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Structure-Based Site-Directed Mutagenesis of the UDP-MurNAc-Pentapeptide-Binding Cavity of the FemX Alanyl Transferase from Weissella viridescens

Antoine P. Maillard ,^1,, Sabrina Biarrotte-Sorin,^1,2, Régis Villet,¹ Stéphane Mesnage,¹ Ahmed Bouhss,³ Wladimir Sougakoff,¹ Claudine Mayer,¹ and Michel Arthur^1*

Laboratoire de Recherche Moléculaire sur les Antibiotiques, INSERM U655, Université Paris 6,¹ Laboratoire de Minéralogie-Cristallographie de Paris, Université Paris 6, Paris,² Enveloppes Bactériennes et Antibiotiques CNRS, UMR 8619, Université Paris 11, Orsay, France³

Received 20 December 2004/ Accepted 23 February 2005

	ABSTRACT

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Weissella viridescens FemX (FemX_Wv) belongs to the Fem family of nonribosomal peptidyl transferases that use aminoacyl-tRNA as the amino acid donor to synthesize the peptide cross-bridge found in the peptidoglycan of many species of pathogenic gram-positive bacteria. We have recently solved the crystal structure of FemX_Wv in complex with the peptidoglycan precursor UDP-MurNAc-pentapeptide and report here the site-directed mutagenesis of nine residues located in the binding cavity for this substrate. Two substitutions, Lys36Met and Arg211Met, depressed FemX_Wv transferase activity below detectable levels without affecting protein folding. Analogues of UDP-MurNAc-pentapeptide lacking the phosphate groups or the C-terminal D-alanyl residues were not substrates of the enzyme. These results indicate that Lys36 and Arg211 participate in a complex hydrogen bond network that connects the C-terminal D-Ala residues to the phosphate groups of UDP-MurNAc-pentapeptide and constrains the substrate in a conformation that is essential for transferase activity.

	INTRODUCTION

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The bacterial cell wall peptidoglycan is a network of glycan strands cross-linked by short peptides of variable composition and complexity (24). Many species of gram-positive bacteria produce branched peptidoglycan precursors resulting from the addition of a side chain to the -amino group of L-Lys in the pentapeptide stem L-Ala-D-iGlu-L-Lys-D-Ala-D-Ala (24). The addition of these amino acids is catalyzed by the Fem transferases, a unique family of peptide bond-forming enzymes that use aminoacyl-tRNAs as aminoacyl donors (21).

The D,D-transpeptidases catalyzing the cross-linking step of peptidoglycan synthesis are the essential target of ß-lactam antibiotics (9). Resistance to ß-lactam antibiotics in gram-positive bacteria is usually due to the production of modified D,D-transpeptidases that are commonly referred to as low-affinity penicillin binding proteins (PBP). Addition of a complete side chain to the peptidoglycan precursors is essential for ß-lactam resistance mediated by such low-affinity PBPs in Staphylococcus aureus (26), Streptococcus pneumoniae (8, 28), and, to a lesser extent, Enterococcus faecalis (6). Transferases of the Fem family are therefore considered as potential targets for the development of novel antibiotics which are active against ß-lactam-resistant bacteria (2, 13).

The Weissella viridescens FemX (FemX_Wv) transferase has been selected as a model enzyme for kinetics and structural analyses because this enzyme catalyzes the transfer of L-Ala onto the cytoplasmic precursor UDP-N-acetyl-muramyl-pentapeptide (UDP-MurNAc-pentapeptide) (11, 18), in contrast to other members of the Fem family, which preferentially (5) or exclusively (16, 25) transfer amino acids to membrane-bound peptidoglycan precursors. Kinetic studies revealed an ordered mechanism involving sequential binding of UDP-MurNAc-pentapeptide and Ala-tRNA to FemX_Wv, transfer of Ala to the -amino group of L-Lys in the pentapeptide stem, and release of the tRNA and UDP-MurNAc-hexapeptide reaction products (10). Site-directed mutagenesis identified Asp108 as a candidate for the catalytic base, as the Asp108Asn substitution decreased the catalytic efficiency (V/K) of the enzyme 230-fold, primarily due to a 60-fold decrease in k_cat (10). More recently, we have reported the structure of FemX_Wv in complex with its UDP-MurNAc-pentapeptide substrate at 1.9-Å resolution (4). In the present report, structure-based design of substitutions in FemX_Wv, combined with deletions of residues in the UDP-MurNAc-pentapeptide substrate, led to the identification of specific substrate-enzyme interactions that are essential for the catalytic activity of the enzyme.

