E. Binda, Department of Biotechnology and Life Sciences, University of Insubria, via J. H. Dunant 3, 21100 Varese, Italy Fax: +39 0332421500 Tel: +39 0332421546 E-mail: email@example.com
VanYn is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. ATCC 39727, which produces the glycopeptide antibiotic A40926, the precursor of the second-generation dalbavancin, which is in phase III of clinical development. VanYn (196 residues) is encoded by the dbv7 gene within the dbv biosynthetic cluster devoted to A40926 production. C-terminal His6-tagged VanYn was successfully expressed as a soluble and active protein in Escherichia coli. The analysis of the sequence suggests the presence of a hydrophobic transmembrane portion and two conserved sequences (SxHxxGxAxD and ExxH) in the extracytoplasmic domain that are potentially involved in coordination of Zn2+ and catalytic activity. The presence of these conserved sequences indicates a similar mechanism of action and substrate binding in VanYn as in VanY, VanX and VanXY Zn2+-dependent d,d-carboxypeptidases and d-Ala-d-Ala dipeptidases acting on peptidoglycan maturation and involved in glycopeptide resistance in pathogens. On substrates mimicking peptidoglycan precursors, VanYn shows d,d-carboxypeptidase and d,d-dipeptidase activity, but lacks d,d-carboxyesterase ability on d-Ala-d-Lac-terminating peptides. VanYn belongs to the metallo-d,d-carboxypeptidase family, but it is inhibited by β-lactams. Its characterization provides new insights into the evolution and transfer of resistance determinants from environmental glycopeptide-producing actinomycetes (such as Nonomuraea sp.) to glycopeptide-resistant pathogens (enterococci and staphylococci). It may also contribute to an early warning system for emerging resistance mechanisms following the introduction into clinics of a second-generation glycopeptide such as dalbavancin.
Database The nucleotide sequence of vanYn is available in the GenBank data base under accession number CAD91202
Glycopeptide antibiotics are drugs of last resort against multiresistant Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, which is a major cause of community-acquired infections and leads to high morbidity and mortality rates in hospital-acquired infections . Vancomycin and teicoplanin have been in clinical use since 1958 and 1988, respectively. The spread of resistance to glycopeptides in enterococci since 1988 and the recent emergence of high-level glycopeptide resistance in clinical isolates of methicillin-resistant S. aureus has prompted the search for second-generation drugs belonging to this chemical class .
Glycopeptide antibiotics interfere with the last steps of peptidoglycan (PG) cell wall synthesis in Gram-positive bacteria. These large molecules, which cannot penetrate the cytoplasmic membrane, form a complex with the C-terminal d-Ala-d-Ala of the late PG UDP-pentapeptide precursors, when they are transported to the external face of the cytoplasmic membrane . As a result, PG precursors can no longer be incorporated into nascent PG through transglycosylation and transpeptidation reactions.
The mechanism of resistance in enterococci and staphylococci relies on reprogramming PG synthesis, which produces UDP-linked precursors ending in d-alanyl-d-lactate (d-Lac) or d-Ala-d-Ser, and concomitantly eliminates precursors ending in d-Ala-d-Ala. The replacement of terminal d-Ala with d-Lac suppresses one of the five hydrogen bonds between the glycopeptides and their target, which leads to a 1000-fold decrease in the binding affinity. The substitution in d-Ser causes a conformational change, which also reduces vancomycin affinity, although not as markedly as with d-Lac [4,5]. In enterococci, six phenotypes of resistance (named VanA, VanB, VanC, VanD, VanE, and VanG, from the name of the d,d-dipeptide ligase) have been described; these differ in the organization of resistance genes (named van genes), the regulation of their expression, and the level of resistance/susceptibility to vancomycin and teicoplanin. In any of these phenotypes, glycopeptide resistance requires the coordinated action of several enzymes encoded by van genes, which are clustered in operons with a conserved basal structure [4,6]. Thus, the resistant bacteria need to synthesize d-Lac [VanH (α-keto acid reductase that reduces pyruvate to d-Lac)] or d-Ser [VanT (serine racemase)] and d-Ala-d-Lac or d-Ala-d-Ser [VanA, VanB, and VanD (ligases that catalyse the formation of d-Ala-d-Lac), or VanC, VanE, and VanG (ligases that catalyse the formation of d-Ala-d-Ser)], and to degrade d-Ala-d-Ala [VanX (d,d-dipeptidase)] or to remove d-Ala from growing precursors [VanY (d,d-carboxypeptidase)] or both [VanXY (d,d-dipeptidase, d,d-carboxypeptidase)] .
