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1 Unité des Agents Antibactériens, Institut Pasteur, Paris, France
2 Centre d'Ingénierie Biomoléculaire, Université Libre de Bruxelles, Brussels, Belgium
3 Unité de Pharmacologie Cellulaire et Moléculaire, Université Catholique de Louvain, Brussels, Belgium
Reprint requests to: Dr. Patrice Courvalin, Unité des Agents Antibactériens, Institut Pasteur, 25, rue du Dr Roux 75724 Paris CEDEX 15, France; e-mail: pcourval{at}pasteur.fr; fax: 33-1-45-68-83-19.
(RECEIVED September 19, 2000; FINAL REVISION January 19, 2001; ACCEPTED January 22, 2001)
Article and publication are at www.proteinscience.org/cgi/doi/10.1110/ps.39101.
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G, G99 R, V241 D, D295 G, P313 L). The potential consequences of the deletion and point mutations on the 3-D structure of the enzyme were evaluated by comparative molecular modeling of the E. faecium enzyme, using the X-ray structure of the homologous Escherichia coli D-Ala:D-Ala ligase DdlB as a template. All mutated residues were found either to interact directly with one of the substrates of the enzymatic reaction (E13 and D295) or to stabilize the position of critical residues in the active site. Maintenance of the 3-D structure in the vicinity of these mutations in the active site appears critical for D-Ala:D-Ala ligase activity. Keywords: D-Ala:D-Ala ligase; glycopeptide-dependence; DNA sequencing; comparative molecular modeling
Abbreviations: D-Ala, D-Alanine • D-Ala:D-Ala ligase, D-Alanine:D-Alanine ligase • 3-D, three-dimensional
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We describe, at both phenotypic and genotypic levels, nine glycopeptide-dependent clinical isolates of E. faecium. We have determined the sequence of their ddl gene. We have analyzed by comparative molecular modeling the possible impact of the mutations on the structure of the active site of the D-Ala:D-Ala ligase to gain more insight into the structure–activity relationship of this key enzyme in bacterial metabolism.
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). Strains BM4480 to BM4486 were isolated from patients receiving vancomycin, and strains BM4487 and BM4488 from patients receiving teicoplanin. The nine strains displayed different restriction patterns by pulse-field gel electrophoresis (Fig. 1) and by susceptibility to antibiotics other than glycopeptides (Table 1), except for isolates BM4482 and BM4483, which came from the same hospital. This suggests that the strains are independently derived except for the two latter ones, which correspond to the same clone.
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). The E. faecium enzyme contained 358 amino acids; that is, 10 more than the E. faecalis enzyme. The sequence identity was very high (78%) between the two enterococcal enzymes, and both were similar to that of E. coli (36% amino acid identity for E. faecium and 37% for E. faecalis). All the residues suspected to play an important role in the enzymatic activity, based on the crystal structure of the DdlB E. coli enzyme, were conserved in the three proteins.
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). The ddl genes in BM4480 and BM4481 had a 5-bp GAGCA repeat between codons 14 and 15, resulting in a frame-shift mutation. The ddl gene of BM4485 had an in-frame deletion of 6 bp at codons 244 and 245, leading to the loss of two residues (Asp 244 and Val 245). The six remaining strains showed point mutations in their ddl genes. Only two mutations, at positions 13 and 295, affected residues known to be involved in the binding of the substrates to the active site of the enzyme (Shi and Walsh 1995). The other mutations affected residues that are conserved among the ligases but not thought to play a key role in enzymatic activity. Residues at positions 99 and 313 are conserved in D-Ala:D-Ala, D-Ala:D-Lac, and D-Ala:D-Ser ligases, and Val 241 is conserved only in D-Ala:D-Ala ligases of various species of enterococci (Evers et al. 1996).
