Functional and Biochemical Analysis of the Chlamydia trachomatis Ligase MurE

Chlamydiae are unusual obligately intracellular bacteria that do not synthesize detectable peptidoglycan. However, they possess genes that appear to encode products with peptidoglycan biosynthetic activity. Bioinformatic analysis predicts that chlamydial MurE possesses UDP-MurNAc-l-Ala-d-Glu:meso-diaminopimelic acid (UDP-MurNAc-l-Ala-d-Glu:meso-A₂pm) ligase activity. Nevertheless, there are no experimental data to confirm this hypothesis. In this paper we demonstrate that the murE gene from Chlamydia trachomatis is capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-l-Ala-d-Glu:meso-A₂pm ligase activity. Recombinant MurE from C. trachomatis (MurE_Ct) was overproduced in and purified from E. coli in order to investigate its kinetic parameters in vitro. By use of UDP-MurNAc-l-Ala-d-Glu as the nucleotide substrate, MurE_Ct demonstrated ATP-dependent meso-A₂pm ligase activity with pH and magnesium ion optima of 8.6 and 30 mM, respectively. Other amino acids (meso-lanthionine, the ll and dd isomers of A₂pm, d-lysine) were also recognized by MurE_Ct. However, the activities for these amino acid substrates were weaker than that for meso-A₂pm. The specificity of MurE_Ct for three possible C. trachomatis peptidoglycan nucleotide substrates was also determined in order to deduce which amino acid might be present at the first position of the UDP-MurNAc-pentapeptide. Relative k_cat/K_m ratios for UDP-MurNAc-l-Ala-d-Glu, UDP-MurNAc-l-Ser-d-Glu, and UDP-MurNAc-Gly-d-Glu were 100, 115, and 27, respectively. Our results are consistent with the synthesis in chlamydiae of a UDP-MurNAc-pentapeptide in which the third amino acid is meso-A₂pm. However, due to the lack of specificity of MurE_Ct for nucleotide substrates in vitro, it is not obvious which amino acid is present at the first position of the pentapeptide.Chlamydiae cause serious respiratory tract and genital infections in humans (9). They are obligately intracellular gram-negative bacteria, with a unique biphasic development cycle. Elementary bodies (EBs) are the infectious form of the organism and invade susceptible host cells. Once internalized, EBs differentiate into reticulate bodies (RBs), which have the capacity to divide (39, 40). The RBs are fragile and pleomorphic, whereas EBs are comparatively rigid and stable (19, 39). After repeated cycles of binary fission, the RBs differentiate into EBs, and the host cell lyses, releasing infectious EBs (1).In contrast to the vast majority of eubacteria, chlamydiae lack detectable amounts of peptidoglycan (PG), an essential polymer. PG is a giant macromolecule composed of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues cross-linked by short peptides. It determines the shape of bacteria, protects the cell from lysis due to internal osmotic pressure, and also plays a role in cell division. However, PG has not been detected in EBs (14, 20) or RBs (5).Although chlamydiae appear to lack PG, they contain penicillin-binding proteins and are sensitive to antibiotics that inhibit PG synthesis (5, 40). The Chlamydia trachomatis genome contains most of the genes coding for proteins involved in, or associated with, PG synthesis (54). Chlamydial MurA, MurC-Ddl, and the MurC domain of the latter fusion protein are active in vitro and complement Escherichia coli mutants deficient in the respective enzymes (23, 32, 33). Furthermore, proteomic analysis reveals that the murE gene product, which was assigned as UDP-MurNAc-l-Ala-d-Glu:meso-diaminopimelic acid ligase (UDP-MurNAc-l-Ala-d-Glu:meso-A₂pm ligase), is expressed in RBs (52).MurE ligases catalyze the addition of the third amino acid residue to the peptide chain of PG. This residue, generally a diamino acid, is usually meso-A₂pm for gram-negative bacteria and bacilli, and l-lysine for gram-positive bacteria, although other amino acids (for example, l-ornithine, meso-lanthionine, ll-A₂pm, l-diaminobutyric acid, or l-homoserine) occur in certain species (6, 50, 57). In many organisms, the third residue of the peptide chain participates in PG cross-linking; consequently, the MurE enzyme is highly specific for the relevant amino acid so as to avoid incorporation of incorrect amino acids into the macromolecule, which could result in deleterious morphological changes and cell lysis (35). Crystallization of MurE from E. coli (MurE_Ec) has permitted analysis of the structural basis for this high specificity (22). Sequence alignments of different MurE orthologues have also revealed the specific consensus sequences DNPR and D(D/N)P(N/A) located in the binding pockets for meso-A₂pm and l-Lys, respectively (11, 17). Chlamydia trachomatis MurE (MurE_Ct) possesses the DNPR motif, which suggests that it adds meso-A₂pm (17). However, there are no experimental data to confirm this prediction.In this paper we report for the first time the overproduction and purification of MurE_Ct, as well as a detailed investigation of its in vivo and in vitro biochemical properties. These studies contribute to our understanding of the nature and properties of the PG biosynthetic enzymes in chlamydiae and do indeed suggest that MurE_Ct has meso-A₂pm ligase activity.     