Enterococci, staphylococci and other Gram-positive bacteria have been shown to exchange van genes, often located on large mobile elements, by recursive and extensive events of horizontal gene transfer under the selective pressure of glycopeptides introduced in clinics and the environment [4–6]. The evolutionary sources of glycopeptide resistance determinants arising in bacterial pathogens are supposed to be the van genes present in glycopeptide-producing soil actinomycetes [4,6,7]. In these microbes, resistance genes play a role in glycopeptide self-resistance, avoiding suicide during antibiotic production [8–10].
We have recently investigated glycopeptide resistance in Nonomuraea sp. ATCC 39727 , an actinomycete producing the teicoplanin-like A40926 , which is used as precursor of the semisynthetic dalbavancin . Dalbavancin is a promising second-generation glycopeptide currently in phase III clinical development . Nonomuraea sp. ATCC 39727 is resistant to vancomycin and to its own product A40926, but vanHAX genes have not been identified in its genome. However, the dbv cluster, which is a contiguous set of 37 ORFs devoted to the production, regulation and transport of A40926, contains a gene (here named vanYn) involved in conferring some level of resistance to the producing strain . On the basis of the first annotation of the deduced product, vanYn from Nonomuraea sp. ATCC 39727 was assigned to the vanY family of d,d-carboxypeptidase genes [11,13]. The tetrapeptides resulting from VanYn activity on PG precursors are poor substrates for glycopeptide binding. Consistent with this, our recent analysis of UDP-linked PG precursors in Nonomuraea sp. ATCC 39727 revealed the predominant presence of the tetrapeptide. Moreover, a vanYn-null mutant showed a significantly reduced level of glycopeptide resistance .
The aim of this work was to determine the role of VanYn by means of its heterologous expression, biochemical characterization, and phylogenetic analysis. A better comprehension of the self-resistance mechanism in the as yet poorly explored Nonomuraea sp. ATCC 39727  may provide new insights into the evolution of resistance genes and their transfer from environmental glycopeptide-producing actinomycetes to pathogens.
Protein sequence analysis
The analysis of the primary sequence of 196-residue VanYn (Fig. 1A) suggests the presence of three domains: a cytoplasmic domain at the N-terminus (the first 20 residues, underlined), followed by a hydrophobic transmembrane portion (20 residues, in bold), and then by the remaining C-terminal domain, which is probably exposed on the external face of the cytoplasmic membrane. The extracellular domain contains motifs typical of VanY and VanX Zn2+-dependent d,d-carboxypeptidases and d,d-peptidases previously characterized in glycopeptide-resistant enterococci [15–22]: the two conserved sequences SxHxxGxAxD and ExxH (Fig. 1A, highlighted) are putatively involved in the coordination of Zn2+ and in the active site. Identification of these motifs suggests that VanYn might be a VanY d,d-carboxypeptidase (such as those studied in VanA-type or VanB-type enterococci) acting on the UDP-pentapeptide PG precursors when they are already translocated on the extracellular side of the cytoplasmic membrane [4,18,19]. In contrast, VanX d-Ala-d-Ala dipeptidases lack any transmembrane domain: they are involved in degrading the cytoplasmic pool of d-Ala-d-Ala [20–22].
