10408
Biochemistry 1991, 30, 10408-10415
Takesue, Y., Tamura, R., & Nishi, Y. (1977) Biochim. Biophys. Acta 483, 375-385. Toll, L., Brandt, S.R., Olsen, C. M., Judd, A. K., & Almquist, R. G. (1991) Biochem. Biophys. Res. Commun. 175, 886-893. Towbin, H., Staehelin, T., & Gordon, J. (1979) Proc. Natf. Acad. Sci. U.S.A. 76, 4350-4354.
Wacker, H., Jaussi, R., Sonderegger, P., Dokow, M., Ghersa, P., Hauri, H.-P., Christen, Ph., & Semenza, G. (1981) FEBS Lett. 136, 329-332. Wong, J. T.-F. (1975) in Kinetics of Enzyme Mechanisms, pp 5-9, Academic Press Inc., New York. Yeh, K. Y.,Yeh, M., & Holt, P. p.(1989) Am. J . Physiol. 256, G604-6 12.
Molecular Basis for Vancomycin Resistance in Enterococcus faecium BM4 147: Biosynthesis of a Depsipeptide Peptidoglycan Precursor by Vancomycin Resistance Proteins VanH and VanAt Timothy D. H. Bugg,l Gerard D. Wright,* Sylvie Dutka-Malen,$ Michel Arthur,$ Patrice Courvalin,l and Christopher T. Walsh*v* Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 021 15, and Unit; des Agents Antibactgriens, Institut Pasteur, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France Received May 17, 1991; Revised Manuscript Received July 31, I991 ABSTRACT: Vancomycin resistance in Enterococcus faecium BM4 147 is mediated by vancomycin resistance proteins VanA and VanH. VanA is a D-alaninemalanine ligase of altered substrate specificity [Bugg, T. D. H., Dutka-Malen, S., Arthur, M., Courvalin, P., & Walsh, C. T. (1991) Biochemistry 30, 2017-20211, while the sequence of VanH is related to those of a-keto acid dehydrogenases [Arthur, M., Molinas, C., Dutka-Malen, S., & Courvalin, P. (1991) Gene (submitted)]. We report purification of VanH to homogeneity, characterization as a D-specific a-keto acid dehydrogenase, and comparison with D-lactate dehydrogenases from Leuconostoc mesenteroides and Lactobacillus leichmanii. VanA was found to catalyze ester bond formation between D-alanine and the D-hydroxy acid products of VanH, the best substrate being D-2hydroxybutyrate ( K , = 0.60 mM). The VanA product ~-alanyl-~-2-hydroxybutyrate could then be incorporated into the UDPMurNAc-pentapeptide peptidoglycan precursor by D- Ala-D- Ala adding enzyme from Escherichia coli or by crude extract from E. faecium BM4147. The vancomycin binding constant of a synthetic modified peptidoglycan analogue N-acetyl-~-alanyl-~-2-hydroxybutyrate (& > 73 mM) was > 1000-fold higher than the binding constant for N-acetyl-D-alanyl-D-alanine(& = 54 pM), partly due to the disruption of a hydrogen bond in the vancomycin-target complex, thus providing a molecular rationale for high-level vancomycin resistance.
%e recent emergence of bacterial resistance to the glycopeptide family of antibiotics in strains of Enterococcusfaecium and Enterococcus faecalis (Courvalin, 1990) has prompted considerable interest in the mechanism of resistance: both because of the increasing use of the glycopeptide vancomycin in treatment of life-threatening Gram-positive bacterial infections and because of the unusual mode of action of the glycopeptide antibiotics. Vancomycin does not appear to penetrate the cell membrane or interact with cellular proteins but functions by complexation of peptidyl-DAla-D-Ala termini on the bacterial cell surface (Barna & Williams, 1984), thereby preventing transglycosylation and cross-linking of the peptidoglycan layer (Reynolds, 1989; Nagarajan, 1991). In view of the apparent ubiquity of the D-Ala-D-Ala terminus of bacterial peptidoglycan, it is difficult to envisage a simple mechanism for vancomycin resistance, which may explain the lack of bacterial resistance for over 30 years of clinical use. How then is this recently emergent vancomycin resistance achieved? 'Supported in part by N S F Grant DMB 8917290, a SERC/NATO postdoctoral fellowship (T.D.H.B.),and a NSERC postdoctoral fellowship (G.D.W.). * Author to whom correspondence should be addressed. Harvard Medical School. 8 lnstitut Pasteur.