The Elucidation of the Structure of Thermotoga maritima Peptidoglycan Reveals Two Novel Types of Cross-link

Thermotoga maritima is a Gram-negative, hyperthermophilic bacterium whose peptidoglycan contains comparable amounts of l- and d-lysine. We have determined the fine structure of this cell-wall polymer. The muropeptides resulting from the digestion of peptidoglycan by mutanolysin were separated by high-performance liquid chromatography and identified by amino acid analysis after acid hydrolysis, dinitrophenylation, enzymatic determination of the configuration of the chiral amino acids, and mass spectrometry. The high-performance liquid chromatography profile contained four main peaks, two monomers, and two dimers, plus a few minor peaks corresponding to anhydro forms. The first monomer was the d-lysine-containing disaccharide-tripeptide in which the d-Glu-d-Lys bond had the unusual γ→ϵ arrangement (GlcNAc-MurNAc-l-Ala-γ-d-Glu-ϵ-d-Lys). The second monomer was the conventional disaccharide-tetrapeptide (GlcNAc-MurNAc-l-Ala-γ-d-Glu-l-Lys-d-Ala). The first dimer contained a disaccharide-l-Ala as the acyl donor cross-linked to the α-amine of d-Lys in a tripeptide acceptor stem with the sequence of the first monomer. In the second dimer, donor and acceptor stems with the sequences of the second and first monomers, respectively, were connected by a d-Ala⁴-α-d-Lys³ cross-link. The cross-linking index was 10 with an average chain length of 30 disaccharide units. The structure of the peptidoglycan of T. maritima revealed for the first time the key role of d-Lys in peptidoglycan synthesis, both as a surrogate of l-Lys or meso-diaminopimelic acid at the third position of peptide stems and in the formation of novel cross-links of the l-Ala¹(α→α)d-Lys³ and d-Ala⁴(α→α)d-Lys³ types.Peptidoglycan (or murein) is a giant macromolecule whose main function is the protection of the cytoplasmic membrane against the internal osmotic pressure. It is composed of alternating residues of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc)² cross-linked by short peptides (1). The composition of the peptide stem in nascent peptidoglycan is l-Ala¹-γ-d-Glu²-X³-d-Ala⁴-d-Ala⁵, where X is most often meso-diaminopimelic acid (meso-A₂pm) or l-lysine in Gram-negative and Gram-positive species, respectively (2, 3). In the mature macromolecule, the last d-Ala residue is removed. Cross-linking of the glycan chains generally occurs between the carboxyl group of d-Ala at position 4 of a donor peptide stem and the side-chain amino group of the diamino acid at position 3 of an acceptor peptide stem (4→3 cross-links). Cross-linking is either direct or through a short peptide bridge such as pentaglycine in Staphylococcus aureus (2, 3). The enzymes for the formation of the 4→3 cross-links are active-site serine dd- transpeptidases that belong to the penicillin-binding protein (PBP) family and are the essential targets of β-lactam antibiotics in pathogenic bacteria (4). Catalysis involves the cleavage of the d-Ala⁴-d-Ala⁵ bond of a donor peptide stem and the formation of an amide bond between the carboxyl of d-Ala⁴ and the side chain amine at the third position of an acceptor stem. Transpeptidases of the ld specificity are active-site cysteine enzymes that were shown to act as surrogates of the PBPs in mutants of Enterococcus faecium resistant to β-lactam antibiotics (5). They cleave the X³-d-Ala⁴ bond of a donor stem peptide to form 3→3 cross-links. This alternate mode of cross-linking is usually marginal, although it has recently been shown to predominate in non-replicative “dormant” forms of Mycobacterium tuberculosis (6).Thermotoga maritima is a Gram-negative, extremely thermophilic bacterium isolated from geothermally heated sea floors by Huber et al. (7). A morphological characteristic is the presence of an outer sheath-like envelope called “toga.” Although the organism has received considerable attention for its biotechnological potential, studies about its peptidoglycan are scarce (8–11), and in particular the fine structure of the macromolecule is still unknown. In their initial work, Huber et al. (7) showed that the composition of its peptidoglycan was unusual for a Gram-negative species, because it contained both isomers of lysine and no A₂pm. Recently, we purified and studied the properties of T. maritima MurE (12); this enzyme is responsible for the addition of the amino acid residue at position 3 of the peptide stem (13, 14). We demonstrated that T. maritima MurE added in vitro l- and d-Lys to UDP-MurNAc-l-Ala-d-Glu. Although l-Lys was added in the usual way, yielding the conventional nucleotide UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys containing a d-Glu(γ→α)l-Lys amide bond, the d-isomer was added in an “upside-down” manner, yielding the novel nucleotide UDP-MurNAc-l-Ala-d-Glu(γ→ϵ)d-Lys. We also showed that the d-Lys-containing nucleotide was not a substrate for T. maritima MurF, the subsequent enzyme in the biosynthetic pathway, whereas this ligase catalyzed the addition of dipeptide d-Ala-d-Ala to the l-Lys-containing tripeptide, yielding the conventional UDP-MurNAc-pentapeptide (12).However, both the l-Lys-containing UDP-MurNAc-pentapeptide and d-Lys-containing UDP-MurNAc-tripeptide were used as substrates by T. maritima MraY with comparable efficiencies in vitro (12). This observation implies that the unusual d-Lys-containing peptide stems are likely to be translocated to the periplasmic face of the cytoplasmic membrane and to participate in peptidoglycan polymerization. Therefore, we have determined here the fine structure of T. maritima peptidoglycan and we have shown that l-Lys- and d-Lys-containing peptide stems are both present in the polymer, the latter being involved in the formation of two novel types of peptidoglycan cross-link.     