In Fig. 1B, a comparison of VanYn sequences that encompass Zn2+ ligand residues, catalytic residues and substrate-binding residues with homologous sequences conserved in VanX, VanY and VanXY is shown. The role of highly conserved residues was previously identified by mutational analysis  and crystallographic determination  of VanX from VanA-type Enterococcus faecium BM4147: His116, Asp123 and His184 (see 5 in Fig. 1B) are involved in Zn2+ binding, Glu181 and Arg71 (3 in Fig. 1B) belong to the catalytic site, and Ser114 (4 in Fig. 1B) is involved in substrate binding [23,24]. These residues are also conserved in the recently partially characterized VanYb from the glycopeptide balhimycin biosynthetic cluster (bal) in Amycolatopsis balhimycina  and in VanYn from Nonomuraea sp. Tyr21 and Asp142 (1 in Fig. 1B), which are crucial for binding α-NH3+of free d-Ala-d-Ala, are present in VanX but not in VanY and VanXY; they both are absent from VanYb, whereas VanYn possesses the Asp residue. Asp68 (2 in Fig. 1B) is an auxiliary residue at the active site that is conserved in VanX proteins whose function – i.e. orientating the Arg in the transition state – is performed by Gln76 in VanY enzymes : both VanYb and VanYn possess the Gln, like VanY enzymes. Trp182 and Trp183 are two additional residues of the active site that are conserved in VanX proteins and present in VanYn (Fig. 1B, conserved sequence EWWH); in VanY proteins, including VanYb, Pro and Trp residues are present in homologous positions (Fig. 1B, conserved sequence EPWH).
Therefore, sequence alignment analyses suggest that VanYn from Nonomuraea sp. ATCC 39727 belongs to the VanY family according to its transmembrane domain and the extracellular localization of the active site, but it also shares some typical active site residues with VanX dipeptidases. To better investigate these aspects, we decided to express and purify the protein and to study its enzymatic activity and structural features.
VanYn was purified from 24 g of cell paste obtained from a 3-L culture batch of BL21 Star(DE3) Escherichia coli cells carrying pET24b(+)-vanYn and grown in TB medium (see Docs S1 and S2 for details of VanYn overexpression in E. coli). In the optimized lysis conditions, 85% of recombinant VanYn was detectable in the soluble fraction after cell sonication. Protein was fractionated from crude extracts by affinity chromatography on a HiTrap Chelating column followed by gel filtration chromatography on PD10 Sephadex. The purification yield was 4.6 mg·L−1 culture (0.6 mg·g−1 cells). SDS/PAGE analysis (see Doc. S2) showed that the final VanYn preparation migrated as a single band of 25 kDa and was > 90% pure.
VanYn enzymatic activity
Enzymatic activity of the purified VanYn was assayed on tripeptides that mimic the terminal portions of PG precursors (Nα,Nε-diacetyl-l-Lys-d-Ala-d-Ala, acetyl-l-Lys-d-Ala-d-Ala, and acetyl-l-Lys-d-Ala-d-Lac) and on the dipeptide d-Ala-d-Ala. Table 1 shows that VanYn cleaved the last d-Ala from both Nα,Nε-diacetyl-l-Lys-d-Ala-d-Ala and acetyl-l-Lys-d-Ala-d-Ala, this activity being only slightly affected by the acetylation level of the Lys. The specific activity was halved if the substrate was the free d-Ala-d-Ala. When the substrate was acetyl-l-Lys-d-Ala-d-Lac, no traces of released d-Lac were detectable. Thus, VanYn showed higher d,d-carboxypeptidase activity (VanY-like) than d,d-peptidase activity (VanX-like), whereas it did not show any activity as a d,d-carboxyesterase. With both acetyl-l-Lys-d-Ala-d-Ala and d-Ala-d-Ala, a strong substrate inhibition effect was observed at concentrations > 10 mm. With the tripeptides, a higher kcat and catalytic efficiency ratio (kcat/Km) were apparent as compared with the dipeptide (Table 1).