	MATERIALS AND METHODS

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Site-directed mutagenesis and enzyme purification. A C-terminal His₆ tag was introduced into FemX_Wv by cloning the femX gene of W. viridescens (CIP 102810 T [formerly designated Lactobacillus viridescens ATCC 12706]; Institut Pasteur Collection) (18) into the vector pTrc-His60 (19), and mutagenesis was performed according to the QuickChange procedure of Stratagene (La Jolla, Calif.). The wild-type and mutated FemX_Wv proteins were produced into Escherichia coli TOP10 (Invitrogen, Cergy Pontoise, France) and purified by affinity chromatography (Ni-NTA agarose; QIAGEN, Hilden, Germany). Protein concentration was determined by using the Bradford reagent (Bio-Rad, Munich, Germany) with bovine serum albumin as a standard. Alanyl-tRNA synthetase was purified as previously described (5), except that an additional anion-exchange chromatographic step was performed to completely eliminate E. coli tRNAs.

Preparation of the substrates. UDP-MurNAc peptides were purified from natural sources as previously described (15). Diphospho-MurNAc-pentapeptide was obtained by periodate treatment of UDP-MurNAc-pentapeptide (14). Phospho-MurNAc-pentapeptide was prepared by treatment of UDP-MurNAc-pentapeptide with nucleotide pyrophosphatase (1). MurNAc-pentapeptide was obtained by acid hydrolysis of UDP-MurNAc-pentapeptide (27). Pentapeptide L-Ala-D-iGlu-L-Lys-D-Ala-D-Ala was purchased from Sigma-Aldrich (St. Quentin Fallavier, France). All the substrates were purified by reverse-phase high-performance liquid chromatography and analyzed by mass spectrometry and tandem mass spectrometry as previously described (6).

The RNA 5'-GGGGCCUUAGCUCAGCUGGGAGAGCGCCUGCUUUGCACGCAGGAGGUCAGCGGUUCGAUCCCGCUAGGCUCCACCA-3', corresponding to the lone tRNA^Ala sequence in the genomic sequence of E. faecalis V583 (http://www.tigr.org/), was prepared by in vitro transcription, as previously described (23). The ethanol-precipitated RNA was further purified by reverse-phase high-performance liquid chromatography to remove nucleotides. Briefly, 150 A₂₆₀ units were loaded onto a Lichrosorb RP-18 column (300 by 7.8 mm, 10 µm; Waters, Mildford, MA) and eluted with an acetonitrile gradient (0 to 20%) in 500 mM ammonium acetate (pH 6.5). RNA was dialyzed against water, lyophilized, and dissolved in 25 mM Tris (pH 7.5) containing 100 mM NaCl and 5 mM MgCl₂. RNA was denatured for 5 min at 70°C, cooled to room temperature, and loaded onto a gel filtration column equilibrated with 25 mM Tris (pH 7.5) containing 100 mM NaCl (Superdex75; Amersham Biosciences, Uppsala, Sweden). The tRNA stock solution concentrated by ultrafiltration (100 µM) was judged to be pure at homogeneity based on denaturing polyacrylamide gel electrophoresis, the absorbance spectrum (, 717,000 M^–1 cm^–1 at 260 nm), and the yield of acylation by purified alanyl-tRNA synthetase (98% ± 5% per mole).

