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Keywords:

  • carbamoylated lysine;
  • chemical rescue;
  • Mur synthetases;
  • peptidoglycan biosynthesis

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Enzymes MurD, MurE, MurF, folylpolyglutamate synthetase and cyanophycin synthetase, which belong to the Mur synthetase superfamily, possess an invariant lysine residue (K198 in the Escherichia coli MurD numbering). Crystallographic analysis of MurD and MurE has recently shown that this residue is present as a carbamate derivative, a modification presumably essential for Mg2+ binding and acyl phosphate formation. In the present work, the importance of the carbamoylated residue was investigated in MurD, MurE and MurF by site-directed mutagenesis and chemical rescue experiments. Mutant proteins MurD K198A/F, MurE K224A and MurF K202A, which displayed low enzymatic activity, were rescued by incubation with short-chain carboxylic acids, but not amines. The best rescuing agent was acetate for MurD K198A, formate for K198F, and propionate for MurE K224A and MurF K202A. In the last of these, wild-type levels of activity were recovered. A complementarity between the volume of the residue replacing lysine and the length of the carbon chain of the acid was noted. These observations support a functional role for the carbamate in the three Mur synthetases. Experiments aimed at recovering an active enzyme by introducing an acidic residue in place of the invariant lysine residue were also undertaken. Mutant protein MurD K198E was weakly active and was rescued by formate, indicating the necessity of correct positioning of the acidic function with respect to the peptide backbone. Attempts at covalent rescue of mutant protein MurD K198C failed because of its lack of reactivity towards haloacids.

Abbreviations
UDP-MurNAc-dipeptide

UDP-N-acetylmuramoyl-l-alanine-d-glutamate

UDP-MurNAc-tripeptide

UDP-N-acetylmuramoyl-l-alanine-γ-d-glutamyl-meso-diaminopimelate

UDP-MurNAc-pentapeptide

UDP-N-acetylmuramoyl-l-alanine-γ-d-glutamyl-meso-diaminopimelyl-d-alanyl-d-alanyl

UMA

UDP-N-acetylmuramoyl-l-alanine

FolC

tetrahydrofolate:l-glutamate γ-ligase or folylpolyglutamate synthetase

Mpl

UDP-N-acetylmuramate:l-alanyl-γ-d-glutamyl-meso-2,6-diaminopimelate ligase

MurC

UDP-N-acetylmuramate:l-alanine ligase

MurD

UDP-N-acetylmuramoyl-l-alanyl:d-glutamate ligase

MurE

UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-2,6-diaminopimelate ligase

MurF

UDP-N-acetylmuramoyl-l-alanyl-γ-d-glutamyl-meso-2,6-diaminopimelate:d-alanyl-d-alanine ligase

The Mur synthetases (MurC, MurD, MurE, MurF and Mpl), which catalyse the assembly of the peptide moiety of peptidoglycan [1], have been defined as a functionally and phylogenetically related superfamily of enzymes [2,3]. Folylpolyglutamate synthetase (FolC) [4], cyanophycin synthetase from cyanobacteria [5,6], and protein CapB from Bacillus anthracis[7] can be included in this superfamily [2,3,5]. All these enzymes share the following common properties: (a) they catalyse the synthesis of an amide or peptide bond with concomitant degradation of ATP to ADP and Pi; (b) they operate through a similar mechanism involving the formation of acyl phosphate ([8], and references therein) and tetrahedral [9,10] intermediates; (c) several residues in their primary structure, as well as the ATP-binding consensus sequence, are conserved [2,3]. Site-directed mutagenesis experiments have shown that these invariant residues are involved in the binding of the substrates and/or the catalytic process [2,3,11]. Over the past few years, the Mur synthetases have gained importance as potential targets in antibacterial research.