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), as their sequences exhibit significant amino acid identity (36%; see Fig. 2
). The degree of accuracy in various stereochemical features of the model was comparable to that of the X-ray template structure. These criteria (Ramachandran plot, peptide bonds planarity, nonbonded interactions, hydrogen bonds, closeness of side chain dihedral angles to ideal values) were consistent with crystal structures determined at a resolution of 2–2.7 Å. For example, the percentage of nonglycine and nonproline residues in most favored regions of the Ramachandran plot was 88% for the E. coli X-ray structure and 72% for the E. faecium model (Fig. 4). The regions of the protein model with the poorest stereochemistry were located between residues 30 and 90, as well as in the last C-terminal 14 residues for which there are no equivalent residues in the template structure and contribute toward 11% of the poorer stereochemistry of the E. faecium model (Fig. 4). These portions, therefore, had to be modeled ab initio, a procedure that is known to yield relatively inaccurate results for insertions larger than seven to eight residues (Sanchez and Sali 1997).
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, Asp 295 occupies a crucial location in the active site. It interacts directly with one of the two Mg2+ ions assisting the phosphate group transfer from ATP and forms a H bond with Arg 293. The latter residue has an important catalytic role in the E. coli ligase. Asp 295 has been proposed to interact with the carboxylate group of D-Ala1 substrate, as it makes contact with the inhibitor P–O group (Shi and Walsh 1995). The replacement of Asp 295 by Gly in BM4482 and BM4483 should grossly perturb the phosphate group transfer because of the loss of a negative charge. Moreover, glycine, because of its lack of side chain, generates larger backbone mobility that could give rise to a local structural rearrangement of the polypeptidic chain. Figure 5B shows that Glu 13 could play a critical role in stabilizing D-Ala1 binding, as it is within hydrogen bond distance to the amino group of the inhibitor. Recent pKa calculations of the E. coli enzyme have indicated that the corresponding E. coli residue, Glu 15, is one of the most probable participants in the deprotonation step of D-Ala2 NH3+ group (Carlson et al. 1999). In addition to this role, a H-bond triad is formed involving Tyr 254, Ser 185, and Glu 13 side chains, whose function, as in the E. coli enzyme (Fan et al. 1994), could be to secure the mobile loop 246–258 on the top of the active site so as to position Lys 253 toward the 
-phosphate of ATP. Given its location in the active site, its interaction with other active site residues, and its conservation in ligase sequences, the mutation of Glu 13 into Gly is expected to strongly impair the enzyme activity by removing a net charge and a number of H-bond partners.
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). The latter residues are in an 
-loop composed of residues 246–258 that is thought to swing over the active site after the substrates bind (Parsons et al. 1988; Healy et al. 2000). Figure 5C shows that in the wild-type protein, residues 243–246 form a turn following an extended segment and preceding the -loop, which adopts a helical conformation. These residues may be important for the hinge-bending motion that will bring the -loop over the active site to occur. The model (Fig. 5C) shows that the deletion grossly affects the position of the -loop, including Phe and Tyr residues, which in turn, could result in a deficiency in the substrate binding. Most importantly, the conformation of Lys 253 (251 in the mutant), a conserved residue that is likely to be involved in the stabilization of transfer of the phosphate group of ATP, is also perturbed by this deletion, suggesting an impairment of activity for the mutant enzyme.
Gly 99 does not interact directly with the inhibitor in the wild-type protein (Fig. 5D); however, the adjacent His 98 makes contact with D-Ala1 side chain. Both residues are invariant in ligases (Evers et al. 1996). The model shows that the positively charged side chain of Arg present at position 99 in the mutant enzyme of BM4486 is close to the NH3+ group of the inhibitor and to one of the two Mg2+ ions. This spatial arrangement could lead to a reduced affinity of D-Ala1 and of the Mg2+ ion for the active site pocket and will likely impair enzyme activity.