Table 1. Substrate specificity and kinetic parameters of recombinant VanYn. Specific activity was determined with 40 μg of His6-tagged VanYn added to 10 mm solutions of the indicated compounds. The activity was assayed at 25 °C as described in Experimental procedures. ND, not determined.
Specific activity (U·mg−1 protein)
As most peptidases and carboxypeptidases are activated by divalent cations, the effect of these on VanYn activity was assayed. The addition of exogenous MgCl2, CaCl2 and ZnCl2 reduced the enzymatic activity (Table 2). Preincubation of VanYn with EDTA totally abolished its enzymatic activity, indicating that metal ions are absolutely required for enzyme functionality. Increasing concentrations of ZnCl2, but not of MgCl2 and CaCl2, reversed the inhibitory effect of EDTA (Table 2). These data, and the identification of a conserved Zn2+-binding site (see above), allow us to conclude that VanYn was copurified with a stoichiometric amount of Zn2+ (tightly bound to the protein), which is required for its full catalytic activity.
Table 2. Metal ion effect on recombinant VanYnd,d-carboxypeptidase activity on acetyl-l-Lys-d-Ala-d-Ala substrate.
Relative activity (%)
a Similar results were obtained when EDTA was used at 2 or 8 mm.
Following preincubation with 4 mm EDTAa
MgCl2 or CaCl2
VanYn activity was inhibited by the addition of increasing concentrations of β-lactams: ID50 values were 0.06 mm and 1 mm with ampicillin and penicillin G, respectively. At increasing antibiotic concentrations (0, 0.025, 0.078 and 0.2 mm), a decrease in kcat was apparent, without affecting the Km for acetyl-l-Lys-d-Ala-d-Ala (data not shown). This behaviour is consistent with a noncompetitive mechanism of inhibition: Ki values of 0.05 mm and 1.02 mm were estimated for ampicillin and penicillin G, respectively.
The CD spectrum for the purified VanYn sample indicated a predominance of β-sheets (∼ 38%) and ∼ 15% of α-helices (Fig. 2, continuous line). Addition of the acetyl-l-Lys-d-Ala-d-Ala substrate significantly altered the CD spectrum of VanYn, increasing to ∼ 48% the β-sheet content (Fig. 2A, dashed line). In contrast, the presence of exogenous Zn2+ slightly influenced the secondary structure content of VanYn, confirming the tight interaction of this metal ion with the protein (Fig. 2B, dashed line). The ability of ampicillin to bind to VanYn was also confirmed by CD measurements: binding of the antibiotic deeply modified the secondary structure content of the protein (Fig. 2A, dotted line).
Despite the high identity of the conserved sequences belonging to the active sites, the overall identity between VanYn and previously described VanY and VanX enzymes in enterococci is moderate: 46% with VanXD from Enterococcus gallinarum , 40% with VanXA from E. faecium , 39% with VanXB from E. faecium (accession no. ADA62242), 34% with VanXB from Enterococcus faecalis , 38% with VanXYC from E. gallinarum , 27% with VanYB from E. faecalis , VanYG from E. faecalis  and VanYB from E. faecium , and 25% with VanYA from E. faecium . The phylogenetic relationships of VanYn are shown in Fig. 3, which was constructed with the neighbour-joining method with 41 full-length protein sequences having > 25% identity (blastp database). VanYn is located in a branch (60% identity) containing three proteins (StAA4_17335, StAA4_14210, and StAA4_13515) of unknown function from Streptomyces sp. AA4 (Birren, B. et al., CONSRTM The Broad Institute Genome Sequencing Platform, Broad Institute Microbial Sequencing Center. Annotation of Streptomyces sp. strain AA4, unpublished). VanYn shows ∼ 48% sequence identity with a d,d-carboxypeptidase VanY/endolysin from Gordonia bronchialis DSM 43247  and with VanYbd,d-carboxypeptidase identified in the bal cluster devoted to the biosynthesis of balhimycin from A. balhimycin DSM 5908 . These last two sequences cluster together with those from other actinomycetes such as Nocardia and Mycobacterium spp. Low-GC Gram-positive bacteria such as enteroccocci, bacilli, paenibacilli and streptococci form a separate branch, and have a lower level of identity with VanYn.