The crystallographic structures of one of the Mur synthetases, MurD, in the presence of various ligands have been solved [12,13]. An interesting observation is that K198 (conserved in MurD, MurE, MurF, FolC and cyanophycin synthetase, but not in MurC, Mpl and CapB) is present as a carbamate derivative. In the MurD–UDP-N-acetylmuramoyl-l-alanine (UMA)–ADP–Mg2+ complex, the carbamate is linked to two water molecules, which also interact with Mg2+ of site 1 [13]. This Mg2+ is presumably the centre of a network including the carboxylate of UMA and the γ-phosphate of ATP (Fig. 1). It has been hypothesized that site 1 plays a role in several steps of the reaction mechanism such as the formation and stabilization of the acyl phosphate intermediate [13]. Very recently, the three-dimensional structure of MurE complexed with its product UDP-N-acetylmuramoyl-l-alanine-γ-d-glutamyl-meso-diaminopimelate (UDP-MurNAc-tripeptide) has also revealed the carbamoylation of the conserved residue K224 [14].

image

Figure 1. The two sites for Mg2+ and the carbamoylated lysine residue of MurD. Two binding sites for Mg2+ have been detected in the MurD–UMA–ADP–Mg2+ complex [13]: in site 1, Mg2+ is co-ordinated with one of the carboxylate oxygens of UMA, one imidazole nitrogen of H183 and four water molecules; in site 2, the classical one, it is co-ordinated by the β-phosphoryl oxygen of ADP, the hydroxyl group of S116, one of the side-chain carboxylate oxygens of E157, and three water molecules. In the presence of all the substrates, as shown, the γ-phosphate of ATP is presumably the sixth ligand for both Mg2+ ions in lieu of water [13].

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The specificity constants of mutant MurD proteins K198A and K198F for the three substrates are markedly decreased [11]. However, as these mutations suppress both the lysine side chain and the carbamic acid, it is impossible to deduce whether the latter is important for enzymatic activity. In this paper, we describe the chemical rescue by aliphatic carboxylic acids of proteins MurD, MurE and MurF mutated on the conserved lysine residue. These results establish the importance of the acidic function and the length of the side chain of the carbamoylated lysine residue in the enzymatic activity of these synthetases. Attempts at covalent rescue for MurD are also described.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Chemicals

UMA [15], [14C]UMA [16], UDP-MurNAc-dipeptide [13], UDP-MurNAc-tripeptide [17], d-[14C]glutamic acid [15] and d-Ala-d-[14C]Ala [18] were prepared according to the published procedures. meso-[14C]Diaminopimelic acid was purchased from CEA (Saclay, France).

Strains and growth conditions

Escherichia coli strain JM83 was used as a host for plasmids as well as for the overproduction of mutant MurD proteins. E. coli strain BMH71-18 mutS defective in mismatch repair was used in site-directed mutagenesis experiments [19]. 2YT [20] was used as a rich medium for growing cells, and growth was monitored by measuring A600. For strains carrying resistance genes, antibiotics were used at the following concentrations: ampicillin (100 µg·mL−1) and tetracycline (15 µg·mL−1).

General DNA techniques and E. coli cell transformation

Small-scale and large-scale plasmid isolations were carried out by the alkaline lysis method [21]. Standard procedures for endonuclease digestion, ligation and agarose electrophoresis were used [21,22]. E. coli cells were made competent for transformation by the method of Dagert & Ehrlich [23] or by electroporation.

Plasmid construction and site-directed mutagenesis

Plasmid pABD16 [11], suitable for the overproduction of wild-type MurD containing a C-terminal Ser-Arg-Ser-(His)6 extension, was used as a target for a site-directed mutagenesis study performed with the Transformer site-directed mutagenesis kit (Clontech, Palo Alto, CA, USA) based on the method of Deng & Nickoloff [19]. MurD K198A/F mutants were prepared as described previously [11]. As far as the MurD K198C mutant was concerned, we followed the same procedure except that the primer used for the introduction of the specific mutation (in bold) was: 5′-CAGTATCGTGCAGCATGCCTGCGCATTTAC-3′. A new SphI restriction site was then created (underlined bases) and used to select, in addition to sequencing, mutated plasmids.

The MurD K198E mutant was prepared by a different procedure. Site-directed mutagenesis was performed on plasmid pABD16 using the primer extension and DpnI digestion approach of the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The oligonucleotide pair used was 5′-CAGTATCGTGCAGCAGAACTGCGCATTTACG-3′ and 5′-CGTAAATGCGCAGTTCTGCTGCACGATACTG-3′ (these primers are complementary and confer the base pair change in bold). Candidate mutant genes were sequenced to verify the presence of the desired mutation without other alterations.

