side opposite to the interface with the penicillin-binding domain. The side chains of these residues seem to float into the solvent. Accordingly, the temperature factors of the side chains are high, and the electron density around them is quite poor. A crown of negatively charged residues (Glu⁷⁶, Asp⁸⁷, Asp¹²¹, Glu²⁵⁰, Glu²⁵¹, Glu²⁵⁴, Asp²⁶³, Asp²⁷², Asp²⁷⁵, and Glu²⁷⁷) surrounds this positively charged head (Fig. 3B). This dipolar surface might be involved in protein-protein interaction. On the opposite side, following 3_II strand, a long unstructured loop (126-153) extends out of domain II. It packs onto the penicillin-binding domain, with residues Trp¹³⁹, Asp¹⁴², and Tyr¹⁴⁷ being part of a specific pocket in the bottom of the active site. A small hairpin connects this loop to domain III.

View larger version (31K): [in this window] [in a new window]   FIG. 4. Structure of R39 domain III. The six -strands of domain III are labeled. Connections with domain II are represented by small red spheres.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The Actinomadura R39 DD-peptidase is encoded by a 1614-nucleotide gene. The 538-amino acid protein precursor presents a 49-amino acid N-terminal signal peptide and, presumably, a 23-amino acid C-terminal extension that are both cleaved to yield a mature soluble protein of 466 residues. When the protein is produced in Streptomyces lividans TK24 grown in YEME (Difco yeast extract, 3 g; Difco bactopeptone, 5 g; Oxoid malt extract, 3 g; glucose, 10 g; sucrose, 340 g) medium, its molecular mass is about 3 kDa higher than the molecular mass exhibited by the protein purified from the culture supernatant of Actinomadura R39. Moreover, the protein produced in S. lividans lacks both DD-peptidase activity and penicillin-binding ability. The mass difference associated with loss of enzymatic activity was first thought to be an incorrect processing of the signal peptide (8), but it might also result from an incorrect processing of the C-terminal extension. A similar situation has been observed with the R61 DD-peptidase, for which the precursor containing the C-terminal extension was shown to be inactive (37). Proteolytic cleavage of a C-terminal fragment has been observed in few other cases, although not necessarily associated with enzymatic activity (38-40). Inspection of the R39 three-dimensional structure reveals that the C-terminal extension might reduce the active site accessibility to peptide substrates or -lactam antibiotics, which would result in a loss of enzymatic activity and -lactam binding ability.

The mechanism of interaction between PBPs and -lactam antibiotics is known to follow a three-step pathway involving the rapid reversible formation of a non-convalent Henri-Michaelis complex followed by formation of a long-lived acyl-enzyme intermediate. Evidence supporting this mechanism is given by the crystallographic structure of a non-covalent PBP--lactam complex described recently (28) and those of acyl-PBP intermediates that have been solved with different PBPs (15, 16, 28-30). The possible acylation mechanism involved in R39 might be a general base-assisted catalytic process where the role of the general base could be played by an unprotonated lysine (Lys⁵² in R39), a residue strictly conserved in all PBPs. Lys⁵² would abstract a proton from the active serine hydroxyl group to promote the nucleophilic attack on the -lactam carbonyl by the activated serine O. The proton would subsequently be back donated by Lys⁵² to the -lactam nitrogen via Ser²⁹⁸. The distances between Ser⁴⁹ O and Lys⁵² N and between Lys⁵² N and Ser²⁹⁸ O are, respectively, 2.8 and 2.98 Å, in agreement with this interpretation. In this case, the unprotonated state of Lys⁵² can be promoted by the active site environment and more precisely by the presence of Lys⁴¹⁰. An alternative interpretation of the acylation mechanism relies upon a concerted one-step process where the covalent bond formation between the -lactam and the active serine Ser⁴⁹ is concomitant with the transfer of the Ser⁴⁹ proton directly to Ser²⁹⁸ and to the transfer of the Ser²⁹⁸ hydroxyl proton to the -lactam nitrogen (41). Charges on both Lys⁵² and Lys⁴¹⁰ support the correct orientation of the Ser²⁹⁸ hydroxyl group. This explanation could prevail for all penicilloyl serine transferases, including enzymes possessing a tyrosine residue instead of a serine in the second conserved active site motif, such as the R61 DD-peptidase.

Compared with most other PBPs, R39 exhibits a very high -lactam binding activity. The value of the second order acylation rate constant on benzylpenicillin is 300 mM^-1 s^-1 (i.e. the highest value to date for a DD-peptidase) and the high k₂/K values obtained for other penicillins and cephalosporins explains the broad spectrum specificity profile of the R39 enzyme (see Ref. 3 for detailed kinetic data on R39). Like class A -lactamases and other low molecular mass PBPs (PBP5 and K15), R39 presents a open active site capable of accommodating most -lactam antibiotics. Many factors may impair the ability of a PBP to bind -lactams. For example, in the highly resistant Staphylococcus aureus PBP2a, there is a narrow binding cavity (the distance between Asn³⁰⁰ O1 and Thr⁴¹³ backbone nitrogen is 9 Å in R39, whereas this distance is 6.7 Å in the apo structure of PBP2a). As a result, this cavity must be enlarged to accommodate the incoming antibiotic and the active site needs undergoing conformational changes at strand 3 and at the helix 2 N terminus for acylation to occur (15). Modification of active site catalytic residues may also reduce acylation rate constants. In K15, the enzyme has a very poor -lactam binding activity, because a cysteine, instead of an asparagine, is present at the position equivalent to Asn³⁰⁰ (6). Nothing prevents an efficient acylation of R39 by -lactams and if R39 is the best -lactam-binding DD-peptidase, this is due to the fact that the active site of other PBPs are less accessible or lack the complete set of amino acids involved in the acylation machinery.