Previous studies showed that vanYn is involved in conferring glycopeptide resistance to Nonomuraea sp. ATCC 39727, which produces the novel glycopeptide A40926 . There is a link between antibiotic production and induction of self-resistance, which is needed in the producing microbe to avoid suicide during production [8,11]. vanY is, in fact, part of the biosynthetic cluster dvb, and its expression is coregulated with A40926 production .
In the present study, we cloned vanYn in E. coli and produced the recombinant full-length VanYn in fairly good amounts, notwithstanding the fact that heterologous production of enzymes belonging to the peptidase family is often limited by the protein toxicity towards the host cells [18,22,23,26,34]. VanYn preferential activity as a d,d-carboxypeptidase confirms its attribution to the VanY family, even though the enzyme showed an unusual d,d-peptidase activity (with a 3.75-fold lower catalytic efficiency) and lacked any d,d-carboxyesterase activity. This pattern of substrate preference differs from those described earlier for VanX and VanY enzymes from resistant VanA-type and VanB-type enterococci. Typically, VanX enzymes have only d,d-peptidase activity (with a much higher kcat/Km of 50–60 mm−1·s−1 on d-Ala-d-Ala) and no carboxypeptidase or esterase activities [20–23], whereas VanY enzymes are d,d-carboxypeptidases (kcat/Km of 8 mm−1·s−1 on acetyl-l-Lys-d-Ala-d-Ala), lack any activity on dipeptides, and have low carboxyesterase activity on d-Lac-terminating tripeptides [16,18,19]. Moreover, in contrast to what was observed for VanYn, the d,d-carboxypeptidase specific activity of VanY from VanA-type E. faecium was significantly reduced by the diacetylation of l-Lys .
The behaviour of VanYn most closely resembles that of VanXYC (190 residues) from VanC-type E. gallinarum, which combines the specificities of VanX and VanY, as it hydrolyses both d-Ala-d-Ala and the PG precursor analogs ending in d-Ala [15,30]. Moreover, the response of VanYn activity to EDTA, Zn2+ and other metal ions was similar to that previously reported for VanXYC . In contrast to VanYn, VanXYC has a cytoplasmic location, and its d,d-peptidase activity is approximately twofold higher than its d,d-carboxypeptidase activity [15,34].
Alignment of Zn2+-binding and active site sequences shows that VanYn shares well-conserved motifs with VanY, VanX and VanXY enzymes from different strains of glycopeptide-resistant enterococci with VanA , VanB [29,32], VanC [15,30], VanD [27,35] and VanG  phenotypes. It is worth noting that VanYn possesses an Asp active site residue that is involved in recognition of the α-NH3+ of d-Ala-d-Ala in VanX and that is not present in known VanY sequences. Structural analyses of VanX from E. faecium BM4147 [23,24] have shown that the active site is a small cavity that accommodates a dipeptide only: this explains the narrow specificity of VanX dipeptidases. Therefore, VanXYC and VanYn presumably have lower active site hindrance and thus accommodate larger substrates .
Quite surprisingly, the activity of VanYn was inhibited by β-lactams in a noncompetitive manner. VanY d,d-carboxypeptidases involved in glycopeptide resistance catalyse the same reaction as the low molecular mass membrane-bound penicillin-binding proteins (PBPs) involved in PG metabolism in most bacteria, but the former are penicillin-insensitive [18,19,21,22]. In fact, VanY sequences lack the classical Ser-X-X-Lys motif found in the active sites of PBPs [36,37]. The only exception is VanYD from VanD-type E. faecium BM4339, which belongs to the PBP family and whose activity is inhibited by β-lactams . We did not find canonical PBP signatures in the VanYn sequence, but the observed inhibitory effect of β-lactams is significant: this aspect needs further investigation.
