Identification and characterization of the metal ion-dependent L-alanoyl-D-glutamate peptidase encoded by bacteriophage T5 Galina V Mikoulinskaia1, Irina V Odinokova2, Andrei A Zimin3, Valentina Ya Lysanskaya3, Sergei A Feofanov1 and Olga A Stepnaya3 Branch of Shemyakin & Ovchinnikov’s Institute of Bioorganic Chemistry RAS, Pushchino, Moscow, Russia Institute of Theoretical and Experimental Biophysics RAS, Pushchino, Moscow, Russia Skryabin’s Institute of Biochemistry and Physiology of Microorganisms RAS, Pushchino, Moscow, Russia Keywords bacteriophage T5; endolysin; Gram-negative; holin; L-alanoyl-D-glutamate peptidase Correspondence G V Mikoulinskaia, Branch of Shemyakin & Ovchinnikov’s Institute of Bioorganic Chemistry RAS, Prospekt Nauki, 6, Pushchino, Moscow Region 142290, Russia Fax: +7 4967 330527 Tel: +7 4967 731780 E-mail: mikulinskaya@fibkh.serpukhov.su (Received 14 August 2009, revised 14 October 2009, accepted 16 October 2009) doi:10.1111/j.1742-4658.2009.07443.x Although bacteriophage T5 is known to have lytic proteins for cell wall hydrolysis and phage progeny escape, their activities are still unknown This is the first report on the cloning, expression and biochemical characterization of a bacteriophage T5 lytic hydrolase The endolysin-encoding lys gene of virulent coliphage T5 was cloned in Escherichia coli cells, and an electrophoretically homogeneous product of this gene was obtained with a high yield (78% of total activity) The protein purified was shown to be an l-alanoyl-d-glutamate peptidase The enzyme demonstrated maximal activity in diluted buffers (25–50 mm) at pH 8.5 The enzyme was strongly inhibited by EDTA and BAPTA, and fully reactivated by calcium ⁄ manganese chlorides It was found that, along with E coli peptidoglycan, peptidase of bacteriophage T5 can lyse peptidoglycans of other Gram-negative microorganisms (Pectobacterium carotovorum, Pseudomonas putida, Proteus vulgaris, and Proteus mirabilis) This endolysin is the first example of an l-alanoyl-d-glutamate peptidase in a virulent phage infecting Gramnegative bacteria There are, however, a great many sequences in databases that are highly similar to that of bacteriophage T5 hydrolase, indicating a wide distribution of endolytic l-alanoyl-d-glutamate peptidases The article discusses how an enzyme with such substrate specificity could be fixed in the process of evolution Introduction There are two ways by which phages lyse host cells They can either cause ‘lysis from without’ – which takes place when the cell is being infected – or induce ‘lysis from within’, which occurs when the phage progeny escape the host cell There can be at least two evolutionary strategies for lysis from within [1,2] The phages whose genome is dsDNA (like lambda phage) usually have a two-protein lysis system: this consists of endolysin, which destroys the bacterial cell wall, and holin, a hydrophobic protein providing access of endolysin to the substrate (peptidoglycan of the cell wall) by forming lesions in the inner membrane Endolysins are proteins with various muralytic activities; as a rule, they are initially accumulated in the cytoplasm before lysis, which is eventually triggered by holin However, some endolysins have an N-terminal translocation domain, and will therefore accumulate in the periplasm; in these cases, holin is not necessary, and lysis will be mediated by the Sec system of the host cell [3] Abbreviations BAPTA, 1,2-bis-(O-aminophenoxy)ethane-N,N,N¢,N¢-tetraacetic acid; DNF, 2,4-dinitrophenyl FEBS Journal 276 (2009) 7329–7342 ª 2009 The Authors Journal compilation ª 2009 FEBS 7329 L-Alanoyl-D-glutamate peptidase of bacteriophage T5 G V Mikoulinskaia et al Endolysins are divided into five classes according to the type of bonds that they cleave in peptidoglycans The classes are as follows: (a) lysozyme-like muramidases, which hydrolyze the glycoside bond between N-acetylmuramic acid and N-acetylglucosamine; (b) lytic transglycosylases, which attack the same bonds as muramidases but additionally catalyze the intramolecular transfer of the O-muramylic residue to the C6 hydroxyl; (c) N-acetyl-b-d-glucosaminidases, which hydrolyze the glycoside bond between N-acetylglucosamine and N-acetylmuramic acid; (d) N-acetylmuramyl-l-alanine amidases, which hydrolyze bonds between N-acetylmuramic acid and l-alanine; and (e) peptidases, which hydrolyze peptide bonds [4,5] Some endolysins have multiple activities: for example, endolysin of bacteriophage B30 is both a muramidase and an endopeptidase [6] Most endolysins have two functional domains: an enzymatically active N-terminal domain, and a C-terminal domain, which is responsible for recognition of the substrate (peptidoglycan) and determines the specificity of the enzyme [4] The antibacterial effect of endolysins is not always associated with their enzymatic activity For example, endolysin of bacteriophage T4 has four amphipathic C-terminal a-helices, whose basic (positively charged) amino acids interact with negatively charged components of the outer membrane, and thus cause its degradation [7] Endolysins may be acid or alkaline proteins with diverse pH and ionic strength optima The spectrum of antibacterial action of endolysins is determined by the enzyme type, composition of the cell wall components of the target bacterium, and configuration of the substrate (peptidoglycan) The range of bacteria sensitive to lysis may be wide, as in the case of endolysin of Lactobacillus helveticus temperate bacteriophage u0303 [8] There are also counterexamples: endolysin of bacteriophage u3626 specifically degrades only cell walls of the bacterium Clostridium perfringens [9] The selectivity of endolysin of bcateriophage C1 for streptococci allows one to forecast its use as a agent against these bacteria colonizing the mucous epithelium of the upper air passages [10] The specificity of another phage endolysin for Bacillus anthracis suggests that it has potential for application in diagnostics [11] Also, endolysins may act synergistically with antibiotics [12] All this makes endolysins potential candidates as antibacterial drugs The lysozyme of bacteriophage T4, for example, was successfully used to protect potato from Bacillus subtilis [13] The system of lytic proteins of bacteriophage T5 was first