Biochemical Characterization of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate ligase (MurE) from Verrucomicrobium spinosum DSM 4136T

Verrucomicrobium spinosum is a Gram-negative bacterium that is related to bacteria from the genus Chlamydia. The bacterium is pathogenic towards Drosophila melanogaster and Caenorhabditis elegans, using a type III secretion system to facilitate pathogenicity. V. spinosum employs the recently discovered l,l-diaminopimelate aminotransferase biosynthetic pathway to generate the bacterial cell wall and protein precursors diaminopimelate and lysine. A survey of the V. spinosum genome provides evidence that the bacterium should be able to synthesize peptidoglycan de novo, since all of the necessary genes are present. The enzyme UDP-N-acetylmuramoyl-l-alanyl-d-glutamate: meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.15) catalyzes a reaction in the cytoplasmic step of peptidoglycan biosynthesis by adding the third amino acid residue to the peptide stem. The murE ortholog from V. spinosum (murE _Vs) was cloned and was shown to possess UDP-MurNAc-l-Ala-d-Glu:meso-2,6-diaminopimelate ligase activity in vivo using functional complementation. In vitro analysis using the purified recombinant enzyme demonstrated that MurE_Vs has a pH optimum of 9.6 and a magnesium optimum of 30 mM. meso-Diaminopimelate was the preferred substrate with a K _m of 17 µM, when compared to other substrates that are structurally related. Sequence alignment and structural analysis using homology modeling suggest that key residues that make up the active site of the enzyme are conserved in MurE_Vs. Our kinetic analysis and structural model of MurE_Vs is consistent with other MurE enzymes from Gram-negative bacteria that have been characterized. To verify that V. spinosum incorporates diaminopimelate into its cell wall, we purified peptidoglycan from a V. spinosum culture; analysis revealed the presence of diaminopimelate, consistent with that of a bona fide peptidoglycan from Gram-negative bacteria.     

Regulation of sulfate uptake by amino acids in cultured tobacco cells

The sulfur requirements of tobacco (Nicotiana tabacum L. var. Xanthi) XD cells grown in chemically defined liquid media can be satisfied by sulfate, thiosulfate, l-cyst(e)ine, l-methionine or glutathione, and somewhat less effectively by d-cyst (e) ine, d-methionine or dl-homocyst (e)ine. Sulfate uptake is inhibited after a 2 hr lag by l-cyst (e)ine, l-methionine, l-homocyst(e)ine or l-isoleucine, but not by any of the other protein amino acids, nor by d-cyst(e)ine. l-cyst(e)ine is neither a competitive nor a non-competitive inhibitor of sulfate uptake. Its action most closely resembles apparent uncompetitive inhibition. Inhibition of sulfate uptake by l-cyst(e)ine can be partially prevented by equimolar l-arginine, l-lysine, l-leucine, l-phenylalanine, l-tyrosine or l-tryptophan, but is little affected by any of the other protein amino acids. The effective amino acids are apparent competitive inhibitors of l-cyst(e)ine uptake after a 2 hr lag. Inhibition of sulfate uptake by l-methionine cannot be prevented, nor can uptake of l-methionine be inhibited by any single protein amino acid. The results suggest the occurrence of negative feedback control of sulfate assimilation by the end products, the sulfur amino acids, in cultured tobacco cells.     

Autolysis of cell walls of Bacillus stearothermophilus B65 and the chemical structure of the peptidoglycan

1. The cell walls of Bacillus stearothermophilus B65 contain glucosamine, muramic acid, alanine, α-diaminopimelic acid (Dap), glutamic acid, aspartic acid, glycine, and serine in the molecular proportions 0.60:0.64:2.30:0.85:1.00:0.11:0.13:0.31. 2. Both d- and l-alanine are present, but glutamic acid and diaminopimelic acid are present only as the d- and meso-isomers respectively. 3. The peptide fragments Ala-Dap, Dap-Ala, and Dap-Ala-Dap have been isolated from a partial acid hydrolysate of the cell walls. 4. The major products of autolysis of the cell wall were d-alanine, a peptide mixture, peptidoglycan material and a peptidoglycan–teichoic acid complex. 5. Separation of the peptide mixture into ten major peptides was achieved by DEAE-Sephadex and paper chromatography, and paper electrophoresis. 6. The structures of these peptides have been determined and they fall into four groups, the individual members of each group differing only in number or position of carboxamide substituents. 7. The structures are I, a tripeptide l-Ala–d-Glu-meso-Dap; II, a pentapeptide made up by the tripeptide (I) linked through the -amino group of its diaminopimelic acid residue to the carboxyterminal of the dipeptide meso-Dap-d-Ala; III, a heptapeptide made up by a similar linkage between the tripeptide (I) and the tetrapeptide l-Ala-d-Glu-meso-Dap-d-Ala; IV, a possible undecapeptide made up by a further tetrapeptide similarly linked to the heptapeptide (III) structure. 8. The structure of the peptidoglycan and the actions of the autolytic enzymes are discussed in terms of these peptide structures.     