Besides the conserved fingerprint signature of the active site between VanX and VanY enzymes, the overall identity is moderate. From the phylogenetic analysis, VanYn branches within the group of high-GC Gram-positive actinomycetes, including VanYb from the glycopeptide producer A. balhimycina, and proteins of unknown function from Nocardia, Streptomyces, Gordonia, Stackebrandtia and Mycobacterium spp. VanX, VanY and VanXY sequences from enterococci with different resistant phenotypes form a separate branch. vanY and vanX genes have been also found in other low-GC Gram-positive environmental bacteria, such as Paenibacillus and Bacillus spp., which may represent intermediate steps along the events of evolution and transfer of glycopeptide resistance determinants from glycopeptide-producing actinomycetes to Gram-positive pathogens . In both environmental bacteria and glycopeptide-producing actinomycetes, when the set of vanHAX is present, it is a well-conserved coregulated operon, whereas the presence and localization of vanY are variable. Some glycopeptide-producing actinomycetes possess, alternatively, vanHAX or vanY genes, and some others combine vanHAX and vanY: this combination perhaps emerged and was selected in pathogens as the most protective solution, whereby VanX and VanY enzymes act in sequence to eliminate PG precursors ending with d-Ala-d-Ala.
Taking together the experimental results and sequence identity analysis, the hypothesis that vanY has evolved independently from vanHAX genes can be advanced. In this evolutionary view, the VanYn investigated in this study seems to be a particularly interesting result of functional convergent evolution: it belongs to the family of VanY or VanXY Zn2+-binding peptidases, but is inhibited by β-lactams, as are PBPs. Low molecular mass PBPs and metalloenzymes are considered to be two unrelated families of d,d-carboxypeptidases that participate in PG metabolism in bacteria . In addition, as β-lactams and glycopeptides are the most common classes of antibiotics used to treat multiresistant Gram-positive bacteria (for serious infections, they are often used in combination), the fact that a protein involved in A40926 self-resistance maintains susceptibility to β-lactams is a promising finding with regard to the prevention of cross-resistance mechanisms following the introduction of new antibiotics into clinics.
Design, synthesis and cloning of cDNA coding for VanYn
The synthetic gene coding for VanYn was prepared by GENEART (Life Technologies Italia, Monza, Italy) on the basis of the previously deposited sequence of dbv7 (GenBank Accession no. AJ564722.1) from Nonomuraea sp. ATCC 39727  (see Doc. S1). The cDNA molecule was cloned into the pET24b(+) expression plasmid (Novagen, Milan, Italy), with NcoI and XhoI (Roche, Milan, Italy) restriction sites in BL21 Star(DE3) E. coli (Invitrogen, Milan, Italy). With this cloning strategy, a His6-tag encoded by the plasmid was added at the C-terminus of the protein. The entire cloned regions were confirmed by sequencing (Primm, Milan, Italy).