found in the process of sequencing of the early region of its genome, which is now deposited in the GenBank database (GenBank accession no AY509815) [14] Analysis of the phage’s primary DNA sequence revealed hol and lys genes, which turned out to be located in the same operon under a common promoter A search of protein databases for homologs of hol and lys gene products provided evidence that they are probably involved in the process of host cell lysis [14] The objectives of the present work included preliminary biochemical characterization of the novel endolysin of bacteriophage T5, determination of the peptidoglycan cleavage site, and analysis of endolysin specificity for bacterial species Results The hol and lys genes are located in the early C region of the bacteriophage T5 genome under a common promoter (Fig 1) Endolysin (GenBank accession no AAS19387; UniProt accession no Q6QGP7) is a polypeptide that consists of 137 amino acids (expected molecular mass of 15.266 kDa) and has a calculated pK value of 8.32 Gene cloning and protein purification The lys gene was cloned into plasmid vector pET3a, and the electrophoretic analysis showed that the Escherichia coli clones selected induced synthesis of a protein product of the expected size (about 15 kDa) The sequence of the lys gene in the plasmid was checked by sequencing both DNA strands Inside the cell, all of the protein product of lys gene was in a soluble state: after centrifugation of cellular homogenate, the pellet fraction did not contain the target protein, and this was confirmed electrophoretically (data not shown) Fig Organization of the bacteriophage T5 gene locus that carries genes coding for lytic proteins Flags indicate promoters; T-like signs indicate transcription terminators Arrows indicate open reading frames Numerals on the sides indicate the distance from the beginning of the genome in bp 7330 FEBS Journal 276 (2009) 7329–7342 ª 2009 The Authors Journal compilation ª 2009 FEBS L-Alanoyl-D-glutamate G V Mikoulinskaia et al peptidase of bacteriophage T5 Enzyme activity, U*10–3 Fig SDS ⁄ PAGE analysis of fractions from bacteriophage T5 endolysin purification steps Lanes: 1, molecular weight markers; 2, crude extract; 3, chromatography on Toyopearl 650M; 4, chromatography on phosphocellulose Wells 2–4 were loaded with lg of total protein each 0 50 100 150 200 250 Buffer concentration, mM Endolysin of bacteriophage T5 was extracted from the cells of the producer strain and purified to an electrophoretically homogeneous state by ion exchange chromatography on two consecutive columns, Toyopearl DEAE 650M and phosphocellulose (Fig 2; Table 1) The activity of the final preparation was calculated to be 8.38 · 103 units per mg of protein Enzyme activity, U*10–3 Storage It was shown that, when stored in Tris ⁄ HCl buffer in the presence of mm EDTA at °C, the protein remained stable BSA (1 mgỈmL)1), 20% glycerol or 0.1% Triton X-100 had a positive effect on the protein storage: the enzyme activity did not decrease over several months 5.5 6.0 6.5 7.0 7.5 pH 8.0 8.5 9.0 9.5 Optimal conditions for enzyme functioning Fig (A) The effect of buffer concentration on enzyme activity The activity was measured in Tris ⁄ HCl (pH 8.2) containing 0.1% Triton X-100; the reaction was initiated by the addition of 0.015 lg of enzyme (B) Effect of pH on enzyme activity The activity was measured in 50 mM Tris ⁄ HCl buffer containing 0.1% Triton X-100; the reaction was initiated by the addition of 0.015 lg of enzyme The activity of bacteriophage T5 endolysin was found to depend strongly on the concentration of the buffer (Fig 3A) The enzyme showed maximal activity in diluted Tris ⁄ HCl buffers (25–50 mm) With increasing pH, the enzyme activity grew, and it reached its maximum at a pH of about 8.5 (Fig 3B) Other components of the buffer also affected the lytic ability of bacteriophage T5 endolysin We measured enzyme activity at two different pH values under the chosen standard conditions, and compared it with the activity observed upon the addition of various substances As can be seen in Fig 4, the enzyme is metal ion-dependent, because it was inhibited by EDTA This was confirmed by the experiments in which a pure peptidoglycan was used as the substrate High buffer concentrations and high divalent metal ion concentrations inhibited the enzyme activity Interestingly, the Table Purification of recombinant endolysin of bacteriophage T5 Specific activity values represent the mean ± standard deviation (n ‡ 3) Fraction volume (mL) Crude extract Chromatography on Toyopearl 650M Chromatography on phosphocellulose Protein concentration (mgỈmL)1) Specific activity (mg)1) Total activity (U) Purification factor (fold) Yield (%) 10.0 33.0 6.0 0.8 1.7 1830 ± 58 4000 ± 105 8380 ± 140 109 800 105 600 85 480 2.2 4.6 100 96 78 FEBS Journal 276 (2009) 7329–7342 ª 2009 The Authors Journal compilation ª 2009 FEBS 7331 L-Alanoyl-D-glutamate peptidase of bacteriophage T5 G V Mikoulinskaia et al Fig The effect of reaction mixture composition on enzyme activity Standard conditions: 50 mM Tris ⁄ HCl containing 0.1% Triton X-100 K-Pi is 50 mM potassium phosphate buffer containing 0.1% Triton X-100 activity depended on the type of buffer used: in the potassium phosphate buffer, the enzyme worked much worse than in the Tris ⁄ HCl buffer, especially at high pH values Perhaps the catalytic function of endolysin requires the presence of ions of some metals whose phosphate salts are poorly soluble We supposed that the cations needed for endolysin functioning might be Ca2+, Mg2+, or Zn2+: these metals form low-solubility phosphate complexes, whose solubility drops further upon alkalization of the medium It is also significant that the enzyme is much more active in the presence of 0.1% Triton X-100 Caldentey and Bamford, [15] whose work suggested to us the idea of using this soft nonionic detergent, considered that it enhanced endolysin activity through action on the cell wall (Triton X-100 probably makes peptidoglycan more accessible or facilitates enzyme binding) We also explored the effects of various cations on the storage of endolysin The presence of 10 mm Mg2+, Ca2+or Mn2+ in the storage medium was shown to have no effect on the enzyme activity Other divalent (Zn2+ and Cu2+) and trivalent (Fe3+ and Cr3+) cations, at a concentration of 10 mm, were found to completely inactivate the enzyme immediately after addition to the storage medium To determine which cations are necessary for the enzyme to function, we used 0.1 mm metal chelators with different affinity for metal ions: EDTA, which binds many cations; 1,2-bis-(O-aminophenoxy)ethaneN,N,N¢,N¢-tetraacetic acid (BAPTA), which selectively binds Ca2+ and, to a lesser extent, Mg2+, Zn2+ [16], and 7332 Fe2+ [17]; 1,10-phenanthroline, which has high affinities for Zn2+, Ni2+, Co2+, and Cd2+, and which is rather effective in binding Mn2+, but is ineffective in binding Mg2+ and Ca2+ [18,19]; and deferoxamine (desferrioxamine) and Tiron (4,5-dihydroxy-1,3-benzene-disulfonic acid), both of which are chelators of Fe3+ EDTA (a broad-spectrum chelator) and BAPTA (a specific chelator for Ca2+) completely inhibited the activity of endolysin at a concentration of 0.1 mm, whereas 0.1 mm 1,10-phenanthroline did not affect the enzyme activity – although the binding constants of phenanthroline for Zn2+ and of BAPTA for Ca2+ are very close [19] A 1-day incubation of the protein in 10 mm 1,10-phenanthroline did not decrease its activity, in contrast to what was observed with 10 mm EDTA or BAPTA It should also be noted that, in the absence of chelators, 0.5 mm ZnCl2 completely inactivated the enzyme We tried to restore activity of the enzyme inhibited with 0.1 mm EDTA or BAPTA by adding various salts at concentrations of 0.1–1 mm (zinc, magnesium, manganese and calcium chlorides) Among those salts, only calcium and manganese chlorides were found to restore the enzyme activity completely (Table 2) Zinc chloride and, in the case of EDTA, magnesium chloride at low (0.1–0.25 mm) concentrations partially restored enzyme activity, probably because of binding of the chelator Increasing the concentration of magnesium or zinc chloride to 0.5 mm resulted in inhibition of the enzyme There was no inhibition of enzyme activity in the presence of Tiron and deferoxamine; moreover, deferoxamine even enhanced the activity by 25%, probably by chelating some endogenous cations that could compete with Ca2+ and thus affect enzyme activity Classification of the enzyme by the type of bond hydrolyzed To determine whether the enzyme studied was a glycosyl hydrolase, peptidase, or amidase, we compared its hydrolytic action with that of egg white lysozyme To this, we analyzed the quantity of reducing and amino groups released after peptidoglycan hydrolysis (to exclude the effect of pre-existing groups, peptidoglycan was either acetylated or reduced prior to hydrolysis) The results of the assay are presented in Table Pre-acetylation of free amino groups in peptidoglycan practically eliminates staining for them in the sample hydrolyzed by egg white lysozyme This is not surprising, because this enzyme is a glycosyl hydrolase, not a peptidase In contrast, the samples treated with FEBS Journal 276 (2009) 7329–7342 ª 2009 The Authors Journal compilation ª 2009 FEBS L-Alanoyl-D-glutamate G V Mikoulinskaia et al peptidase of bacteriophage T5 Table Restoration of lytic activity of the enzyme by cations after its inhibition with EDTA or BAPTA The activity was measured turbidimetrically (see Experimental procedures) Before measurements, 0.1 mM EDTA or BAPTA and enzyme (0.015 lg) were added to the cell suspension The reaction was initiated by the addition of a chloride of the corresponding metal The activity was calculated as a percentage of the initial activity, measured in the absence of chelators Values represent the mean ± standard deviation (n ‡ 3) ND, not determined Concentration of the metal ion (mM) Control Zn2+ Mg2+ Mn2+ Ca2+ Relative activity (%) 0.000 0.110 0.125 0.250 0.500 0.110 0.125 0.250 0.500 0.110 0.125 0.250 0.500 1.000 0.110 0.125 0.250 0.500 1.000 2.8 51.5 53.7 31.32 10.8 11.7 14.1 26.4 6.5 106.6 116.5 111.5 91.9 68.1 120.5 117.6 84.6 79.5 69.8 EDTA BAPTA ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.2 4.1 4.5 1.7 0.8 0.8 1.0 2.1 0.3 2.9 5.4 4.9 5.7 5.7 13.6 8.3 6.9 6.0 10.8 3.2 ± 47.0 ± 42.9 ± 21.9 ± 5.5 ± ND 1.1 ± 4.2 ± 3.8 ± 105.6 ± 106.9 ± 122.9 ± 101.1 ± 66.2 ± 92.1 ± 103.0 ± 101.6 ± 95.8 ± 90.8 ± 0.2 3.2 3.0 1.8 0.5 0.2 0.3 0.3 1.2 1.7 8.4 7.5 5.4 7.8 9.5 8.7 6.3 10.0 Table Release of amino and reducing groups during enzymatic hydrolysis of E coli peptidoglycan Values represent the mean ± standard deviation (n ‡ 3) Fig Analysis of amino acids released by peptidase of bacteriophage T5 TLC was performed on Kieselgel 60 F254 plates as described in Experimental procedures Lanes: 1, DNF–alanine (Sigma); 2, DNF–glutamic acid (Sigma); 3, control peptidoglycan; 4, peptidoglycan hydrolyzed by bacteriophage T5 peptidase; 5, peptidoglycan hydrolyzed by egg white lysozyme The thin lines at the bottom and the top indicate the initial and final eluent fronts, respectively Table Content of Glu relative to Ala in the samples of enzymatically hydrolyzed peptidoglycan of Ps putida The contents of individual amino acids were determined after acidic hydrolysis of peptidoglycan samples using an amino acid analyzer (see Experimental procedures) Peptidoglycan Substrate Enzyme Reducing groups (nmolỈmg)1) Acetylated peptidoglycan Reduced peptidoglycan T5 endolysin Egg white lysozyme T5 endolysin Egg white lysozyme 23.06 36.09 19.02 77.04 ± ± ± ± 2.8 2.9 1.6 5.9 Amino groups (nmolỈmg)1) 229.2 15.7 550.6 382.0 ± ± ± ± 13.3 0.6 27.8 22.1 bacteriophage T5 endolysin show the appearance of free amino groups, suggesting that the enzyme has a peptidase activity The peptide subunit of E coli peptidoglycan is l-alanoyl-d-glutamyl-l-diaminopimelinoyl-d-alanoyl-dalanine [20], and about one-third of the total number of subunits are involved in the formation of intersubunit cross-bridges between diaminopimelic acid and d-alanine [21] A TLC analysis of ether-extracted 2,4-dinitrophenyl (DNF)–amino acids (see Experi- Component Untreated Treated with egg white lysozyme Ala Glu 1.00 0.57 1.00 0.54 Treated with bacteriophage T5 endolysin 1.00 0.17 mental procedures) showed the presence of DNF– glutamic acid in the bacteriophage T5 hydrolysate (Fig 5) Accordingly, as revealed with an amino acid analyzer, the material remaining after extraction showed about 70% loss in the glutamic acid content (Table 