Bacteriocin Protein BacL1 of Enterococcus faecalis Targets Cell Division Loci and Specifically Recognizes l-Ala2-Cross-Bridged Peptidoglycan

Bacteriocin 41 (Bac41) is produced from clinical isolates of Enterococcus faecalis and consists of two extracellular proteins, BacL₁ and BacA. We previously reported that BacL₁ protein (595 amino acids, 64.5 kDa) is a bacteriolytic peptidoglycan d-isoglutamyl-l-lysine endopeptidase that induces cell lysis of E. faecalis when an accessory factor, BacA, is copresent. However, the target of BacL₁ remains unknown. In this study, we investigated the targeting specificity of BacL₁. Fluorescence microscopy analysis using fluorescent dye-conjugated recombinant protein demonstrated that BacL₁ specifically localized at the cell division-associated site, including the equatorial ring, division septum, and nascent cell wall, on the cell surface of target E. faecalis cells. This specific targeting was dependent on the triple repeat of the SH3 domain located in the region from amino acid 329 to 590 of BacL₁. Repression of cell growth due to the stationary state of the growth phase or to treatment with bacteriostatic antibiotics rescued bacteria from the bacteriolytic activity of BacL₁ and BacA. The static growth state also abolished the binding and targeting of BacL₁ to the cell division-associated site. Furthermore, the targeting of BacL₁ was detectable among Gram-positive bacteria with an l-Ala-l-Ala-cross-bridging peptidoglycan, including E. faecalis, Streptococcus pyogenes, or Streptococcus pneumoniae, but not among bacteria with alternate peptidoglycan structures, such as Enterococcus faecium, Enterococcus hirae, Staphylococcus aureus, or Listeria monocytogenes. These data suggest that BacL₁ specifically targets the l-Ala-l-Ala-cross-bridged peptidoglycan and potentially lyses the E. faecalis cells during cell division.     

Amino Acid and vitamin requirements of several bacteroides strains

Nutritional studies were performed on nine Bacteroides strains, by use of the methodology and media of anaerobic rumen microbiology. Ristella perfoetens CCI required l-arginine hydrochloride, l-tryptophan, l-leucine, l-histidine hydrochloride, l-cysteine hydrochloride, dl-valine, dl-tyrosine, and the vitamin calcium-d-pantothenate, since scant turbidity developed in media without these nutrients. R. perfoetens was stimulated by glycine, dl-lysine hydrochloride, dl-isoleucine, l-proline, l-glutamic acid, dl-alanine, dl-phenylalanine, dl-methionine, and the vitamins nicotinamide and p-aminobenzoic acid, since maximal turbidity developed more slowly in media without these nutrients than in complete medium. Medium A-23, which was devised for R. perfoetens, contained salts, 0.0002% nicotinamide and calcium d-pantothenate, 0.00001% p-aminobenzoic acid, 0.044% l-tryptophan, 0.09% l-glutamic acid, and 0.1% of the other 13 amino acids listed above. Zuberella clostridiformis and seven strains of R. pseudoinsolita did not require vitamins, and showed no absolute requirement for any one amino acid. Various strains produced maximal turbidity more slowly in media deficient in l-proline, glycine, l-glutamic acid, dl-serine, l-histidine hydrochloride, dl-alanine, or l-cysteine hydrochloride, than in complete medium. These eight strains grew optimally in medium A-23 plus 0.1% dl-serine but without vitamins.     

A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily: PRODUCTION,FUNCTIONAL CHARACTERIZATION,AND STRUCTURE MODELING*

Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of l-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of l-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of l-arginine, l-ornithine, l-2,4-diaminobutyric acid, and l-alanine. d-Lysine is bound 45 times more weakly than its l-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010–1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for l-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed.     

Structural and Functional Characterization of VanG d-Ala:d-Ser Ligase Associated with Vancomycin Resistance in Enterococcus faecalis

d-Alanyl:d-lactate (d-Ala:d-Lac) and d-alanyl:d-serine ligases are key enzymes in vancomycin resistance of Gram-positive cocci. They catalyze a critical step in the synthesis of modified peptidoglycan precursors that are low binding affinity targets for vancomycin. The structure of the d-Ala:d-Lac ligase VanA led to the understanding of the molecular basis for its specificity, but that of d-Ala:d-Ser ligases had not been determined. We have investigated the enzymatic kinetics of the d-Ala:d-Ser ligase VanG from Enterococcus faecalis and solved its crystal structure in complex with ADP. The overall structure of VanG is similar to that of VanA but has significant differences mainly in the N-terminal and central domains. Based on reported mutagenesis data and comparison of the VanG and VanA structures, we show that residues Asp-243, Phe-252, and Arg-324 are molecular determinants for d-Ser selectivity. These residues are conserved in both enzymes and explain why VanA also displays d-Ala:d-Ser ligase activity, albeit with low catalytic efficiency in comparison with VanG. These observations suggest that d-Ala:d-Lac and d-Ala:d-Ser enzymes have evolved from a common ancestral d-Ala:d-X ligase. The crystal structure of VanG showed an unusual interaction between two dimers involving residues of the omega loop that are deeply anchored in the active site. We constructed an octapeptide mimicking the omega loop and found that it selectively inhibits VanG and VanA but not Staphylococcus aureus d-Ala:d-Ala ligase. This study provides additional insight into the molecular evolution of d-Ala:d-X ligases and could contribute to the development of new structure-based inhibitors of vancomycin resistance enzymes.     