Activity measurements. The standard assay mixture for FemX_Wv transferase activity contained Tris-HCl (50 mM, pH 7.5), alanyl-tRNA synthetase of E. faecalis (800 nM), E. coli inorganic pyrophosphatase (10 U ml^–1; Sigma, Steinheim, Germany), ATP (7.5 mM), MgCl₂ (12.5 mM), and [¹⁴C]Ala (50 µM, 3,700 Bq/nmol; ICN, Orsay, France). The assay mixture also contained FemX_Wv, tRNA, and UDP-MurNAc-pentapeptide at the concentrations specified in the text. The reaction was performed at 30°C for 10 min with a preincubation of 10 min in the absence of FemX for synthesis of Ala-tRNA by the auxiliary system. The reaction was stopped at 95°C for 10 min and analyzed by descending paper chromatography (Whatman 4MM, Elancourt, France) with isobutyric acid-ammonia, 1 M (5:3 per vol). Radioactive spots were identified by autoradiography, cut out, and counted by liquid scintillation. The concentration of wild-type FemX_Wv and mutated proteins was adjusted to obtain initial velocities under conditions where transferase activity was rate limiting. Determination of [¹⁴C]Ala-tRNA in aliquots of the reactions was performed by precipitation with 5% trichloroacetic acid and 0.5% Casamino Acids, filtration on glass fiber filters, and liquid scintillation counting.

CD spectra. The circular dichroism (CD) spectra were measured at a protein concentration of 0.5 mg/ml in 20 mM phosphate buffer (pH 7.5) with a Jasco J-810 spectropolarimeter at 20°C in a quartz cell with a light path of 0.1 cm. For all measurements, a 1.0-nm bandwidth and a 0.25-s time constant were used, and scans were accumulated from 195 to 260 nm at the speed of 50 nm/min.

Crystallographic characterization. FemX_WvLys36Met (7.5 mg/ml) was crystallized in presence of UDP-MurNAc-pentapeptide (4) in a 1:2 ratio by vapor diffusion at room temperature against 30% polyethylene glycol 6000, 300 mM NaCl, and 100 mM cacodylate (pH 6.5). The crystals belong to the P21 space group, with unit cell parameters a = 42.33 Å, b = 101.30 Å, c = 46.33 Å, and ß = 103.02°.

Crystals were flash frozen directly from the drop with an additional 30% glycerol. Diffraction data were collected to 1.95 Å at 100 K using a MAR X-ray detection system on the French Beamline for Investigation of Proteins-Beam Magnet 30A (FIP-BM30A) at the European Synchrotron Radiation Facility, Grenoble, France (22). Data were processed with the programs MOSFLM (20) and SCALA from the CCP4 program suite (1994). X-ray data statistics are given in Table 1. The structure of FemX_WvLys36Met was solved by molecular replacement using AMORE (17) and native FemX_Wv (Protein Data Bank accession number 1NE9) as the search model. Refinement was performed using a crystallography and NMR system (7). The final model has an R factor of 19.6% and an R_free of 23.3% calculated for 5% randomly selected data. It includes 335 protein residues, 367 water molecules, and 1 magnesium ion.

	RESULTS

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Steady-state kinetic parameters for wild-type FemX_Wv and design of an assay for mutant proteins. The initial velocity was determined for all combinations of five concentrations of UDP-MurNAc-pentapeptide (20, 40, 80, 200, and 400 µM) and tRNA (0.6, 1.2, 3.0, 6.0, and 12.0 µM). The resulting double reciprocal plots were intersecting (data not shown), as previously found by Hegde and Blanchard (10) and in agreement with the proposed sequential kinetic mechanism (10). In comparison to that previous study (10), we observed a lower K_m for Ala-tRNA (1.6 versus 15 µM), similar K_m values for UDP-MurNAc-pentapeptide (70 versus 42 µM), and a slightly higher k_cat (1,600 versus 660 min^–1). Use of an extensively purified tRNA preparation in our study as opposed to crude in vitro transcription reaction products (10) could account for the difference between the K_m values for Ala-tRNA. In our assay, the tRNA was completely acylated by [¹⁴C]Ala (>90%) at the onset of the FemX_Wv reaction and remained fully acylated during the entire 10-min reaction, as judged by direct determination of [¹⁴C]Ala-tRNA, which was not reported in that previous study (10).