Residues Pro 313 (replaced by Leu in strain BM4487) and Gly 314 are invariant in ligases (Evers et al. 1996). As shown in Figure 5E, Pro 313 does not directly interact with the inhibitor, but the NH backbone of Gly 314 forms a H bond with the P–O group. Pro 313 also makes hydrophobic contacts with the side chain of Arg 293, an important catalytic residue. The model shown in Figure 5E for the Pro to Leu mutant located the Leu side chain on the opposite side of the backbone relative to Arg 293, leaving the latter less constrained. In addition, the side chain of Asn 310, a conserved residue (Evers et al. 1996) that stabilizes one of the Mg2+ ions via its carbonyl side chain group, is moved away from this ion. This may have a detrimental effect on the phosphate transfer step from ATP to the D-ala substrate. It thus seems that the Pro 313 to Leu mutation produces a protein with a higher local flexibility that affects the enzyme activity.
Finally, the Val 241 residue (mutated into Asp in E. faecium BM4488) is located in a region close to the active site pocket but that exhibits only a weak similarity with the E. coli sequence (see Fig. 2). As correct sequence alignments are crucial for the production of reliable 3-D models constructed by comparative modeling, the quality of the model in this protein region is uncertain. Nevertheless, in the mutant enzyme, Asp can possibly repel the Glu 222 side chain, which establishes H bonds with the ribose of ADP in the wild-type enzyme (Fig. 5F), and therefore, Asp can affect the binding of one of the substrates to the enzyme. Moreover, the absence of positive charge to stabilize the Asp residue could perturb the correct position of the -loop, as in the 244–245 deletion.
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) would appear to indicate a low production of resistance proteins (Arthur et al. 1996b). This was experimentally confirmed in vancomycin-dependent E. faecalis mutants and attributed to the exclusive synthesis, by these strains, of precursors ending in D-Ala-D-Lac, making the production of the resistance enzymes removing D-Ala-D-Ala-containing precursors superfluous (Van Bambeke et al. 1999).
Considering the molecular mechanism involved in glycopeptide dependence, there is growing evidence that the appearance of this phenotype follows the generation of mutations in the gene encoding the D-Ala:D-Ala ligase that lead to enzyme inactivation (Baptista et al. 1997; Fraimow et al. 1994; Van Bambeke et al. 1999). Glycopeptide-dependent mutants may, therefore, be considered as useful tools to refine structure–activity relationships for this enzyme, which may represent an attractive target for new antibiotics. First described by Neuhaus in 1960, D-Ala:D-Ala ligase plays a key role in cell wall biosynthesis (Walsh 1989). Yet it has only been purified from a few bacterial species (Neuhaus 1962; Daub et al. 1988; Zawadzke et al. 1991), and its 3-D structure has only been established for the E. coli enzyme (Fan et al. 1994). In a previous study (Prévost et al. 2000), we showed that molecular modeling applied to D-Ala:D-Ala ligase E. faecalis mutants (Baptista et al. 1997; Van Bambeke et al. 1999) could help in understanding changes in a 3-D enzyme structure that lead to its inactivation. In the absence of X-ray data, we have applied this approach to the D-Ala:D-Ala ligase of E. faecium. The deduced sequence of the ddl gene in E. faecium showed high sequence similarity with the corresponding E. faecalis enzyme, as well as with that of E. coli. In particular, and as described for the E. faecalis enzyme (Prévost et al. 2000), all the residues involved in the ATP-dependent ligation of the carboxyl group of one substrate to the amino function of another substrate, which is the common feature of the enzymes belonging to the ATPgrasp family (Galperin and Koonin 1997), are also conserved in the E. faecium enzyme. Likewise, the E. faecium, E. faecalis, and E. coli enzymes possess an organization of their ATP-binding site similar to that of cAMP-dependent protein kinase, a member of an unrelated family of protein kinases (Denessiouk et al. 1998). The mutated amino acids in the glycopeptide-dependent mutants are all conserved in D-Ala:D-Ala ligases (Evers et al. 1996); interestingly, however, they were not necessarily predicted to be directly involved in substrate recognition (Fig. 2). Although the degree of enzyme impairment was not measured biochemically, the lack of growth of the mutants