Physiology and Substrate Specificity of Two Closely Related Amino Acid Transporters,SerP1 and SerP2, of Lactococcus lactis

The serP1 and serP2 genes found adjacently on the chromosome of Lactococcus lactis strains encode two members of the amino acid-polyamine-organocation (APC) superfamily of secondary transporters that share 61% sequence identity. SerP1 transports l-serine, l-threonine, and l-cysteine with high affinity. Affinity constants (K_m) are in the 20 to 40 μM range. SerP2 is a dl-alanine/dl-serine/glycine transporter. The preferred substrate appears to be dl-alanine for which the affinities were found to be 38 and 20 μM for the d and l isomers, respectively. The common substrate l-serine is a high-affinity substrate of SerP1 and a low-affinity substrate of SerP2 with affinity constants of 18 and 356 μM, respectively. Growth experiments demonstrate that SerP1 is the main l-serine transporter responsible for optimal growth in media containing free amino acids as the sole source of amino acids. SerP2 is able to replace SerP1 in this role only in medium lacking the high-affinity substrates l-alanine and glycine. SerP2 plays an adverse role for the cell by being solely responsible for the uptake of toxic d-serine. The main function of SerP2 is in cell wall biosynthesis through the uptake of d-alanine, an essential precursor in peptidoglycan synthesis. SerP2 has overlapping substrate specificity and shares 42% sequence identity with CycA of Escherichia coli, a transporter whose involvement in peptidoglycan synthesis is well established. No evidence was obtained for a role of SerP1 and SerP2 in the excretion of excess amino acids during growth of L. lactis on protein/peptide-rich media.     

Molecular Basis of Vancomycin Dependence in VanA-Type Staphylococcus aureus VRSA-9

The vancomycin-resistant Staphylococcus aureus VRSA-9 clinical isolate was partially dependent on glycopeptide for growth. The responsible vanA operon had the same organization as that of Tn1546 and was located on a plasmid. The chromosomal d-Ala:d-Ala ligase (ddl) gene had two point mutations that led to Q260K and A283E substitutions, resulting in a 200-fold decrease in enzymatic activity compared to that of the wild-type strain VRSA-6. To gain insight into the mechanism of enzyme impairment, we determined the crystal structure of VRSA-9 Ddl and showed that the A283E mutation induces new ion pair/hydrogen bond interactions, leading to an asymmetric rearrangement of side chains in the dimer interface. The Q260K substitution is located in an exposed external loop and did not induce any significant conformational change. The VRSA-9 strain was susceptible to oxacillin due to synthesis of pentadepsipeptide precursors ending in d-alanyl-d-lactate which are not substrates for the β-lactam-resistant penicillin binding protein PBP2′. Comparison with the partially vancomycin-dependent VRSA-7, whose Ddl is 5-fold less efficient than that of VRSA-9, indicated that the levels of vancomycin dependence and susceptibility to β-lactams correlate with the degree of Ddl impairment. Ddl drug targeting could therefore be an effective strategy against vancomycin-resistant S. aureus.Methicillin-resistant Staphylococcus aureus (MRSA) bacteria that have acquired the vancomycin resistance vanA operon from glycopeptide-resistant enterococci are designated vancomycin-resistant S. aureus (VRSA) (29). Vancomycin acts by binding to the C-terminal acyl-d-Ala-d-Ala of the undecaprenol-diphosphate MurNAc-pentapeptide intermediate and inhibits transglycosylation and transpeptidation reactions in cell wall peptidoglycan polymerization and cross-linking (30). d-Ala-d-Ala is synthesized by the ATP-dependent d-Ala:d-Ala ligase (Ddl) (EC 6.3.2.4) before its incorporation in peptidoglycan precursors (26, 35). VanA-type vancomycin resistance results from the incorporation into peptidoglycan intermediates of a d-alanyl-d-lactate (d-Ala-d-Lac) depsipeptide, synthesized by a d-Ala:d-Lac ligase, which is responsible for diminished binding affinity of glycopeptides for their target. Kinetic analyses of Ddls have established two subsites in the active site for d-Ala binding (24, 27). The reaction mechanism culminates in the transfer of the γ-phosphoryl of ATP to the carboxyl group of d-Ala₁ to produce an acylphosphate and ADP. The acyl carbon atom of the acylphosphate then reacts with the amino group of d-Ala₂ to yield a tetrahedral intermediate. Finally, the intermediate releases phosphate to yield d-Ala-d-Ala.Mutants of Enterococcus faecium (8, 14), Enterococcus faecalis (34), and S. aureus (23) with an impaired Ddl are able to grow because they use the vancomycin resistance pathway for cell wall synthesis. Since resistance is inducible by the drug, these bacteria require the presence of vancomycin in the culture medium for growth. Ddls from vancomycin-dependent enterococci (14) have mutations affecting amino acids highly conserved in the d-Ala:d-Ala ligase superfamily (10). Molecular modeling based on the X-ray structure of Escherichia coli DdlB (11) revealed that all the mutated residues interact directly with one of the substrates of the enzymatic reaction or stabilize the position of critical residues in the active site. However, the degree of enzyme impairment was not evaluated biochemically. Recently, we reported the mechanism of vancomycin dependence in VanA-type S. aureus VRSA-7 and showed that the chromosomal Ddl had the single mutation N308K, which probably affects the binding of the transition-state intermediate, leading to a 1,000-fold decrease in activity relative to that of the wild-type enzyme (23). Glycopeptide-dependent mutants could therefore be considered useful tools to explore structure-activity relationships of the Ddl, which represents an attractive target for designing new drugs. Here we describe the partially vancomycin-dependent VanA-type S. aureus strain VRSA-9 and report the biochemical and structural characterization of its mutated Ddl.     