View larger version (36K): [in this window] [in a new window]   FIG. 5. Comparison between structures of R39 and E. coli PBP5. The penicillin-binding domain of R39 is green, and domain II is yellow. PBP5 is cyan.

The most distinctive feature of the R39 active site is the pocket located in the bottom of the active site and formed by residues Trp¹³⁹, Asp¹⁴², Tyr¹⁴⁷, Arg³⁵¹, and Met⁴¹⁴. Trp¹³⁹, Asp¹⁴², and Tyr¹⁴⁷ belong to domain II, a unique domain only found in class C PBPs. This pocket could be specific to accommodate the N-terminal portion of the diaminopimelic acid component typical of the Actinomadura cell wall peptidoglycan. In R61, such a specific subsite, different from the R39 one, has been shown to be responsible for the high affinity of R61 for its cell wall peptidoglycan (43).

As with most PBPs, the deacylation step in R39 is extremely slow compared with the efficiency of the deacylation step in class A -lactamases (a factor up to 10⁸). In the latter a glutamic acid of the -loop defining the bottom of the active site (Glu¹⁶⁶) activates a conserved water molecule that attacks the acyl-enzyme carbonyl, resulting in very high deacylation rates. In R39, the 343-353 loop in the bottom of the active site is folded like its counterpart in PBP5 but lacks the conserved Asp¹⁵⁴ and His¹⁵¹ residues that have been hypothesized to be responsible for the relatively high deacylation rate of PBP5 (44). The absence of residue capable of activating a deacylating water molecule as well as the absence of such a deacylating water molecule in the nitrocefin-R39 acyl-enzyme crystal structure would explain the poor deacylation rate of R39.

Low molecular mass PBPs exhibit a high carboxypeptidase activity, but class C low molecular mass PBPs also have an `endopeptidase' activity that is generally not encountered in other PBPs. Where and when class C low molecular mass PBPs might require one of these enzymatic activities remains unclear, and their physiological role is still unknown. It has been suggested that the endopeptidase activity of E. coli PBP4 is involved in breaking peptide cross-links to allow the insertion of new glycan strands into the murein sacculus (45). Recent investigations have assigned a role to this PBP in determining a uniform morphology thanks to the same activity, i.e. removal of inappropriate cross-links initiated by PBP5. In vivo the enzyme seems to behave as a DD-carboxypeptidase rather than a transpeptidase (46, 47). In B. subtilis, it was shown that PBP4a is expressed during the late exponential phase but is dispensable for the synthesis of spore peptidoglycan (10, 48).

The structure of R39, with its two additional non-penicillin binding domains, suggests that R39 and more generally class C PBPs could be part of a proteic complex. The resemblance of the R39 domain II topology with E. coli d-Tyr-tRNA^Tyr deacylase and barley 1,3-1,4--glucanase is astonishing but seems unlikely to be associated with the functional properties of these enzymes. Although B. subtilis PBP4a was shown to be absent from the septum site (49), it is interesting that interactions between proteins of the septation machinery and minor peptidoglycan-modifying enzymes have been shown to be responsible for significant aspects of bacterial shape determinations (50). Although very limited, the resemblance of R39 domain II to the N-terminal domain of MinC and the 1A region of FtsA, two proteins involved in the regulation of septum formation, suggests that class C low molecular mass PBPs could interact with a protein of the septation machinery. The interacting partner of domain II, if any, remains to be identified if one is to understand the physiological importance of this element of class C low molecular mass PBPs.

	FOOTNOTES

 
This paper is dedicated to the memory of J.-M. Ghuysen who initiated the study of R39 more than thirty years ago.

The atomic coordinates and structure factors (code 1W79, 1W8Q, and 1W8Y) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by the Belgian Program on Inter-university Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Science Policy Programming (Grant PAI P5/33), by the Fonds National de la Recherche Scientifique (Grants IISN 4.4505.00, FRFC 9.45/9.99, FRFC 2.4.508.01 [EC] .F, FRFC 9.4.538.03 [EC] .F, and FRFC 2.4.524.03 [EC] ), and by the University of Liège (Fonds spéciaux, Crédit classique, 1999). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A fellow of the French Fondation Recherche Médicale.

^¶ Chercheur Qualifié of the Fonds National de la Recherche Scientifique (Brussels, Belgium).

To whom correspondence should be addressed: Tel.: 32-43-66-36-20; Fax: 32-43-66-33-64; E-mail: eric.sauvage{at}ulg.ac.be.

¹ The abbreviations used are: PBP, penicillin-binding protein; K15, Streptomyces K15 DD-peptidase; R61, Streptomyces R61 DD-transpeptidase-carboxypeptidase; R39, Actinomadura sp. strain R39 DD-peptidase; PBP5fm, E. faecium PBP5; SaG, Streptomyces albus G class A -lactamase; ESRF, European Synchrotron Radiation Facility; SAD, single anomalous diffraction.

	ACKNOWLEDGMENTS

 
We thank the staff of beamlines ID29 and BM30a at ESRF for assistance in data collection and J. A. Kelly for critical reading of the manuscript.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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