The impact of substitutions in FemX_Wv was analyzed by comparing the initial velocity of the reaction with various concentrations of wild-type or mutant transferases at fixed concentrations of tRNA (0.6 µM) and UDP-MurNAc-pentapeptide (50 µM) (Fig. 1A). Under such conditions, the initial velocity of the transfer reaction (v) was proportional to the concentration of the enzyme (E) for concentrations of wild-type FemX_Wv ranging from 0.1 to 1 nM (data not shown). The ratio v/E was determined from the slope of the curve obtained by plotting v versus E (235 ± 28 min^–1 for wild-type FemX_Wv). The mutant proteins were similarly tested at various concentrations (0.1 to 500 nM), and the ratio v/E was deduced from the linear portion of the plot of v versus E. The ratio v/E was used as an estimate of the catalytic efficiency of the enzyme because the relevant mutant proteins could not be saturated by UDP-MurNAc-pentapeptide (Table 2).

View larger version (37K): [in this window] [in a new window]   FIG. 1. Structure of the UDP-MurNAc-pentapeptide-binding cavity of FemXWv. (A) Schematic representation of the nine FemXWv residues in contact with the substrate through hydrogen (dashed lines) or stacking (arrows) interactions. The estimates of the transferase activity (v/E) were obtained for the mutant proteins with a single amino acid substitution as described in the text. (B) Close view of the superimposition of the FemXWv UDP-MurNAc-pentapeptide-binding cavity in the structures of the apo wild-type enzyme (4), UDP-MurNAc-pentapeptide:apo wild-type enzyme complex (4), and Lys36Met mutant protein (this work). The bound substrate and secondary structures of FemXWv are colored in magenta and dark blue, respectively. The FemXWv side chains of Trp32, Lys36, Trp39, Arg211, Tyr215, and Tyr256 are colored in yellow for the apo wild-type enzyme, in cyan for the complex, and in orange for the Lys36Met mutant.

Interactions of FemX_Wv with the uracil ring of the substrate did not appear to be critical for activity, because the Tyr103Phe substitution did not decrease activity (v/E) and FemX_WvPhe70Leu retained 65% of the activity of the wild-type enzyme (Fig. 1A). Likewise, interactions of Thr209 with D-iGlu in the pentapeptide stem and of Asn38 and Trp39 with the ß-phosphate group of UDP appeared to be largely dispensable for activity. In contrast, the hydrogen network involving Lys36, Arg211, and Tyr215 was clearly critical for the transferase activity of FemX_Wv. Lys36 makes hydrogen interactions with both phosphates of UDP, and the Lys36Met substitution reduced FemX_Wv activity below detectable levels. The substitution of Lys with Arg at the same position led to a 160-fold reduction of the activity (Fig. 1A), which could be partially accounted for by a 5.4-fold increase in the apparent K_m for UDP-MurNAc-pentapeptide (Table 2). Arg211 interacts with Tyr256 and Tyr215 of FemX_Wv, both phosphate groups of UDP, and the two D-Ala residues of the pentapeptide stem (Fig. 1A). The replacement of Arg211 by Met decreased the transferase activity below detectable levels, whereas the Arg211Lys substitution led to a 17,000-fold reduction in activity (Table 2). The latter substitution did not affect the apparent K_m for Ala-tRNA, whereas the apparent K_m for UDP-MurNAc-pentapeptide was greater than the highest concentration of substrate that could be tested (500 µM) (Table 2). The combination of Arg211Lys with Lys36Arg (above) further depleted activity below detectable levels (data not shown). The Tyr215 side chain interacts with the carboxyl end of the pentapeptide stem through hydrogen bonding and with Arg211 through stacking between the phenyl ring and the guanidium group. The Tyr215Phe substitution led to a 25-fold decrease in the transferase activity (Fig. 1A) which was associated with an increase in the K_m for UDP-MurNAc-pentapeptide (Table 2). The contribution of the stacking interaction to FemX_Wv activity could not be studied, as the Tyr215Leu substitution led to an insoluble protein. Finally, the Tyr256Phe substitution that suppressed the hydrogen interactions with Arg211 and with the C-terminal D-Ala of the substrate had a less pronounced effect on enzyme activity (2.6-fold reduction) (Fig. 1A).