Amino Acid Isomerization in the Production of l-Phenylalanine from d-Phenylalanine by Bacteria

To establish an advantageous method for the production of l-amino acids, microbial isomerization of d- and dl-amino acids to l-amino acids was studied. Screening experiments on a number of microorganisms showed that cell suspensions of Pseudomonas fluorescens and P. miyamizu were capable of isomerizing d- and dl-phenylalanines to l-phenylalanine. Various conditions suitable for isomerization by these organisms were investigated. Cells grown in a medium containing d-phenylalanine showed highest isomerization activity, and almost completely converted d- or dl-phenylalanine into l-phenylalanine within 24 to 48 hr of incubation. Enzymatic studies on this isomerizing system suggested that the isomerization of d- or dl-phenylalanine is not catalyzed by a single enzyme, “amino acid isomerase,” but the conversion proceeds by a two step system as follows: d-pheylalanine is oxidized to phenylpyruvic acid by d-amino acid oxidase, and the acid is converted to l-phenylalanine by transamination or reductive amination.     

Deficiency in l-Serine Deaminase Interferes with One-Carbon Metabolism and Cell Wall Synthesis in Escherichia coli K-12

Escherichia coli K-12 provided with glucose and a mixture of amino acids depletes l-serine more quickly than any other amino acid even in the presence of ammonium sulfate. A mutant without three 4Fe4S l-serine deaminases (SdaA, SdaB, and TdcG) of E. coli K-12 is unable to do this. The high level of l-serine that accumulates when such a mutant is exposed to amino acid mixtures starves the cells for C₁ units and interferes with cell wall synthesis. We suggest that at high concentrations, l-serine decreases synthesis of UDP-N-acetylmuramate-l-alanine by the murC-encoded ligase, weakening the cell wall and producing misshapen cells and lysis. The inhibition by high l-serine is overcome in several ways: by a large concentration of l-alanine, by overproducing MurC together with a low concentration of l-alanine, and by overproducing FtsW, thus promoting septal assembly and also by overexpression of the glycine cleavage operon. S-Adenosylmethionine reduces lysis and allows an extensive increase in biomass without improving cell division. This suggests that E. coli has a metabolic trigger for cell division. Without that reaction, if no other inhibition occurs, other metabolic functions can continue and cells can elongate and replicate their DNA, reaching at least 180 times their usual length, but cannot divide.The Escherichia coli genome contains three genes, sdaA, sdaB, and tdcG, specifying three very similar 4Fe4S l-serine deaminases. These enzymes are very specific for l-serine for which they have unusually high K_m values (3, 32). Expression of the three genes is regulated so that at least one of the gene products is synthesized under all common growth conditions (25). This suggests an important physiological role for the enzymes. However, why E. coli needs to deaminate l-serine has been a long-standing problem of E. coli physiology, the more so since it cannot use l-serine as the sole carbon source.We showed recently that an E. coli strain devoid of all three l-serine deaminases (l-SDs) loses control over its size, shape, and cell division when faced with complex amino acid mixtures containing l-serine (32). We attributed this to starvation for single-carbon (C₁) units and/or S-adenosylmethionine (SAM). C₁ units are usually made from serine via serine hydroxymethyl transferase (GlyA) or via glycine cleavage (GCV). The l-SD-deficient triple mutant strain is starved for C₁ in the presence of amino acids, because externally provided glycine inhibits GlyA and a very high internal l-serine concentration along with several other amino acids inhibits glycine cleavage. While the parent cell can defend itself by reducing the l-serine level by deamination, this crucial reaction is missing in the ΔsdaA ΔsdaB ΔtdcG triple mutant. We therefore consider these to be “defensive” serine deaminases.The fact that an inability to deaminate l-serine leads to a high concentration of l-serine and inhibition of GlyA is not surprising. However, it is not obvious why a high level of l-serine inhibits cell division and causes swelling, lysis, and filamentation. Serine toxicity due to inhibition of biosynthesis of isoleucine (11) and aromatic amino acids (21) has been reported but is not relevant here, since these amino acids are provided in Casamino Acids.We show here that at high internal concentrations, l-serine also causes problems with peptidoglycan synthesis, thus weakening the cell wall. Peptidoglycan is a polymer of long glycan chains made up of alternating N-acetylglucosamine and N-acetylmuramic acid residues, cross-linked by l-alanyl-γ-d-glutamyl-meso-diaminopimelyl-d-alanine tetrapeptides (1, 28). The glucosamine and muramate residues and the pentapeptide (from which the tetrapeptide is derived) are all synthesized in the cytoplasm and then are exported to be polymerized into extracellular peptidoglycan (2).In this paper, we show that lysis is caused by l-serine interfering with the first step of synthesis of the cross-linking peptide, the addition of l-alanine to uridine diphosphate-N-acetylmuramate. This interference is probably due to a competition between serine and l-alanine for the ligase, MurC, which adds the first l-alanine to UDP-N-acetylmuramate (7, 10, 15). As described here, the weakening of the cell wall by l-serine can be overcome by a variety of methods that reduce the endogenous l-serine pool or counteract the effects of high levels of l-serine.     