VanYn expression and purification
For VanYn overexpression conditions in E. coli, see Doc. S2. For protein purification, E. coli cells containing pET24b(+)–vanYn were grown overnight at 37 °C and 200 r.p.m. in LB medium supplemented with 50 μg·mL−1 kanamycin. Starter cultures (initial A600nm = 0.1) were inoculated in 2-L Erlenmeyer flasks containing 750 mL of TB medium, and incubated at 37 °C and 200 rpm. Protein expression was induced by the addition of 0.5 mm isopropyl thio-β-d-galactoside during the early exponential phase of growth (A600nm ∼ 0.8). Cells were harvested after 4 h at 25 °C, and sonicated for 10 cycles of 30 s each on ice. His6-tagged VanYn was purified from soluble fractions containing 300 mm NaCl by chromatography on a HiTrap chelating affinity column (1.6 × 2.5 cm) (GE Healthcare Sciences, Milan, Italy) equilibrated with 50 mm phosphate buffer (pH 7.0) containing 30 mm imidazole and 300 mm NaCl, according to the manufacturer’s instructions. The recombinant protein was eluted with 50 mm phosphate buffer (pH 7.0) containing 300 mm NaCl and 250 mm imidazole, and loaded onto a PD10 Sephadex G25 column (GE Healthcare Sciences) equilibrated with 50 mm phosphate buffer (pH 7.0). Protein purity was checked by western blot analysis (see Fig. S2) and SDS/PAGE with 15% polyacrylamide gels and staining with Coomassie brilliant blue. The protein concentration was estimated by use of the extinction coefficient at 280 nm (45 258 m−1·cm−1) determined by urea denaturation and the theoretical extinction coefficient based on the amino acid sequence.
d,d-Dipeptidase and d,d-carboxypeptidase assays
Enzymatic activity was assayed by measuring the release of d-Ala from Nα,Nε-diacetyl-l-Lys-d-Ala-d-Ala, acetyl-l-Lys-d-Ala-d-Ala and d-Ala-d-Ala in a d-amino acid oxidase/peroxidase-coupled colorimetric assay or by reaction with a fluorescent reagent. All substrates were purchased from Sigma-Aldrich (Milan, Italy). One unit of d,d-carboxypeptidase activity is defined as the amount of enzyme that produced 1 μmol d-Ala min−1.
d-Amino acid oxidase/peroxidase assay
Reaction mixtures contained different concentrations of the substrates Nα,Nε-diacetyl-l-Lys-d-Ala-d-Ala, acetyl-l-Lys-d-Ala-d-Ala, or d-Ala-d-Ala (range: 0–40 mm), 5 mm of the peroxidase colorimetric substrate 4-aminoantipyrine (Sigma-Aldrich), 3 U·mL−1d-amino acid oxidase from Rhodotorula gracilis , 7.5 U·mL−1 horseradish peroxidase (Sigma-Aldrich) and 6 mm phenol in 50 mm 1,3-bis[tris(hydroxymethyl)methylamino]propane (pH 7.4) in a final volume of 1 mL. At 25 °C, a variable amount of His6-tagged VanYn (range: 0–0.14 mg) was added to the reaction mixture, and the increase in absorbance (ΔA per min) was measured at 510 nm for the test sample and for the control to which no VanYn was added. A molar extinction coefficient for chinonemine of 6.58 mm−1·cm−1 was used.
Fluorimetric o-phthaldialdehyde method
Reaction mixtures contained 10 mmNα,Nε-diacetyl-l-Lys-d-Ala-d-Ala and 40 μg of His6-tagged VanYn in 50 mm phosphate buffer (pH 7.0) in a final volume of 200 μL. After 10 min at 25 °C, the reaction was stopped by addition of 50 μL of 250 mm HCl followed by 750 μL of water . Enzymatically released d-Ala was detected by the addition of 100 μL of fluoraldehyde (o-phthaldialdehyde) solution (Sigma-Aldrich) to 100 μL of the reaction mix, and this was followed by incubation at room temperature for 5 min. Eight hundred microliters of water was added, and 200 μL was removed: the fluorescence intensity was measured (λex, 340 nm; λem, 455 nm) in a fluorescence microplate reader (Tecan Infinite 200 Pro, Milan, Italy). Assay results were quantified from a standard curve prepared with known amounts of d-Ala.