4) Thus, we can conclude that the enzyme studied hydrolyzes the bond between l-alanine and d-glutamic acid; that is, it is an l-alanoyl-d-glutamate peptidase FEBS Journal 276 (2009) 7329–7342 ª 2009 The Authors Journal compilation ª 2009 FEBS 7333 L-Alanoyl-D-glutamate peptidase of bacteriophage T5 G V Mikoulinskaia et al Amino acid sequence analysis A psi-blast analysis of bacteriophage T5 l-alanoyld-glutamate peptidase showed that there are many similar proteins, of both phage and bacterial origin (217 hits after the third iteration) The best hits with low E-values ( 10)45–10)30) were sequences of pro- 100 Phage ST64T 79 Phage PS3 83 Proteus mirabilis Yersinia bercovieri 53 88 Phage RB43 Phage T5 100 Phage EPS7 Haemophilus influenzae 20 Brevundimonas sp Phage phiEcoM 4 Chloroherpeton thalassium 17 Aliivibrio salmonicida 100 96 teins from Gram-negative bacteria of the genera Yersinia, Shigella, Escherichia, Photorhabdus, Providencia, Agrobacterium, Proteus, Serratia, Hahella, Haemophilus, Chromobacterium, Vibrio, Vibrionales, Aliivibrio, Brevundimonas, Erwinia, and Bordetella, as well as bacteriophage proteins In the E-value region of 10)27– 10)20, one can find protein sequences of Gram-positive bacteria On the basis of multiple alignment of 53 sequences, which were selected among 95 of the best hits after removing repetitions, a phylogenetic tree was constructed under conditions of minimal evolution using the program mega v 4.0 (Fig 6) On this tree, the proteins of enterobacteria and close species, as well as their bacteriophages, form a branch separated from the branches of Gram-positive bacteria and their bacteriophages Figure shows an alignment of seven sequences constructed with the program clustalx The sequences belong to the branch of proteins that Vibrio fischeri Phage Xp15 Bordetella bronchiseptica 100 Bordetella parapertussis 37 Photorhabdus asymbiotica 29 Erwinia tasmaniensis Providencia stuartii 36 19 Chromobacterium violaceum Yersinia enterocolitica 85 Yersinia mollaretii 84 Yersinia pseudotuberculosis 95 Yersinia pestis Pestoides F Proteus penneri Vibrio splendidus 22 99 Vibrionales bacterium Phage PY100 78 Yersinia frederiksenii Agrobacterium tumefaciens Hahella chejuensis 50 20 Phage phiJL001 100 Phage RB49 26 Phage Phi1 Serratia proteamaculans 36 Phage phiP27 70 29 Shigella boydii Escherichia coli 99 Phage A500 87 99 Listeria innocua 40 Phage A006 17 Phage P35 Phage SPO1 37 Exiguobacterium sibiricum 88 47 90 Geobacillus sp Geobacillus kaustophilus 41 Anoxybacillus flavithermus Clostridium acetobutylicum 15 Exiguobacterium sp Bacillus cereus 73 Phage B025 79 Listeria monocytogenes 87 0.1 7334 99 Phage A118 Fig Phylogenetic tree of amino acid sequences of bacterial and phage endolysins constructed by the minimum evolution method Numbers at the nodes represent the bootstrap values with 500 replications The scale bar indicates an evolutionary distance of 0.1 amino acid substitutions per site Bootstrap values are indicated above or below the branches GenBank accession numbers are as follows: bacteriophage ST64T, NP_720320.1; bacteriophage PS3, CAA09701.1; P mirabilis, YP_002151708.1; Y bercovieri, ZP_ 00822433.1; bacteriophage RB43, YP_239135.1; bacteriophage T5, YP_006868.1; bacteriophage EPS7, YP_001836966.1; Haemophilus influenzae, YP_248988.1; Brevundimonas sp., YP_002588905.1; bacteriophage phiEcoM, YP_001595416.1; Chloroherpeton thalassium, YP_001995193.1; Aliivibrio salmonicida, YP_002262290.1; Vibrio fischeri, YP_205400.1; bacteriophage Xp15, YP_239293.1; Bordetella bronchiseptica, NP_889694.1; Bordetella parapertussis, NP_885037.1; Photorhabdus asymbiotica, CAR67777.1; Erwinia tasmaniensis, YP_001907932.1; Providencia stuartii, ZP_02961079.1; Chromobacterium violaceum, NP_903215.1; Yersinia enterocolitica, AAT90759.1; Yersinia mollaretii, ZP_00825275.1; Yersinia pseudotuberculosis, YP_001399405.1; Yersinia pestis pestoides F, YP_ 001161675.1; Proteus penneri, ZP_03801884.1; Vibrio splendidus, ZP_00991905.1; Vibrionales bacterium, ZP_01814817.1; bacteriophage PY100, CAJ28446.1; Yersinia frederiksenii, ZP_00828831.1; Agrobacterium tumefaciens, NP_353494.2; Hahella chejuensis, YP_ 435691.1; bacteriophage phiJL001, YP_224014.1; bacteriophage RB49, NP_891673.1; bacteriophage Phi1, YP_001469446.1; Serratia proteamaculans, YP_001471697.1; bacteriophage phiP27, NP_ 543082.1; Shigella boydii, YP_001880486.1; E coli, ZP_03049236 1; bacteriophage A500, YP_001468411.1; Listeria innocua, NP_ 469473.1; bacteriophage A006, YP_001468860.1; bacteriophage P35, YP_001468812.1; bacteriophage SPO1, YP_002300379.1; Exiguobacterium sibiricum, YP_001814297.1; Geobacillus sp., ZP_ 02914525.1; Geobacillus kaustophilus, YP_145852.1; Anoxybacillus flavithermus, YP_002315045.1; Clostridium acetobutylicum, NP_ 347645.1; Exiguobacterium sp., ZP_02992216.1; Bacillus cereus, YP_002446097.1; bacteriophage B025, YP_001468664.1; L monocytogenes, ZP_03669234.1; bacteriophage A118, NP_463486.1 FEBS Journal 276 (2009) 7329–7342 ª 2009 The Authors Journal compilation ª 2009 FEBS L-Alanoyl-D-glutamate G V Mikoulinskaia et al are the closest to bacteriophage T5 peptidase; they are encoded by the genomes of bacteriophages ST64T, PS3, phiEcoM, and RB43, and the bacteria Yersinia bercovieri and Proteus mirabilis In multialignment, we did not use the bacteriophage EPS7 protein sequence, which is the most similar to the sequence of bacteriophage T5 peptidase, because it seems to be shortened The high similarity of two bacterial proteins (from Y bercovieri and P mirabilis) with the phage proteins could indicate their coding by prophages As a result of the analysis, we found the amino acid sequence motifs HXXXXXXD and DXXH, which are typical for peptidases of the C subfamily of family M15 (http://merops.sanger.ac.uk/) The former sequence is a part of the phage consensus sequence SK(R)HI(L,M)T(S)GD(N)AI(V,L)DI(L,F)I(L,A,Y),P, consisting of 13 amino acids, six of which are identical and the others of which are highly conserved Testing the lytic ability of endolysin on heterologous microorganisms To assess the spectrum of bacteriolytic action of bacteriophage T5 endolysin, we tested its ability to hydrolyze various substrates prepared from the cells of selected bacteria according to a standard protocol (Table 5) Among the