in the absence of vancomycin (which induces expression of the structural gene for the D-Ala:D-Lac ligase) strongly suggests a complete lack of D-Ala:D-Ala ligase activity. This is indeed what has been found previously in vancomycin-dependent mutants of E. faecalis, which produced 0% of precursors ending in D-Ala-D-Ala (Van Bambeke et al. 1999). The consequences of two mutations, Asp 295 Gly (in strains BM4482 and BM4483) and Glu 13 Gly (in strain BM4484), can be explained on the basis of homology with the E. coli enzyme (Fan et al. 1994; Shi and Walsh 1995) because they affect residues directly involved in the binding of the substrate (see Fig. 5A,B). This confirms the importance of Asp 295 in catalysis, observed for point mutants of E. faecalis (Asp Val [Van Bambeke et al. 1999]) and of E. coli (Asp Asn [Shi and Walsh 1995]). We also demonstrate the critical role of Glu 13, as was surmised from the studies with E. coli (where a mutation of the corresponding Glu [at position 15] to Gln causes a 100-fold decrease in activity [Shi and Walsh 1995]). In addition, we suggest that the H bond established between Glu 13 and the amino group of the inhibitor (and, therefore, most likely with D-Ala1) is critical for binding. The other point mutations, Gly 99 Arg, Pro 313 Leu, and Val 241 Asp, affect residues that do not interact directly with the substrates and were, therefore, only rationalized by molecular modeling. As discussed in the study of the E. faecalis enzyme (Prévost et al. 2000), comparative modeling provides accurate models in regions homologous with those of the E. coli template. This was clearly the case in two of the three regions studied corresponding to mutations at positions 99 (strain BM4486; Fig. 5D) and 313 (strain BM4487; Fig. 5E). The regions of lowest structural confidence were those corresponding to substitution at position 241 (strain BM4488; Fig. 5F) and the deletion at positions 244–245 (in strain BM4485; Fig. 5C), as the sequence homology in these regions with the E. coli template is weak (Sanchez and Sali 1997; Prévost et al. 2000). The latter deletion is, however, of interest because the deleted residues did not appear to enter in direct contact with the substrate. The model showed that these four mutations may indirectly affect the conformation of the active site. This type of mutant is rarely studied by site-directed mutagenesis, as opposed to active site residues for which functional groups are spatially proximal to the bound substrate or to the reaction intermediates. An important aspect of the active site is that invariance extends to a number of amino acids that do not themselves make contact with the substrate but that, rather, provide substructure or scaffold on which amino acids contacting the substrate are supported. The present data bear some similarity to those obtained with the ketosteroid isomerase of Pseudomonas putida and with the Influenza virus neuraminidase (Varghese et al. 1992), which showed that a H-bond network provides the structural organization needed by the enzyme to maintain the active site geometry optimized for both function and stability (Kim et al. 2000). Other studies have also pointed to the involvement of flexible surface loops, such as that affected in strain BM4485 (Fig. 5C), in the mechanism of enzyme-catalyzed reactions (First and Fersht 1993). Certain residues located in these loops may not be directly involved in the enzyme catalytic mechanism but seem essential for the movement of the loop that brings the active site residues close to the substrate.
Finally, several mutations (Asp 295 Gly, Gly 99 Arg, Glu 13 Gly, Val 241 Asp) are accompanied by the gain or the loss of a charged residue or by a modification in the orientation of a charged residue (in the mutant having a deletion of residues 244 and 245). The subsequent perturbation of electrostatic interactions could restrict or even possibly prevent the access to the active site or the correct positioning in the active site of one of the substrates.
The present data indicate that point mutations offer a larger potential for enzyme inactivation than could be deduced from the examination of the substrate binding site itself. Maintenance of the geometry of the catalytic pocket thus appears critical for enzymatic activity. These conclusions may open interesting perspectives for the understanding of the mechanism of action of enzymes belonging to the same family as well as for the design of new antibiotics.