Amino Acid Racemization in Pseudomonas putida KT2440

d-Amino acids have been shown to play an increasingly diverse role in bacterial physiology, yet much remains to be learned about their synthesis and catabolism. Here we used the model soil- and rhizosphere-dwelling organism Pseudomonas putida KT2440 to elaborate on the genomics and enzymology of d-amino acid metabolism. P. putida KT2440 catabolized the d-stereoisomers of lysine, phenylalanine, arginine, alanine, and hydroxyproline as the sole carbon and nitrogen sources. With the exception of phenylalanine, each of these amino acids was racemized by P. putida KT2440 enzymes. Three amino acid racemases were identified from a genomic screen, and the enzymes were further characterized in vitro. The putative biosynthetic alanine racemase Alr showed broad substrate specificity, exhibiting measurable racemase activity with 9 of the 19 chiral amino acids. Among these amino acids, activity was the highest with lysine, and the k_cat/K_m values with l- and d-lysine were 3 orders of magnitude greater than the k_cat/K_m values with l- and d-alanine. Conversely, the putative catabolic alanine racemase DadX showed narrow substrate specificity, clearly preferring only the alanine stereoisomers as the substrates. However, DadX did show 6- and 9-fold higher k_cat/K_m values than Alr with l- and d-alanine, respectively. The annotated proline racemase ProR of P. putida KT2440 showed negligible activity with either stereoisomer of the 19 chiral amino acids but exhibited strong epimerization activity with hydroxyproline as the substrate. Comparative genomic analysis revealed differences among pseudomonads with respect to alanine racemase genes that may point to different roles for these genes among closely related species.     

l-Lysine Catabolism Is Controlled by l-Arginine and ArgR in Pseudomonas aeruginosa PAO1

In comparison to other pseudomonads, Pseudomonas aeruginosa grows poorly in l-lysine as a sole source of nutrient. In this study, the ldcA gene (lysine decarboxylase A; PA1818), previously identified as a member of the ArgR regulon of l-arginine metabolism, was found essential for l-lysine catabolism in this organism. LdcA was purified to homogeneity from a recombinant strain of Escherichia coli, and the results of enzyme characterization revealed that this pyridoxal-5-phosphate-dependent decarboxylase takes l-lysine, but not l-arginine, as a substrate. At an optimal pH of 8.5, cooperative substrate activation by l-lysine was depicted from kinetics studies, with calculated K_m and V_max values of 0.73 mM and 2.2 μmole/mg/min, respectively. Contrarily, the ldcA promoter was induced by exogenous l-arginine but not by l-lysine in the wild-type strain PAO1, and the binding of ArgR to this promoter region was demonstrated by electromobility shift assays. This peculiar arginine control on lysine utilization was also noted from uptake experiments in which incorporation of radioactively labeled l-lysine was enhanced in cells grown in the presence of l-arginine but not l-lysine. Rapid growth on l-lysine was detected in a mutant devoid of the main arginine catabolic pathway and with a higher basal level of the intracellular l-arginine pool and hence elevated ArgR-responsive regulons, including ldcA. Growth on l-lysine as a nitrogen source can also be enhanced when the aruH gene encoding an arginine/lysine:pyruvate transaminase was expressed constitutively from plasmids; however, no growth of the ldcA mutant on l-lysine suggests a minor role of this transaminase in l-lysine catabolism. In summary, this study reveals a tight connection of lysine catabolism to the arginine regulatory network, and the lack of lysine-responsive control on lysine uptake and decarboxylation provides an explanation of l-lysine as a poor nutrient for P. aeruginosa.Decarboxylation of amino acids, including lysine, arginine, and glutamate, is important for bacterial survival under low pH (2, 7, 19). Lysine is abundant in the rhizosphere where fluorescent Pseudomonas preferentially resides, and serves as a nitrogen and carbon source to these organisms (28). In microbes, lysine catabolism can be initiated either through monooxygenase, decarboxylase, or transaminase activities. The monooxygenase pathway has been considered the major route for l-lysine utilization in Pseudomonas putida, and davBATD encoding enzymes for the first four steps of the pathway have been characterized (25, 26). In contrast, Pseudomonas aeruginosa cannot use exogenous l-lysine efficiently for growth (5, 24). It has been reported that enzymatic activities for the first two steps of the monooxygenase pathway are not detectable in P. aeruginosa, and no davBA orthologs can be identified from this organism (24, 25).Mutants of P. aeruginosa with improved growth on l-lysine and a high level of lysine decarboxylase activity can be isolated by repeated subcultures in l-lysine (5). This suggests that in P. aeruginosa, l-lysine utilization might be mediated by the lysine decarboxylase pathway with cadaverine and 5-aminovalerate as intermediates (Fig. ​(Fig.1).1). Alternatively, conversion of l-lysine into 5-aminovalerate may also be accomplished by a coupled reaction catalyzed by AruH and AruI. The AruH and AruI enzymes were reported as arginine:pyruvate transaminase and 2-ketoarginine decarboxylase, respectively (36). Interestingly, transamination by AruH using l-lysine as an amino group donor can also be detected in vitro (35). The reaction product α-keto-ɛ-aminohexanonate can potentially be decarboxylated into 5-aminovalerate by AruI, providing an alternative route for lysine degradation.Open in a separate windowFIG. 1.Lysine catabolic pathways. l-lysine decarboxylase pathway is shown at center. Broken arrows represent lysine monooxygenase pathway from P. putida which is not present in P. aeruginosa.In this study, we showed that the lysine decarboxylase pathway is the main route for lysine utilization under arginine control. Expression of the ldcAB operon encoding l-lysine decarboxylase and a putative lysine/cadaverine antiporter was analyzed regarding its response to l-lysine, l-arginine, and the arginine-responsive regulator ArgR. Enzyme characterization was performed to verify the function of LdcA as l-lysine decarboxylase. Arginine control on lysine incorporation was also investigated by genetic studies and uptake experiments. The peculiar role of ArgR controlling arginine and lysine uptake and catabolism provides the explanation for poor growth in lysine, and it implies a higher level of complexity in metabolic networks of pseudomonads.     