bacteria selected were Grampositive and Gram-negative species, which differed in the structure of peptidoglycan and the composition of the cell wall All of the tested Gram-negative bacteria were subjected to rapid lysis Among the Gram-positive cells peptidase of bacteriophage T5 examined, only B subtilis cells were lysed relatively well; however, the rate of their lysis was about 104-fold lower than that of Gram-negative cells In Listeria cells, the rate of lysis was an order of magnitude lower than that in bacilli (Table 5) The rest of the Grampositive cells were not lysed The specificity of bacteriophage T5 endolysin towards Gram-negative bacteria suggests that it might be used as a selective antibacterial agent However, to make peptidoglycan accessible to the enzyme, it would be necessary to perturb the outer cell membrane We therefore used polymyxin B, an antibiotic that can bind phospholipids of the outer membrane and destroy membrane integrity, as a putative destructive agent Figure shows that, at a concentration of 40 lgỈmL)1, polymyxin B inhibited the growth of cells but did not lyse them (the attenuance of the cell mixture did not decrease) At the same time, endolysin with polymyxin B lysed the cells completely The nontreated and endolysin-treated E coli cells grew well on a nutrient agar medium, forming a dense bacterial lawn (Fig 8A,D); treatment of cells with polymyxin B resulted in difficulties in cell growth (Fig 8B); and combined endolysin ⁄ polymyxin B treatment led to complete lysis of all living cells – there was no further growth (Fig 8C) Discussion In this study, we cloned the gene of a novel enzyme and then purified and characterized this enzyme, which turned out to be endolysin of bacteriophage T5, a component of the phage cell lysis system Our experi- Fig Multiple alignment of protein sequences of bacteriophages T5, ST64T, PS3d, RB43, and phiEcoM, and the bacteria Y bercovieri and P mirabilis, constructed using the program CLUSTAL X Amino acids common to all the sequences are marked in gray; conservative amino acids, which participate in metal ion binding and catalysis, are marked in black FEBS Journal 276 (2009) 7329–7342 ª 2009 The Authors Journal compilation ª 2009 FEBS 7335 L-Alanoyl-D-glutamate peptidase of bacteriophage T5 G V Mikoulinskaia et al ments have shown that the protein is a metal iondependent l-alanoyl-d-glutamate peptidase The enzyme is strongly inhibited by EDTA and BAPTA, and completely reactivated by Ca2+ and Mn2+ Considering BAPTA as a specific chelator of Ca2+, we calculated the concentration of free Ca2+ in the medium, using the program cabuf, and concluded that, to cleave peptidoglycan, the enzyme would require free Ca2+ at a concentration of 0.05–0.4 mm Probably, in vitro, Ca2+ can be replaced with Mn2+ It is known that bacteriophage T5 requires 0.1 mm Ca2+ to produce phage progeny in E coli cells [22] Calcium ions play a key role at certain stages of phage development: upon infection, when the phage DNA penetrates into cells [22]; and during the synthesis of phage RNA [23] and proteins [24] Interestingly, the enzyme that is directly involved in the final stage of development (lysis) is also activated by the same concentrations of Ca2+ There is another example of such activation: aminopeptidase A (EC 3.4.11.7), a Zn2+containing enzyme (gluzincin) of metallopeptidase family M1, is also activated by Ca2+ and can be reactivated by Ca2+ and Mn2+ [25] The Ca2+, which binds to the enzyme through aspartic acids [26,27], contributes to its substrate specificity: it forms a bridge between an aspartic acid of the enzyme and an acidic N-terminal amino acid of the substrate [26] The affinity of bacteriophage T5 endolysin for d-glutamic acid of peptidoglycan and the ability of endolysin to be activated by Ca2+ may be related as well On the basis of data on enzyme metal ion dependence, we can include l-alanoyl-d-glutamate peptidase of bacteriophage T5 in the sub-subclass of metalloendopeptidases [EC 3.4.24 (probable); the enzyme can be listed in EC after publication of evidence that it catalyzes this reac- Table The effect of bacteriophage T5 peptidase on heterologous microorganisms The rate of cell lysis was measured turbidimetrically (see Experimental procedures), using autoclaved bacterial cells as a susbstrate Values represent the mean ± standard deviation (n ‡ 3) Organism Relative rate of lysis (mg)1 enzyme) E coli K-12 Pe carotovorum Ps putida P vulgaris P mirabilis B subtilis L monocytogenes S aureus C xerosis M luteus (1.12 ± 0.12) · (1.01 ± 0.15) · (1.35 ± 0.18) · (1.19 ± 0.20) · (1.04 ± 0.15) · 0.48 ± 0.05 0.016 ± 0.001 0.0 0.0 0.0 7336 104 104 104 104 104 A B C D Fig Analysis of E coli cell viability after endolysin action Fragments of plates containing cells preincubated with: (A) pure endolysin (40 lgỈmL)1); (B) polymyxin B (40 lgỈmL)1); (C) polymyxin B (40 lgỈmL)1) and pure endolysin (40 lgỈmL)1); and (D) control cells tion)] The analysis of the primary amino acid sequence of bacteriophage T5 peptidase revealed conserved amino acids (His66, Asp73, Asp130, and His133) that are typical for metallopeptidases of the M15 family and take part in the binding of the metal ion in the process of catalysis [28] This is the first example of an l-alanoyl-d-glutamate peptidase to be found in a virulent phage infecting Gram-negative bacteria Enzymes of this class, Ply118 and Ply500, were first found in two temperate phages, A118 and A500, which infect Gram-positive rods of the Listeria genus [29] Since then, there has been only one l-alanoyl-d-glutamate peptidase – from the Grampositive bacteria B subtilis – whose existence has been proved biochemically [30] It is interesting that Listeria and Bacillus are not close relatives of E coli, but their peptidoglycan is also of the A1c type Ply118 and Ply500 show rather high substrate specificities: apart from Listeria, they affect only three species of the Bacillus genus (which also have peptidoglycan of the A1c type) [29] l-Alanoyl-d-glutamate peptidase of bacteriophage T5 is similar to the N-terminal, enzymatically active domain of Ply118 (25% identity) The C-terminal domain of Ply118 is responsible for substrate recognition [31] The enzyme of bacteriophage T5 is much shorter than Ply118 (137 and 281 amino