*
0006-2960/91/0430- 10408$02.50/0
High-level vancomycin resistance in E . faecium BM4 147, a class A vancomycin-resistant strain (Schlaes et al., 1991), is associated with production of a 38-kDa membrane-associated protein VanA, whose amino acid sequence has been determined (Dutka-Malen et al., 1990). Sequence similarity was found with Gram-negative D-Ala-D-Ala ligases, which are cytoplasmic enzymes responsible for synthesis of the D-AlaD-Ala dipeptide for peptidoglycan assembly (Walsh, 1989). VanA has been purified, and shows D-Ala-DAla ligase activity, but has substantially modified substrate specificity, compared with Gram-negative D-Ala-D-Ala ligases (Bugg et al., 1991). VanA is able to synthesize a number of mixed dipeptides including D-Ala-D-Met and D-Ala-D-Phe in preference to D-Ala-D-Ala, suggesting that its cellular role may be synthesis of a D-Ala-X dipeptide, which is incorporated into peptidoglycan and is able to be cross-linked but is not recognized by vancomycin. Sequencing of an open reading frame adjacent to the vanA gene on plasmid PIP816 that is also required for resistance has identified a further vancomycin resistance protein named VanH (Arthur et al., 1991). The amino acid sequence of VanH shows 19-30% sequence identity with sequences of three D-specific a-keto acid reductases: D-hydroxyisocaproate dehydrogenase, D-3-phosphoglycerate dehydrogenase, and Derythronate phosphate dehydrogenase. This raises the pos0 1991 American Chemical Society
Vancomycin Resistance Mechanism
Biochemistry, Vol. 30, No. 43, 1991 10409
Table I: Purification of VanH from JM83/pSD8 purificaact.‘ (unit/mL) proteinb (ma/mL) tot Drotein (mg) SD. act. (units/mn) tion (fold) 19.4 388 0.020 1 .o crude extract 0.39 1.86 195 0.037 25-6076 (NHd)ZS04 0.91 26.0 0.187 9.3 48.0 0.28 1 .50 72 AcA54 gel filtration 0.45 15.0 0.40 0.03 13.5 673 2’.5’-ADP agarose 0.0675 67.8 3,390 Mono-Q FPLC 7.5 0.61 0.009 “Determined by NADH-dependent reduction of pyruvate at pH 5.6 (see Materials and Methods). One unit is defined as the activity required to convert 1 pmol of substrate to product per minute. bDetermined by Bradford protein assay (Bradford, 1976). vol (mL) 20.0 7.5
sibility that VanH synthesizes a D-a-hydroxycarboxylic acid, which may be an alternative substrate for VanA. If VanA is able to condense a D-hydroxy acid instead of a D-amino acid with D-alanine, binding of the subsequently resulting peptidoglycan precursor to vancomycin would be directly affected, since one of the hydrogen bonds formed between vancomycin and N-acetyl-D-Ala-D-Ala is with the N H of the terminal D-alanine residue (Barna & Williams, 1984). In order to examine this mechanistic proposal, we have purified VanH and report characterization of VanH as a D-specific a-keto acid reductase, coupling with VanA, and experimental support for this mechanism of vancomycin resistance. MATERIALS AND METHODS Materials. The following materials were obtained from Sigma Chemical CO:~.: tris(hydroxymethy1)aminomethane base (Tris), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), ampicillin, D-alanine, vancomycin, Dlactate, L-lactate, 2-ketocarboxylic acids, DL-2-hydroxycarboxylic acids, Leuconostoc mesenteroides D-lactate dehydrogenase, Lactobacillus leichmanii D-lactate dehydrogenase, and rabbit muscle L-lactate dehydrogenase. The following materials were obtained from Boehringer Mannheim Biochemicals: nicotinamide adenine dinucleotides (NADH, NAD+, NADPH), ATP, and isopropyl thiogalactoside (IPTG). 4 4 2-Hydroxyethy1)-1-piperazineethanesulfonicacid (HEPES) was purchased from U S . Biochemicals. [UJ4C]D-alanine (40 mCi/mmol) and [ l-14C]pyruvic acid (35 mCi/mmol) were purchased from Amersham. Protected amino acids, N,N’-diacetyl-L-lysyl-D-alanyl-D-alanine and N,N’-diacetyl-L-lysyl-D-alanyl-D-lactate were from Bachem Bioscience. Isoprenyl chloroformate was from Fluka. Other chemicals and solvents were of reagent grade. Samples of UDPMurNAc-tripeptide and UDPMurNAc-pentapeptide were provided by Dr. J. van Heijenoort (University of Paris). VanA (Bugg et al., 1991), Salmonella typhimurium D-AlaD-Ala ligase (Knox et al., 1989), and Escherichia coli DAla-D-Ala ligase B (Zawadzke et al., 1991) were prepared as previously described. E . coli D-Ala-D-Ala adding enzyme was purified from E. coli strain JM105 transformed with recombinant plasmid pTB3 containing the murF gene under the control of the tac promoter (T. D. H. Bugg and C. T. Walsh, unpublished results). Purification of VanH (see Table I ) . VanH was purified from JM83/pSD8, which contains the vanH gene under the control of the lac promoter ( S . Dutka-Malen, unpublished results). All steps were carried out at 4 O C unless otherwise specified. Enzyme activity was monitored by decrease in absorbance of NADPH at 340 nm in the presence of 10 mM pyruvate in 50 mM sodium phosphate buffer (pH 5.6). Protein concentration was determined by the method of Bradford (1976). The standard column buffer consisted of 50 mM Tris (pH 7.4), 1 mM EDTA, and 1 mM DTT. JM105/pSD8 was grown at 37 OC in 1 L of Luria Broth (1% tryptone, 1% NaCl, 0.5% yeast extract) containing am-