Plasmid pMLD117, which is suitable for the overproduction of wild-type MurE containing a C-terminal Arg-Ser-(His)6 extension, was constructed as described by Gordon et al. [14]. A plasmid suitable for the overproduction of wild-type MurF containing a C-terminal His-tag extension was constructed as follows: PCR primers were designed to incorporate a BspHI site (in bold) 5′ to the initiation codon (underlined) of murF, 5′-TGATTTCATGATTAGCGTAACCCTTAGCC-3′ and a BamHI site (in bold) 3′ to the genes without its stop codon, 5′-CAGCCGGATCCACATGTCCCATTCTCCTG-3′. These primers were used to amplify the murF gene from the E. coli chromosome. The resulting material was treated with BspHI and BamHI and was ligated between the compatible NcoI and BglII sites in vector pTrcHis60 [24]. The plasmid obtained was designated pMLD116 and contained the murF gene coding for an enzyme tagged by a C-terminal Gly-Ser-(His)6 extension.

To obtain the alanine replacement of K224 and K202 in MurE and MurF, respectively, we performed site-directed mutagenesis as described for MurD K198C except that pMLD117 and pMLD116 were used as target plasmids. The specific mutations (in bold) were introduced using the following primers: 5′-CACTACGAAGCCGCGGCATGGCTGCTTTATTCT-3′ for murE and 5′-GGTGTCGCGAAAGCCGCGGGTGAAATCTTTAGC-3′ for murF. In both cases, a new SacII restriction site was created (underlined bases) and used to select, in addition to sequencing, mutated plasmids.

Overproduction and purification of His-tagged wild-type and mutant MurD, MurE and MurF proteins

Wild-type and mutant MurD proteins were prepared as described previously [11], except that the harvested cells were washed in cold 20 mm potassium phosphate/1 mm dithiothreitol, pH 7.2 (buffer A). For the MurE and MurF proteins, the following procedure was employed. JM83 cells carrying either the pMLD117 or the pMLD116 (wild-type or mutated) plasmid were grown exponentially at 37 °C in 2YT/ampicillin medium (500-mL cultures). When the absorbance of the culture reached 0.2, isopropyl thio-β-d-galactoside was added at a final concentration of 2 mm, and growth was continued for 5–6 h up to an absorbance of 2.0–2.5. Cells were harvested in the cold and washed in cold buffer A. The cell pellet was suspended in 10 mL buffer A and sonicated in the cold. The resulting suspension was centrifuged at 4 °C for 30 min at 200 000 g in a Beckman TL100 centrifuge, and the pellet was discarded. The supernatant was mixed with 0.5 mL Ni2+/nitrilotriacetate/agarose previously equilibrated with buffer A for 2 h at 4 °C. The resin was recovered by centrifugation and washed with increasing concentrations of imidazole in buffer A (20 mm three times, 40, 60, 100, 150, 200 mm; the 20 mm washings contained 0.3 m KCl). Elution of MurE and MurF started at 40 mm imidazole but the proteins were homogeneous only in the 60 and 100 mm fractions, which were pooled and dialysed against buffer A.

Standard enzyme assays

(a) MurD: formation of UDP-MurNAc-dipeptide was monitored in an assay mixture containing 0.1 m Tris/HCl buffer (pH 9.4), 5 mm MgCl2, 5 mm ATP, 25 µm UMA, 25 µmd-[14C]Glu (0.88 kBq), and enzyme (25 µL of an appropriate dilution in buffer A. (b) MurE: formation of UDP-MurNAc-tripeptide was monitored in an assay mixture containing 0.1 m Tris/HCl buffer (pH 8.6), 0.1 m MgCl2, 5 mm ATP, 100 µm UDP-MurNAc-dipeptide, 100 µmmeso-[14C]diaminopimelic acid (0.88 kBq), and enzyme [15 µL of an appropriate dilution in buffer A containing 15% (v/v) glycerol]. (c) MurF: formation of UDP-MurNAc-pentapeptide was monitored in an assay mixture containing 0.1 m Tris/HCl buffer (pH 8.6), 0.1 m MgCl2, 5 mm ATP, 85 µm UDP-MurNAc-tripeptide, 75 µm d-Ala-d-[14C]Ala (0.88 kBq), and enzyme (15 µL of an appropriate dilution in buffer A).