acids, respectively); evidently, in bacteriophage T5 endolysin, the functions of substrate binding and catalysis FEBS Journal 276 (2009) 7329–7342 ª 2009 The Authors Journal compilation ª 2009 FEBS G V Mikoulinskaia et al are carried out by a single domain Interestingly, a recent crystallographic examination of the Ply500 enzyme [28] showed the presence of Zn2+ in the active center – a phenomenon that is typical for a number of proteins of the peptidase M15 family [32] The inhibition of bacteriophage T5 peptidase by the specific Ca2+ chelator BAPTA and by ZnCl2 does not agree with those data, and nor does the absence of an inhibitory effect of 1,10-phenanthroline, which has a high affinity for zinc However, the above-mentioned Zn2+containing Ca2+-activated aminopeptidase A is inhibited by Zn2+ too [33], and such a feature can also be found among other Zn2+-containing metalloenzymes [34] In addition, the failure to observe inhibition with a particular chelating agent need not be an absolute gauge of the absence of Zn2+ [19] Perhaps the question of which cation is bound at the active center of bacteriophage T5 endolysin will be answered after crystallographic examination Nevertheless, the stimulatory effect of Ca2+ on the enzyme is evident The blast analysis shows that there are more than 100 proteins – both from phages and from bacteria (mainly Gram-negative bacteria) – that are much more similar to bacteriophage T5 endolysin than Ply500 and Ply118 Probably, all of them are l-alanoyl-d-glutamate peptidases and have a common origin, and bacteriophage T5 peptidase is a typical enzyme of the bacterial cell lysis system It is possible that such a wide distribution of endolytic l-alanoyl-d-glutamate peptidases is related to the frequent occurrence of the l-alanine–d-glutamate bond in peptidoglycan The hypothesis that phage l-alanoyl-d-glutamate peptidases have a common origin is supported by the results of comparison of phage holin sequences In most cases, holin sequences demonstrate little similarity to each other, and these proteins are considered to have evolved independently of endolysins [35] However, holin of bacteriophage T5 shows significant similarity to holins of bacteriophages RB43 (E = · 10)16) and RB49 (E = · 10)21), as does bacteriophage T5 l-alanoyl-d-glutamate peptidase towards RB43 and RB49 putative endolysins (E = · 10)19 and E = · 10)16, respectively) It is interesting that bacteriophages RB43 and RB49 belong to the group of pseudo T-even phages Holin of bacteriophage T4, a product of gene t, also resembles holin of bacteriophage T5 (E = · 10)13) However, endolysin E of bacteriophage T4 is a muramidase, and has no relation to bacteriophage T5 peptidase It is possible that there might have been horizontal gene transfer between pseudo-T-even phages and bacteriophage T5 It should be noted that in bacteriophages RB43, RB49, and T4, L-Alanoyl-D-glutamate peptidase of bacteriophage T5 endolysin and holin are located in different genome regions – not in the same operon under a common promoter, as in bacteriophage T5 It can be supposed, therefore, that the variant present in bacteriophage T5 (colocalization of the lytic system genes) brings more evolutionary advantages in terms of their coordinated expression In addition to holin and endolysin, some dsDNA phages have another pair of proteins that are involved in cell lysis and provide a competitive advantage to the phage under unfavorable growth conditions (analogs of the products of the Rz and Rz1 genes of bacteriophage k) [36] One of these proteins is a lipoprotein, and the other is a transmembrane protein; defects in their genes result in Mg2+-dependent disorders in the process of lysis Analogs of these proteins have recently been found in quite a large number of phages, including T5, T4, RB32, RB43, RB49, and RB69 [36] All of these proteins are located separately from holin and endolysin The products of genes T5p045 (Rz analog) and T5p044 (Rz1 analog) show a slight resemblance to the products of genes PseT.3 and PseT.2 of bacteriophages T4 and RB32 Interestingly, the operon in which genes T5p045 and T5p044 are located in is the upstream neighbor of the holin–endolysin gene locus (Fig 1), but the coding sequences of these genes are included in the early transcript At the same time, the bacteriophage T5 holin–endolysin promoter is probably late, although it is located in the early genome region It contains more GC pairs, particularly in the region +10 to +20, which is typical for late promoters of bacteriophage T5 [37]; in addition, the conservative region )33 contains a TTnAnA sequence (typical for late bacteriophage T5 promoters) and does not contain TTGCTn, which is a sign of early promoters [38] It should be noted that holin T and endolysin E of bacteriophage T4 are late proteins, although gene e is located in the early region [39] To assess the spectrum of bacteriolytic action of bacteriophage T5 endolysin, we tested the bacteria of Gram-positive and Gram-negative species, which differ in the structure of peptidoglycan and the composition of the cell wall For example, Gram-negative cells contains peptidoglycan of the A1c type (not amidated, with c-mesodiaminopimelic acid in the third position); in B subtilis, the peptidoglycan is of the same type but amidated; Listeria monocytogenes also has peptidoglycan of the A1c type, but its cell wall structure has some features (for instance, teichoic acid of the Listeria cell wall contains two substituents, N-acetylglucosamine and dirhamnosyl, whereas teichoic acids of the other Gram-positive bacteria include only one type of substituting group) [40]; Staphylococcus aureus has an FEBS Journal 276 (2009) 7329–7342 ª 2009 The Authors Journal compilation ª 2009 FEBS 7337 L-Alanoyl-D-glutamate peptidase of bacteriophage T5 G V Mikoulinskaia et al A3a-type peptidoglycan (with l-lysine in the third position); the type of peptidoglycan of Micrococcus luteus is A2 (l-lysine in the third position; teichuronic acids instead of teichoic acids); and the cell walls of corynebacteria contain lipids, like those of Gram-negative microorganisms [20] The action of bacteriophage T5 endolysin seemed to be directed towards peptidoglycan type A1c, although the cell wall composition of Gram-positive bacteria (L monocytogenes