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. Glycopeptide-dependent strains were recovered from patients undergoing vancomycin therapy, with the exception of strains BM4487 and BM4488, which were isolated from patients receiving teicoplanin. The clinical isolates were grown in brain heart infusion (BHI, Difco Laboratories) supplemented with 10 µg/mL vancomycin. The nondependent strains were grown in nonsupplemented BHI.
Phenotypic and genotypic characterization of the strains
The MICs of vancomycin and teicoplanin were determined after 48 h of incubation by the method of Steers et al. (1959) with 105 CFU per spot on BHI agar or BHI agar supplemented with 10 µg/mL vancomycin (to determine the MICs of teicoplanin for the vancomycin-dependent strains). Susceptibility to antibiotics other than glycopeptides was determined by disk agar diffusion on BHI supplemented with 10 µg/mL vancomycin. The species of enterococci and the glycopeptide resistance genotype were determined by PCR (Dutka-Malen et al. 1995) with DNA from E. faecium BM4147 (vanA) and E. faecalis BM4281 (vanB) as positive controls (Leclercq et al. 1988; Quintiliani and Courvalin 1996). All strains were found to be E. faecium.
Pulse-field gel electrophoresis of genomic DNA
Glycopeptide-dependent strains were analyzed by pulse-field gel electrophoresis. Genomic DNA embedded in agarose plugs (Miranda et al. 1991) was digested overnight at 30°C with 40 U of SmaI endonuclease. The restriction fragments were separated by using a clamped homogenous electric field with a CHEF-DR II system (Bio-Rad Laboratories) with a pulse ranging from 2 to 22 s for 16 h.
Amplification, cloning, and sequencing of the ddl gene
Purified total DNA was used as a template for amplification by PCR using pfu polymerase and primers 5'-GAGTAAATCACTGAACGATT and 5'-GGTTACGCAATCACTCCAGCCT designed from the sequence of the ddl gene from E. faecium BM4339 (Casadewall and Courvalin 1999). Amplification was carried out in a 100–-µL volume containing 50 pmol of each primer, 50 nmol of each deoxynucleotide, reaction buffer, 100 ng of DNA, and 10 U of pfu DNA polymerase. A total of 30 cycles were run; each cycle consisted of 1 min denaturation at 94°C, 1 min annealing at 54°C, and 2 min elongation at 72°C. The PCR products were purified from agarose gels (Sephaglas kit, Pharmacia), cloned into pCR-Blunt (Invitrogen Leek), and sequenced by the dideoxychain termination method (Sanger et al. 1977) using T7 DNA polymerase (Sequenase kit, U.S. Biochemical) and [
-35S]dATP (Amersham Radiochemical Center). Two independent PCR products were sequenced for each strain studied.
Comparative molecular modeling
The procedure followed was that used for building the model of the E. faecalis enzyme (Prévost et al. 2000). In brief, the sequence of the target enzyme was aligned with that of the DdlB of E. coli, which displays 
36% identity with the E. faecium enzyme (Evers et al. 1996; this work). The model was produced for the entire enzyme in the presence of ADP and methylphosphinophosphate in the active site. The latter molecule is an irreversible inhibitor of the enzyme replacing D-Ala-D-Ala in the active site of the E. coli enzyme (Parsons et al. 1988; Fan et al. 1994). Using Modeller4 software (Sali and Blundell 1993), the 3-D structure was built based on the satisfaction of spatial restraints extracted from the alignment and on the optimization of the interaction energy of the molecule. The model obtained was then validated by appropriate programs (Modeller; Procheck [Laskowski et al. 1993]), as described previously for the E. faecalis model (Prévost et al. 2000).
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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References |
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F. Depardieu, P. E. Reynolds, and P. Courvalin VanD-Type Vancomycin-Resistant Enterococcus faecium 10/96A Antimicrob. Agents Chemother., January 1, 2003; 47(1): 7 - 18. [Abstract] [Full Text] [PDF]
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) values are represented by filled circles. Nonglycine residues whose (