In all cases, the mixtures (final volume 50 µL) were incubated at 37 °C for 30 or 60 min, and the reactions were stopped by the addition of acetic acid (10 µL). The mixtures were lyophilized and taken up in the HPLC elution buffer (100 µL). The radioactive substrate and product were separated by HPLC on a Nucleosil 5C18 column (150 × 4.6 mm; Alltech France, Templemars, France) using 50 mm ammonium formate buffers (pH 4.75 for MurD and MurF, 3.90 for MurE) at a flow rate of 0.6 mL·min−1. Detection was performed with a radioactive flow detector (model LB506-C1; EG & G Wallac/Berthold, Evry, France) using the Quicksafe Flow 2 scintillator (Zinsser Analytic, Maidenhead, Berks., UK) at a flow rate of 0.6 mL·min−1. Quantification was carried out with the winflow software (EG & G Wallac/Berthold).

Chemical rescue experiments

The assays in the presence of aliphatic acids and amines were conducted as described above. The stock solutions of acids and amines were titrated to the pH of the assay with KOH and HCl, respectively.

Determination of the MurD kinetic constants

MurD activity was assayed as described above with various concentrations of one substrate and fixed saturating concentrations of the others. When the UMA concentration was much lower than that of d-Glu because of the large difference in their respective Km values, [14C]UMA was used as the labelled substrate in lieu of d-[14C]Glu. Data were fitted to the equation v = VA/(K + A) using the MDFitt software developed by M. Desmadril (UMR 8619 CNRS, Orsay, France).

Incubation of MurD K198C with iodobutyrate

MurD K198C (500 µg) was incubated for 24 h at room temperature in a reaction mixture (1.25 mL) containing 100 mm bicine buffer, pH 8.6, and 50 mm iodobutyrate. A control without iodobutyrate was performed. Reactions were stopped by the addition of dithiothreitol (19 mg). Enzymatic assays showed no loss of activity. The solutions were dialysed against 10 mm ammonium bicarbonate, pH 8.5, and lyophilized. The lyophilized products were submitted to carboxymethylation under reducing and denaturating conditions [25], then to acid hydrolysis [6 m HCl containing 1 : 2000 (v/v) 2-mercaptoethanol; 105 °C; 24 h]. The hydrolysates were analysed with a Biotronik LC2000 amino acid analyser: 7.21 ± 0.36 and 7.07 ± 0.23 mol S-carboxymethylcysteine/mol enzyme were found for the incubation with iodobutyrate and for the control, respectively (mean ± SD from three analyses).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Mutants proteins MurD K198A/F, MurE K224A and MurF K202A displayed very low enzymatic activity in their respective standard assays (less than 1.5% relative to wild-type in all cases; Table 1). The decrease in activity could be due to the loss of either lysine (amino function) or carbamate (acidic function). These hypotheses were tested by two approaches: (a) attempts to rescue the mutant proteins by incubation with short-chain aliphatic acids or amines; (b) attempts to recover an active enzyme by introduction of an acidic residue in place of the conserved lysine residue.

Table 1. Specific activity of wild-type and mutant MurD, MurE and MurF proteins in the presence of acids or amines (0.5 m). The values represent the mean of at least two experiments; the standard error is ≤ 15% in all cases. ND, Activity not detectable.
Compound addedSpecific activity (nmol·min−1·mg−1)
Wild-type MurDMurD K198AMurD K198FMurD K198EMurD K198CWild-type MurEMurE K224AWild-type MurFMurF K202A
None16700.0830.0520.220.5312404.69127015.7
Formate4890.990.561.859.59126026.693052.0
Acetate3922.690.0200.0642.70125054.5882511
Propionate3931.030.0170.0360.16113077.9756769
Butyrate3250.240.00730.0360.1170412.21240437
Methylamine1290.030NDND0.0682402.2554816.3
Ethylamine1630.0098NDND0.0643362.0779945.7
Propylamine134NDNDND0.0301641.8971332.1
Butylamine106NDNDNDND152ND7216.04