and B subtilis) protected them from rapid lysis Thus, our preliminary results suggest that the peptidase of bacteriophage T5 is specific for the cell walls of Gramnegative microorganisms, containing A1c peptidoglycan and lacking either teichoic or teichuronic acids, which are typical of the cell walls of the Gram-positive bacteria Some Gram-negative microorganisms are pathogenic for animals and plants (Pseudomonas, Klebsiella, Proteus, and Agrobacterium), making bacteriophage T5 peptidase a potential candidate for the development of drugs that could be of use in biotechnology and plant cultivation Successful application of bacteriophage T5 endolysin as a selective lytic agent against Gram-negative cells requires the presence of factors that disturb outer membrane permeability There are a number of these: EDTA, sodium tripolyphosphate, heat, pH [41], ultrasound [42], peptidolipids [43], and polymyxin B [44] In this work, we have demonstrated that E coli cell lysis by bacteriophage T5 endolysin is possible after polymyxin B treatment This constitutes an example proving the effectiveness of this approach, and other permeabilizing agents could also be applicable Experimental procedures Materials E coli strains B, Z85 and BL21(DE3) and bacteriophage T5+ were taken from the collection of the Laboratory of Molecular Microbiology of the Institute of Biochemistry and Physiology of Microorganisms (IBPM RAS) Strains of E coli K12, B subtilis, M luteus, Pseudomonas putida, S aureus, Pectobacterium carotovorum, Proteus vulgaris, P mirabilis, L monocytogenes and Corynebacterium xerosis were obtained from the All-Russian Collection of Microorganisms (IBPM RAS) Plasmid pET3a was provided by Novagen (Madison, WI, USA) Bacteria and phages were grown either in liquid LB broth or on agarized LB medium Selection of clones was performed on plates with ampicillin (50 lgỈmL)1) Crystalline egg white lysozyme was purchased from Serva (Heidelberg, Germany) Restriction endonucleases were from Fermentas (Vilnius, Lithuania) 7338 All other chemicals were purchased, unless otherwise stated, from either ICN (Irvine, CA, USA) or Sigma (St Louis, MO, USA) Cloning of the lys gene The lys gene of bacteriophage T5 was amplified by PCR with primers LysF (5¢-gtcgagacATATGAGTTTTAAAT TTGGT-3¢) and LysR (5¢-ctggatccATTAAACTAGTTCG ACATG-3¢), which contain sites hydrolyzed by restriction endonucleases NdeI and BamHI The PCR fragment was cloned into plasmid vector pET3a (into the region controlled by the promoter of gene 10 of bacteriophage T7), using standard molecular biology techniques The clones carrying the insert were selected after treatment with restriction endonucleases and electrophoresis in 1% agarose The construct obtained was named pT5lys Plasmid pT5lys was further used to transform cells of E coli strain BL21(DE3) The synthesis of endolysin was induced with 0.5 mm isopropyl-thio-b-d-galactoside at a culture density corresponding to attenuance D550 nm = 1.0; the cells were harvested by centrifugation (6000 g, 10 min) 2.5 h later Isolation and purification of endolysin Cells of E coli BL21(DE3) from 200 mL of culture (1.1 g) carrying plasmid pT5lys were suspended in 10 mL of 25 mm Tris ⁄ HCl (pH 8.0), containing 40 mm NaCl and mm EDTA, and disrupted by sonication for (two 30 s treatments at a power of 75 W) The suspension was centrifuged at 20 000 g for 30 The supernatant (9.5 mL) was passed through an 11.4 mL column with Toyopearl DEAE 650M (TosoHaas, Stuttgart, Germany) and then applied to a 10 mL phosphocellulose column equilibrated with the same buffer Proteins were eluted by a linear gradient of sodium chloride (0.05–0.50 m) in 25 mm Tris ⁄ HCl buffer (pH 8.0) containing mm EDTA (total volume, 100 mL) Fractions (2 mL) were analyzed by PAGE in a 15% polyacrylamide gel The target protein was eluted with 0.3 m NaCl Enzyme activity assay The substrate for enzyme activity assay was prepared as follows An overnight culture of E coli B cells was treated with chloroform (added to 5% of total volume) for 15 min, and the cells were then were washed twice with water and stored frozen Just before the measurement, the cells were suspended in a reaction buffer (50 mm Tris ⁄ HCl, pH 8.2, containing 0.1% Triton X-100) The activity was determined spectrophotometrically, by the decrease of attenuance at 450 nm, in cm acrylic cuvettes at room temperature An activity unit was defined as the quantity of enzyme that provides the rate of attenuance decrease of 1.0 FEBS Journal 276 (2009) 7329–7342 ª 2009 The Authors Journal compilation ª 2009 FEBS G V Mikoulinskaia et al optical unitỈmin)1 All activity data were calculated from three independent measurements Inhibition of enzyme activity by chelators The substrate-providing cells were suspended in the reaction buffer (50 mm Tris ⁄ HCl, pH 8.2, containing 0.1% Triton X-100), and the chelators were added The reaction was initiated by the addition of a standard amount of the enzyme (15 ng mL)1) Extraction of peptidoglycan Peptidoglycan was extracted by Streshinskaya’s method [45] E coli or Ps putida cells (7 g, wet weight) were resuspended in 14 mL of 10 mm Tris-HCl (pH 8.0), and 0.4 mg of DNase was then added After addition of 20 mL of 4% SDS, the mixture was boiled for 30 and kept at °C for 12 h The suspension was then sonicated (75 W, min) and centrifuged at 20 000 g for h The pellet was resuspended in 14 mL of water and, after addition of 4% SDS (20 mL), the suspension was boiled for 15 and incubated at °C for 12 h After centrifugation at 20 000 g for h, the pellet was washed three times with distilled water, resuspended in 10 mm Tris ⁄ HCl (pH 8.0), treated with trypsin (100 lgỈmL)1) for h at 37 °C, boiled for 15 min, and centrifuged at 20 000 g for h The pellet was resuspended in 0.5 m trichloroacetic acid (1 : 100, v ⁄ v) and incubated at °C under stirring for 48 h; this was followed by centrifugation at 15 000 g for 30 The pellet was resuspended in 0.5 m trichloroacetic acid (1 : 100, v ⁄ v), incubated at 37 °C for 48 h, and centrifuged at 15 000 g for 30 The pellet was washed five times with water and lyophilized Cell walls were acetylated with acetic anhydride as described previously [46] The oxidized redox groups were reduced with NaBH4 by Ward’s method [47] Peptidoglycan hydrolysis The