Enzymatic activity was assayed by measuring the release of d-Lac from acetyl-l-Lys-d-Ala-d-Lac by a fluorimetric-coupled enzymatic reaction . Reaction mixtures contained acetyl-l-Lys-d-Ala-d-Lac (range: 0–50 mm), 100 mm KCl, 0.0004% (v/v) Triton X-100, 5 mm NAD+, 48 μm resazurin, 1 U·mL−1 diaphorase and 7 U·mL−1d-lactate dehydrogenase in 75 mm Tris/HCl buffer (pH 8.9). All reagents were from Sigma-Aldrich. The reaction was started by addition of 10 μL of d-Lac (range: 0–50 mm) for control reactions or His6-tagged VanYn (40 μg) to 100 μL of the reaction mix. After incubation for 30 min at room temperature, the fluorescence intensity was measured (λex, 540 nm; λem, 590 nm) in a fluorescence microplate reader. Assay results were quantified from a standard curve prepared with known amounts of d-Lac.
Effect of divalent cations and EDTA on d,d-dipeptidase and d,d-carboxypeptidase activity
The effect of divalent ions on the VanYn reaction was assessed by using the d-amino acid oxidase/peroxidase assay and adding ZnCl2, MgCl2 and CaCl2 (0, 0.05, 0.1 and 1 mm). To test the effect of EDTA on enzymatic activity, His6-tagged VanYn (40 μg) was preincubated with 0, 1, 4 or 12 mm EDTA at 25 °C in 50 mm 1,3-bis[tris(hydroxymethyl)methylamino]propane (pH 7.4) in a final volume of 100 μL: residual enzymatic activity was assessed after 10 min of incubation. Alternatively, 2, 4, 8 or 12 mm ZnCl2, MgCl2 and CaCl2 was added to the mixture following protein preincubation with EDTA, and the activity was determined as above.
Effect of antibiotics on d,d-carboxypeptidase activity
The effect of ampicillin and penicillin G (Sigma-Aldrich) on VanYn activity was investigated by adding the antibiotics – at concentrations of 0.025, 0.078 and 0.2 mm for ampicillin and 0.5, 1 and 2 mm for penicillin G – to the reaction mixture for the d-amino acid oxidase/peroxidase assay containing concentrations ranging from 0 to 10 mm acetyl-l-Lys-d-Ala-d-Ala tripeptide.
Far-UV CD spectra were recorded with a Jasco J-715 (Jasco Europe, Cremella, Italy) spectropolarimeter in the 195–250 nm wavelength range. Measurements were made in quartz cuvettes of pathlength 1 mm, employing protein solutions of 0.1 mg·mL−1, and were corrected for buffer contribution. Secondary structure fractions were calculated from deconvolution of the CD spectra with the program k2d2 (http://www.ogic.ca/projects/k2d2/) .
Identification of putative protein domains was performed with tmhmm Server 2.0, i.e. for the prediction of transmembrane helices. Alignments of DNA and protein sequences were conducted with blastn and blastp, respectively (http://www.ncbi.nlm.nih.gov/blast/). Homologous sequences were obtained from the blastp nr database [nonredundant GenBank CDS translations + Protein Data Bank (PDB) + SwissProt + PIR + PRF, excluding those in env_nr] and the blastn nr database (all GenBank + EMBL + DDBJ + PDB sequences ‘nonredundant’), and aligned by use of the clustalw program in UniProt (http://www.uniprot.org/), followed by manual adjustments . The phylogenetic tree was constructed with the neighbour-joining algorithm  in UniProt mega 4.0 (http://www.uniprot.org/), with its reliability being assessed by 1000 bootstrap repetitions.
The authors acknowledge M. Terrak from the University of Liege for initial advice on sequence analysis, and G. Molla from the University of Insubria for help with gene cloning. We also thank L. Caldinelli for technical assistance. This work was supported by grants from Fondo di Ateneo per la Ricerca to F. Marinelli, and Progetto Cariplo: Promuovere Capitale Umano d’Eccellenza to E. Binda and G. L. Marcone, and by an MIUR fellowship to G. L. Marcone. Support from Consorzio Interuniversitario per le Biotecnologie (CIB) is also acknowledged.