When the mutant proteins were incubated with short-chain aliphatic acids at a concentration of 0.5 m, enzyme activity increased considerably (Table 1). Control assays with the wild-type enzymes showed that the acids either inhibited the activity or had no effect; in no case was a wild-type enzyme activated by the acids. The activating effect of the acids on the mutant proteins was not due to the increase in ionic strength because a series of salts (KF, KCl, KBr, KI, KHCO3, KCN, NaN3) tested at a concentration of 0.5 m resulted in more or less pronounced inhibition (data not shown). The greatest activation was observed with acetate for MurD K198A (32-fold), formate for MurD K198F (11-fold), and propionate for MurE K224A (17-fold) and MurF K202A (49-fold). The corresponding recoveries of activity were 0.7, 0.1, 7 and 100%, respectively, of the wild-type value in the presence of the same acid at a concentration of 0.5 m. Therefore, whereas the chemical rescue of the MurD activity was far from complete in the standard assay (vide infra), that of the MurF activity was total.

To test the alternative hypothesis, namely the correlation between the fall in activity and the loss of the ε-amino function, the mutant proteins were incubated with short-chain aliphatic amines at a concentration of 0.5 m (Table 1). No activation was found for MurD and MurE mutant proteins, the amines being inhibitory instead. For MurF K202A, two of them produced slight activation (2.9-fold and twofold for ethylamine and propylamine, respectively). However, this effect is much weaker than that of propionate (49-fold).

Further experiments were carried out, taking MurD as a model, to study thoroughly the chemical rescue of the synthetase activity. Dose–response curves for formate and acetate were constructed (Fig. 2). No evidence of saturation was observed, at least up to 0.5 m, indicating a very weak free energy of interaction. Curiously, no activation by formate was detected below 0.1 m (Fig. 2A,C). Whether this threshold effect was due to formate unavailability at low concentration or reveals a sigmoidal pattern is not known. An unexplained sigmoidal curve has been observed by Perona et al. [26] in the rescue of D189S mutant trypsin by acetate.

image

Figure 2. Effect of the concentration of the acids on the MurD mutant proteins. All the assays were performed at an ionic strength of 0.55 (adjusted with KCl). (A) K198A + formate; (B) K198A + acetate; (C) K198F + formate.

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The effect of the best-activating acid on the kinetic constants of the mutant proteins was determined (Table 2). The specificity constant kcat/Km for each substrate was increased in the presence of acetate or formate. This effect was essentially due to a large increase in the kcat value (26-fold and 12-fold for K198A by acetate and K198F by formate, respectively). The effects on the Km values were less marked: no modification of the Km for ATP, moderate increase in the Km for UMA, and slight decrease in the Km for d-Glu. Control experiments on the wild-type protein showed a decrease in kcat, which was in agreement with the inhibition observed in the standard assay (Table 1).

Table 2. Effect of acids (added at a concentration of 0.5 m) on the kinetic constants of wild-type and mutant MurD proteins.
ProteinCompound added k cat (min−1) inline imagem) inline imagem) inline imagem)
Wild-typeNone400 ± 1760 ± 65 ± 272 ± 23
Formate109 ± 6138 ± 165 ± 240 ± 4
Acetate89 ± 788 ± 155 ± 2132 ± 23
K198ANone0.65 ± 0.04215 ± 35165 ± 19283 ± 55
Acetate16.9 ± 0.7216 ± 62256 ± 25188 ± 74
K198FNone0.44 ± 0.01119 ± 35122 ± 53688 ± 38
Formate5.37 ± 0.30144 ± 28559 ± 93485 ± 64

It should be mentioned that, on examination of the kcat values (determined at saturating concentration of all the substrates), the efficiency of the rescue of the MurD mutant proteins was improved relative to that calculated from the specific activity in the standard assay (determined at subsaturating concentrations of some substrates, in particular for the mutant proteins): the kcat values of rescued K198A and K198F were 19% and 4.9%, respectively, of the wild-type value in the presence of the most efficient acid.