CD spectra of inactive mutant proteins with the Lys36Met and Arg211Met substitutions. The CD spectra of wild-type FemX_Wv and of the mutant proteins were indistinguishable (Fig. 2), indicating that impaired protein folding could not account for the lack of transferase activity. Thus, the Lys36Met and Arg211Met substitutions suppressed enzyme-substrate interactions that were essential for activity.

View larger version (11K): [in this window] [in a new window]   FIG. 2. Comparison of CD spectra of wild-type (WT) FemXWv, FemXWvLys36Met, and FemXWvArg211Met. The spectra were recorded under the same experimental conditions (see Materials and Methods). Ten scans were accumulated from 195 to 260 nm at the speed of 50 nm/min with a 0.025-nm/point resolution.

The density of the Met36 side chain is totally well defined. In contrast, the -amino group of the Lys36 was not defined in the electronic density map of the wild-type apo form but stabilized in the complex. Stabilization of the methyl group of Met36 involves a van der Waals contact (3.8 Å) with N of Trp39. Small deviations are found in the proximity of the mutated residue. The presence of the methionine in the binding pocket has slightly expanded the cavity, which is caused by the concomitant shrinkage of Arg211, Tyr215, Tyr256, and Trp32 in the binding pocket. Among these residues, Arg211 is disordered in the Lys36Met mutant, whereas it is well defined in the structures of the apo wild-type enzyme and the binary complex.

Comparison of the apo wild-type and complex structures revealed that binding of the substrate is also associated with movements of Tyr215 and Tyr256 (4). In the FemX_WvLys36Met structure, the positions of Tyr256 and Tyr215 are very similar to that found in the apo wild type. Together, these results indicate that gross alterations of the conformation of the binding pocket cannot account for the loss of activity associated with the Lys36Met substitution.

Transferase activity of FemX_Wv with analogues of the substrate. The impact of deletions of residues in the UDP-MurNAc-pentapeptide substrate on the catalytic activity of wild-type FemX_Wv was estimated by using a fixed concentration (100 µM) of the substrate analogues (Table 3). FemX_Wv was tested at various concentrations (0.1 to 500 nM), and the ratio v/E was deduced from the portion of the curve in the plots of v versus E that was a straight line. The ratio v/E was used as an estimate of the catalytic efficiency of the enzyme because FemX_Wv could not be saturated by analogues of UDP-MurNAc-pentapeptide that were available in limited amounts.

Extraction of the cytoplasmic peptidoglycan precursors of W. viridescens revealed that the peptide stem of the nucleotide precursor contains a C-terminal D-lactyl (D-Lac) residue instead of D-Ala (data not shown). This is a common feature of lactic acid bacteria that are intrinsically resistant to glycopeptide antibiotics (see reference 3 for a review). The natural pentadepsipeptide-containing substrate of FemX_Wv has not been tested in vitro in previous studies. Substitution of the NH group in the D-Ala-D-Ala peptide bond by an oxygen atom in the D-Ala-D-Lac ester bond had very little impact, if any, on FemX_Wv activity (Table 3). Finally, replacement of L-Lys by a meso-diaminopimelyl residue, whose -carbon is substituted by both a carboxyl and an amino group, yielded a molecule that was not a substrate of FemX, as previously described (11).