substrate was acetylated or reduced peptidoglycan from E coli (2 mgỈmL)1); enzymes (egg white lysozyme or bacteriophage T5 endolysin) were added at a concentration 0.1 lgỈmL)1; the reaction was performed in 0.05 m Tris ⁄ HCl buffer (pH 8.2) at room temperature After hydrolysis, the samples were centrifuged at 8000 g for min, and the supernatant was assayed for reducing and amino groups released L-Alanoyl-D-glutamate peptidase of bacteriophage T5 nide (0.5 g of potassium ferricyanide per liter, stored in a dark glass) and carbonate cyanide (5.3 g of sodium carbonate and 0.65 g of KCN per liter) reagents (200 lL each) The sample was boiled in a water bath for 15 min, and mL of the ferric iron solution (1.5 g of ferric ammonium sulfate and g of SDS per liter of 0.025 m H2SO4) was added After 15 min, absorbance was measured at 690 nm The quantity of reducing groups was calculated using a calibration curve constructed for glucose The control was the same acetylated ⁄ reduced peptidoglycan, except that it was not hydrolyzed but was rather supplemented with 0.1 lgỈmL)1 bacteriophage T5 endolysin just before the measurements Determination of free NH2 groups The quantity of free NH2 groups was measured according to Ghuysen’s protocol [49] An ethanol solution of 2,4-dinitrofluorobenzene (13 lL per mL of ethanol) was added to a sample of cell wall hydrolysate (1 : 10, v ⁄ v) The mixture was kept in a water bath at 60 °C for 30 Then, four volumes of m HCl were added, and absorbance was measured at 420 nm The quantity of free amino groups was calculated from an alanine-based calibration curve The control was the same acetylated ⁄ reduced peptidoglycan, except that it was not hydrolyzed but rather supplemented with 0.1 lgỈmL)1 bacteriophage T5 endolysin just before the measurements Determination of enzyme specificity of bacteriophage T5 endolysin Peptidoglycan from Pseudomonas sp (2 mg) was suspended in mL of 15 mm Tris ⁄ HCl (pH 8.2) Three 200 lL aliquots were taken: the first was supplemented with bacteriophage T5 peptidase (0.2 lg); the second was supplemented with the same amount of egg white muramidase; and the third was used as a control After 24 h, the samples were treated with dinitrofluorobenzene, and then subjected to acid decomposition and ether extraction, as described above [49] DNF derivatives of amino acids were analyzed by TLC on Kieselgel 60 F254 plates (Merck, Darmstadt, Germany) in a system of chloroform ⁄ methanol ⁄ benzyl alcohol ⁄ concentrated ammonia ⁄ water (30 : 30 : 30 : : 2) [49] The qualitative and quantitative analysis of amino acids remaining in the solution was performed with a Microtechna T-339 amino acid analyzer (Microtechna, Prague, Czech Republic) Determination of reducing groups Testing the lytic ability of endolysin on heterologous microorganisms The quantity of reducing groups was determined by Park and Johnson’s method [48] A 40 lL aliquot of the syspension of hydrolyzed peptidoglycan (see above) was placed in a test-tube, and this was followed by the addition of ferricya- Cells of overnight cultures of bacteria were killed by autoclaving, washed, and suspended in the reaction buffer (50 mm Tris ⁄ HCl, pH 8.2, containing 0.1% Triton X-100) to a concentration of  · 108 cellsỈmL)1 The reaction was initiated FEBS Journal 276 (2009) 7329–7342 ª 2009 The Authors Journal compilation ª 2009 FEBS 7339 L-Alanoyl-D-glutamate peptidase of bacteriophage T5 G V Mikoulinskaia et al by addition of enzyme (15 ng for the Gram-negative cells; 15 lg for the Gram-positive cells) The lytic activity was determined by the decrease in attenuance at 450 nm, in cm acrylic cuvettes at room temperature, as described above Analysis of viability of endolysin-treated E coli cells Cells of the E coli overnight culture were suspended in the reaction buffer (50 mm Tris ⁄ HCl, pH 8.2, containing 0.1% Triton X-100) to a concentration of  · 108 cellsỈmL)1 The first sample was supplemented with polymyxin B (final concentration, 40 lgỈmL)1); the second sample was supplemented with pure endolysin (final concentration, 40 lgỈmL)1); and the third sample was supplemented with both polymyxin B and endolysin at the same concentrations Untreated cells were used as a control All samples were incubated for 24 h at 37 °C, and the attenuance was then measured Aliquots corresponding to 107 initial cells were grown on LB agar plates overnight Standard analytical techniques The concentration of protein was determined by the Bradford method [50], using egg white lysozyme as the standard Protein samples were analyzed by denaturing electrophoresis in a 12% polyacrylamide gel by the Laemmli method [51], using a standard kit of marker proteins containing b-lactalbumin (14.2 kDa), soybean trypsin inhibitor (20.1 kDa), carboanhydrase (29.0 kDa), ovalbumin (45.0 kDa), BSA (66.0 kDa), and phosphorylase B (94.0 kDa) Electrophoresis was performed for 1.0 h at room temperature (field intensity, 15 VỈcm)1) Gels were stained with Coomassie Brilliant Blue G-250 (Sigma, St Louis, MO, USA) and washed with distilled water Software Nucleotide and amino acid sequences were analyzed using gene runner (v 3.0) (Hastings Software, Inc., Hastings, NY, USA) and clustalx software [52] For analysis of amino acid sequences, we used the blast search program [53] from the server of the National Center of Biotechnology Information (National Library of Medicine, USA; http:// www.ncbi.nlm.nih.gov/blast/) Free and total cation concentrations in the presence of multiple ligands were calculated by the cabuf program (http://www.kuleuven.be/fysio/trp/cabuf) The phylogenetic analysis was conducted with the 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Asp130, and His133) that are typical for metallopeptidases of the M15 family and take part in the binding of the metal ion in the process of catalysis [28] This is the first example of an l-alanoyl-d-glutamate. .. number of proteins of the peptidase M15 family [32] The inhibition of bacteriophage T5 peptidase by the specific Ca2+ chelator BAPTA and by ZnCl2 does not agree with those data, and nor does the. .. bacteriophage T5 peptidase; they are encoded by the genomes of bacteriophages ST64T, PS3, phiEcoM, and RB43, and the bacteria Yersinia bercovieri and Proteus mirabilis In multialignment, we did not use the