As the carbamic acid participates in site 1 for Mg2+[13], the mutation of Lys198 should modify the binding of this cation. Dose–response curves for Mg2+ were constructed for the wild-type enzyme and the K198A mutant protein. The optimal (total) Mg2+ concentration for the wild-type enzyme was 5 mm, in agreement with our previous work [27]; that for the K198A mutant protein was 17.5 mm, a moderate 3.5-fold increase relative to wild-type. However, when free Mg2+ concentrations were considered, i.e. Mg2+ not complexed with ATP and therefore available to site 1, these values were 1.1 (wild-type) and 12.6 mm (K198A), an 11-fold difference (Fig. 3). Unfortunately, it was impossible to draw a conclusion from the dose–response curves with rescued K198A protein: in the presence of 0.5 m acetate, the curves with both wild-type and mutant proteins (not shown) were flattened and the optimal Mg2+ concentrations were shifted to higher values (25 and 50 mm total Mg2+, respectively), presumably owing to an ionic strength effect.

image

Figure 3. Effect of the free Mg2+ concentration on the MurD wild-type (○) and K198A (▪) proteins. MurD activity was determined in the presence of various concentrations of MgCl2. The free Mg2+ concentration was calculated using a stability constant of 2.97 mm−1 for the ATP4––Mg2+ complex [28]. The specific activity for each protein is expressed as percentage of the maximum value (1670 and 0.88 nmol·min−1·mg−1, respectively).

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As mentioned above, another approach to demonstrating a functional role for the carbamic acid is to replace the relevant lysine residue with an acidic amino acid. Such a rescue would, in principle, be more efficient than the one brought about by short-chain carboxylic acids, which yields noncovalent species and needs high concentrations of the rescuing agent. Two experiments were attempted: (a) preparation of the MurD K198E mutant protein; (b) alkylation of the MurD K198C mutant protein in order to generate a new acidic residue with the appropriate side-chain length.

The replacement of a carbamoylated lysine residue by an acidic residue was not expected to be the best method because the only possible replacements (Asp and Glu) have much shorter side chains. This was confirmed by the results obtained with the MurD K198E mutant protein (Table 1): it showed low specific activity (0.013% relative to wild-type); furthermore, it was rescued by formate (8.4-fold), revealing the inability of the γ-carboxylate group to occupy the position of the carbamic acid.

The other experiment, namely the chemical modification of a mutant protein to generate a new acidic residue [29], seemed more promising, as different lengths can be obtained. Mutant protein MurD K198C was prepared, which was weakly active (0.032% relative to wild-type) and rescued by formate and acetate (Table 1). It was preincubated with 50 mm haloacids (bromopropionate, bromobutyrate or iodobutyrate) at two pH values (8.6 or 9.4), and the enzymatic activity determined. Preincubation with a haloamine (bromoethylamine) was also performed. However, no effect on the activity was observed relative to a control without halogenated compound up to 24 h, whatever the pH (data not shown). This could be due to either alkylation without any effect on the activity or lack of reactivity of the cysteine residue in position 198. To check these hypotheses, 500 µg MurD K198C was incubated with 50 mm iodobutyrate at pH 8.6 for 24 h, and then cysteine was quantified by amino-acid analysis after carboxymethylation under reducing and denaturating conditions [25]: no loss of cysteine was detected relative to a control without iodobutyrate, showing that the thiol groups of the native mutant protein, in particular that of C198, are not reactive towards alkyl halides.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