Partial Purification and Properties of l-Glutamine d-Fructose 6-Phosphate Amidotransferase from Phaseolus aureus

l-Glutamine d-fructose 6-phosphate amidotransferase (EC 2.6.1.16) was extracted and purified 600-fold by acetone fractionation and diethylaminoethyl cellulose column chromatography from mung bean seeds (Phaseolus aureus). The partially purified enzyme was highly specific for l-glutamine as an amide nitrogen donor, and l-asparagine could not replace it. The enzyme showed a pH optimum in the range of 6.2 to 6.7 in phosphate buffer. Km values of 3.8 mm and 0.5 mm were obtained for d-fructose 6-phosphate and l-glutamine, respectively. The enzyme was competitively inhibited with respect to d-fructose 6-phosphate by uridine diphosphate-N-acetyl-d-glucosamine which had a Ki value of 13 μm. Upon removal of l-glutamine and its replacement by d-fructose 6-phosphate and storage over liquid nitrogen, the enzyme was completely desensitized to inhibition by uridine diphosphate-N-acetyl-d-glucosamine. This indicates that the inhibitor site is distinct from the catalytic site and that uridine diphosphate-N-acetyl-d-glucosamine acts as a feedback inhibitor of the enzyme.     

Structure-Function Analysis of Staphylococcus aureus Amidase Reveals the Determinants of Peptidoglycan Recognition and Cleavage

The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium''s peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 Å resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen.     

Commentary on Urinary l-erythro-β-hydroxyasparagine: a novel serine racemase inhibitor and substrate of the Zn2+-dependent d-serine dehydratase

The analysis of the urine contents can be informative of physiological homoeostasis, and it has been speculated that the levels of urinary d-serine (d-ser) could inform about neurological and renal disorders. By analysing the levels of urinary d-ser using a d-ser dehydratase (DSD) enzyme, Ito et al. (Biosci. Rep.(2021) 41, BSR20210260) have described abundant levels of l-erythro-β-hydroxyasparagine (l-β-EHAsn), a non-proteogenic amino acid which is also a newly described substrate for DSD. The data presented support the endogenous production l-β-EHAsn, with its concentration significantly correlating with the concentration of creatinine in urine. Taken together, these results could raise speculations that l-β-EHAsn might have unexplored important biological roles. It has been demonstrated that l-β-EHAsn also inhibits serine racemase with K_i values (40 μM) similar to its concentration in urine (50 μM). Given that serine racemase is the enzyme involved in the synthesis of d-ser, and l-β-EHAsn is also a substrate for DSD, further investigations could verify if this amino acid would be involved in the metabolic regulation of pathways involving d-ser.     

A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated d-Asp Cross-bridge,Identified in Lactobacillus casei Bacteriophage Endolysins

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-l-alanine amidase, whereas Lc-Lys-2 is a γ-d-glutamyl-l-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with d-Ala⁴→d-Asx-l-Lys³ in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting d-Ala⁴→l-Ala-(l-Ala/l-Ser)-l-Lys³; moreover, they do not lyse the L. lactis mutant containing only the nonamidated d-Asp cross-bridge, i.e. d-Ala⁴→d-Asp-l-Lys³. In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 l-Lys³-d-Asn-l-Lys³ bridges replacing the wild-type 4→3 d-Ala⁴-d-Asn-l-Lys³ bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly d-Asn but not PG with only the nonamidated d-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the d-Asn interpeptide bridge of PG.