	DISCUSSION

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Three lines of evidence indicate that a complex hydrogen bond network involving Lys36, Arg211, and, to a lesser extent, Tyr215 is critical for the transferase activity of FemX_Wv. First, the Lys36Met and Arg211Met substitutions depleted (>47,000-fold) activity below detectable levels (Fig. 1). Second, comparison of the CD spectra (Fig. 2) and crystal structures in the case of the Lys36Met substitution (Table 1) indicated that conformational changes in the mutant proteins cannot account for the loss of activity. Thus, changes in the chemical nature of the residues at positions 36 and 211 were responsible for the loss of activity. Third, analysis of substrate analogues indicated that a loss of the -phosphate or of the C-terminal D-Ala residue severely depleted activity, whereas compounds lacking both phosphates or both D-Ala residues were not used as substrates by FemX_Wv (Table 3). Together, these results indicate that hydrogen interactions between two residues of FemX_Wv (Lys36 and Arg211) and two regions of UDP-MurNAc-pentapeptide (both phosphate groups and both D-Ala residues) constrain the substrate in a bent conformation which is essential for activity (Fig. 1).

In spite of their essential role in activity, Lys36 and Arg211 are not highly conserved in members of the Fem family (see reference 4 for a structure-connected sequence alignment). In particular, position 211 can be occupied by Arg, Lys, Phe, and His, although the Arg211Lys substitution was poorly tolerated in FemX_Wv (17,000-fold reduction in activity) (Table 2). At position 36, Arg and Lys are present with similar frequencies in members of the Fem family, although the Lys36Arg substitution led to a 160-fold reduction in FemX_Wv activity. In Fem proteins, Arg at position 36 is frequently associated with Lys at position 211, instead of Lys36 with Arg211 in FemX_Wv, suggesting that Arg and Lys could be exchangeable at these positions to a certain extent. However, this was not the case for FemX_Wv, since combination of the Lys36Arg and Arg211Lys substitutions led to an inactive protein. The Tyr215Phe substitution in FemX_Wv reduced the transferase activity 25-fold, and Tyr or Phe is found at the homologous position in Fem proteins. Hydrogen interactions involving this residue are therefore important but not essential for the activity. FemX_WvTyr215Leu was insoluble, suggesting that the stacking interaction between Tyr215 (or Phe215) and Arg211 plays a role in protein folding.

Hegde and coworkers (10, 11) performed site-directed mutagenesis of FemX_Wv based on the alignment of the primary sequence of Fem proteins and surprisingly found that substitutions at invariant or highly conserved positions had a rather moderate (<20-fold) impact on activity, except for the putative catalytic base Asp108 (230-fold). Conservation of the former residues may reflect other functions of the protein such as interactions with components of the peptidoglycan precursor synthesis machinery.

	ACKNOWLEDGMENTS

 
This work was supported by the Ministère de la Recherche (grant ACI252 "Molécules et Cibles Thérapeutiques").

We thank Didier Blanot for preparation of diphospho-MurNAc-pentapeptide and Harald Putzer for helpful discussion.

	FOOTNOTES

 
* Corresponding author. Mailing address: LRMA INSERM U655, Université Paris 6, 15 rue de l'Ecole de Médecine, 75270 Paris Cedex 06, France. Phone: 33 1 43 25 00 33. Fax: 33 1 43 25 68 12. E-mail: michel.arthur{at}bhdc.jussieu.fr.

Present address: Faculty of Medicine, Department of Biochemistry, University of British Columbia, V6T 1Z3 Vancouver, Canada.

A.P.M. and S.B.-S. contributed equally to this work.

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