A carbamoylated lysine residue (or, less commonly, a carbamoylated N-terminus) has been found in certain proteins, e.g. haemoglobin [30], urease [31], and ribulosebisphosphate carboxylase [32]. In general, the carbamic acid is linked to, and stabilized by, a metal ion. In the MurD–UMA–ADP–Mg2+ complex, it is not directly linked to Mg2+ of site 1, but to two water molecules, which also interact with the metal ion (Fig. 1); interestingly, it is still present, together with the two water molecules, in the MurD complexes without metal [13]. As the carbamic acid participates in site 1, it is difficult to imagine that its removal has no deleterious consequence on the enzymatic activity. However, without any evidence of its functional role, its occurrence as a mere chemical modification during purification and/or crystallogenesis cannot be ruled out. We first tried to disrupt the carbamate by submitting MurD to slightly acidic conditions (pH 5.5). However, total precipitation of the protein upon acidification impeded any further functional assay. We therefore decided to assess the role of the carbamate by chemical rescue experiments. This methodology was first used by Toney & Kirsch [33] to demonstrate the importance of the ε-amino group of a lysine residue of aspartate aminotransferase. Since then, it has been extended to other functional groups including carbamates: the lysine residue bearing the carbamate is changed into alanine, yielding a practically inactive mutant protein, and the catalytic activity is restored upon incubation with short-chain carboxylic acids [34,35]. In the present work, we show that MurD, MurE and MurF proteins mutated on the conserved lysine residue are rescued by formate, acetate, propionate or butyrate, according to the case. The specificity of the chemical rescue is substantiated by three observations: (a) the wild-type enzymes are not activated by the carboxylic acids at all; (b) the mutant enzymes are not, or almost not, activated by short-chain aliphatic amines; (c) in MurD, for which several mutant proteins have been prepared, the degree of activation depends both on the volume of the residue replacing lysine and on the length of the carbon chain of the acid: with alanine (volume of the residue 91 Å3[36]), acetate produced the greatest activation, followed by propionate and formate; with bulkier cysteine (106 Å3), the acid that produced the greatest activation was formate, followed by acetate; with the very bulky glutamic acid (155 Å3) or phenylalanine (203 Å3), only formate succeeded in activating the mutant proteins. The activation factor did not increase on preincubation of the MurD mutant proteins with the aliphatic acids for 0–24 h (data not shown), ruling out a time-dependence of the chemical rescue and validating the present correlation.

Although the present results demonstrate the importance of the carbamic acid in the enzymatic activity, its precise role (affinity for a substrate, stabilization of a transition state, catalysis) is difficult to assess. However, the increase in the optimal Mg2+ concentration for mutant protein MurD K198A with respect to wild-type protein shows that the carbamoylated lysine residue contributes, at least in part, to the affinity of site 1 for Mg2+. In agreement with the structural data [13], it can be hypothesized that the carbamate is important for spatial stabilization of the two water molecules, which correctly position the Mg2+ of site 1, which is itself involved in acyl phosphate formation.

The weak activity of mutant protein MurD K198E shows that the presence of a carboxylate group at residue 198 is not a sufficient condition for significant enzymatic activity; the acidic function must be placed at the correct distance from the peptide backbone, the distance corresponding to the length of the (CH2)4-NH side chain. In this regard, it would have been interesting to prepare chemically modified mutant enzymes possessing residues that mimick the carbamoylated lysine; it was attempted, but failed because of the lack of reactivity of mutant protein K198C with haloacids.

As Lys198 is conserved in MurE and MurF, it was tempting to speculate that it was carbamoylated in these enzymes as well. The chemical rescue of the MurE and MurF activities by aliphatic acids was demonstrated in this study, supporting this hypothesis. Recently, the presence of a carbamic acid on Lys224 has been deduced from X-ray crystallographic analysis of the MurE–UDP-MurNAc-tripeptide complex [14]. Similarly, carbamoylation of the relevant lysine residue (K185) has been observed in two structures of FolC [37], another member of the Mur synthetase superfamily. A structure has been published for MurF, but no carbamate was found [38]. However, in this structure, MurF was in an ‘open’ conformation. ‘Open’ forms of MurD [39] and FolC [40,41] have also been described; interestingly, none of them are carbamoylated. Therefore, as the carbamate seems to be present only in the ‘closed’ forms, where it is presumably stabilized by interaction with functional groups of the proteins and with water molecules, its absence from the published MurF structure does not contradict the complete rescue of MurF K202A by propionate observed in the present study. It will be of great interest to solve the structure of a ‘closed’ form of MurF. Another point deserves consideration: as mentioned in the introduction, sequence alignment of the members of the Mur synthetase superfamily revealed that the carbamoylated lysine residue is not conserved in MurC, Mpl and CapB; instead, an aromatic residue (Phe or Tyr) is present [11]. Site-directed mutagenesis and crystallographic studies will be necessary to elucidate its role.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by grants from the Centre National de la Recherche Scientifique (UMR 8619) and the Ministère de l'Education Nationale, de la Recherche et de la Technologie (PRFMMIP). S. D. is recipient of a scholarship from the Ministère de l'Education Nationale, de la Recherche et de la Technologie. We thank M. Desmadril (UMR 8619 CNRS, Orsay, France) for helpful discussions.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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