picillin (100 pg/mL) to ASg5of 0.6, whereupon 100 mM IPTG (10 mL) was added to induce the lac promoter. Cells were then grown for a further 5 h at 37 OC and harvested by centrifugation at 8000 g for 10 min. The cell pellet (4.4 g) was resuspended in 20 mL of 100 mM Tris (pH 7.4) containing 1 mM EDTA and 5 mM DTT and was passed twice through a French press at 1000 psi. Cell debris was removed by centrifugation at 25000g for 30 min. Powdered ammonium sulfate was gradually added to the supernatant to a final concentration of 25% saturation, and the solution was stirred for 1 h. The solution was cleared by centrifugation at 12000g for 30 min, and ammonium sulfate was added to the supernatant to a concentration of 60% saturation. After the solution was stirred for a further 1 h, the precipitate was removed by centrifugation at 12OOOg for 30 min. The precipitate was resuspended overnight in 5 mL of column buffer. The resuspended pellet was loaded onto an Ultrogel AcA54 gel filtration column (2.5 X 108 cm) and eluted at 60 mL/h with column buffer. Active fractions were pooled, and the pool was loaded onto an adenosine 2’,5’-diphosphate agarose column (Sigma; 1.6 X 10 cm) and eluted at 1.0 mL/min with a 300-mL gradient of 0-2 M KCl in column buffer. Enzyme activity eluted at 0.6 M KCl: active fractions were pooled and dialyzed overnight into Tris column buffer. The pool was then loaded onto an HR 5/5 Mono-Q column (Pharmacia) and eluted at 0.5 mL/min with a 100-mL gradient of 0 4 . 5 M KCI in column buffer. Enzyme activity eluted in a single sharp peak at 140 mM KCl, giving 68 pg of VanH of specific activity 68 units/mg which was homogeneous by SDS-PAGE. An N-terminal sequence M-N-N-I-G-I-T-V-Y-G was obtained from an electroblot of VanH onto PVDF membrane (carried out by Dr.W. Lane, Harvard Microchemistry Facility), which is identical to the predicted N-terminal amino acid sequence (Arthur et al., 1991). Kinetic Analysis of VanH and D-Lactate Dehydrogenases. VanH and D-lactate dehydrogenases from L. mesenteroides and L. leichmanii were assayed by the decrease in absorbance of NADPH at 340 nm ( 6 = 6.2 X lo3 M-’ cm-’ ) in 50 mM sodium phosphate buffer (pH 5.6) with varying concentrations of a-keto acid substrates. pH 5.6 was found to be the optimum pH for the forward reaction of VanH. K , values were determined by varying substrate concentration from O.2Km (minimum 2 pM) to 5Km (maximum 80 mM), assays being carried out in duplicate and K , and k,,, being determined by Lineweaver-Burk plots. Determination of Stereospecificity of VanH. A 2 0 0 4 mixture of VanH (0.05 unit), pyruvate (5 pmol), and NADH (5 pmol) in 50 mM potassium phosphate buffer (pH 5.6) was incubated for 24 h at 25 “C, heated for 5 min at 70 “C, spun for 2 min at 13OOOg, filtered through a plug of activated charcoal, and stored at 4 O C . The mixture was then injected onto an Interaction ION-300 Organic Acids HPLC column and eluted in 5 mM H2S04at 0.5 mL/min. Pyruvic acid was found to elute at 27 min and lactic acid at 38 min. Lactic acid
10410 Biochemistry, Vol. 30, No. 43, 1991
Bugg et al.
DHicDH VanH
Sequence alignment of N-terminal sequences of L. leichmanii and L. mesenteroides D-lactate dehydrogenases (Lac D-LDH and Leu D-LDH,respectively) with D-hydroxyisocaproate dehydrogenase (DHicDH) and VanH. Identical conserved residues are boxed. FIGURE 1 :
product was collected, neutralized with NaOH, and added to an assay monitored at 340 nm containing 5 mM NAD+ in 100 mM HEPES buffer (pH 8.6), to which was added either L. mesenteroides D-lactate dehydrogenase or rabbit muscle Llactate dehydrogenase. No increase in absorbance was observed with L-lactate dehydrogenase, but an increase in absorbance of 0.023 was observed with D-lactate dehydrogenase. As controls, authentic D-lactate was oxidized uniquely by D-lactate dehydrogenase, and L-lactate by L-lactate dehydrogenase. Identical large scale reductions of pyruvate were carried out using D-lactate dehydrogenase and L-lactate dehydrogenase, giving after HPLC purification lactic acid products which were oxidized by D-lactate dehydrogenase and L-lactate dehydrogenase, respectively. These results imply that VanH carries out a D-specific reduction of pyruvate, to the extent of >96% enantiomeric excess. Determination of N- Terminal Sequences of D-Lactate Dehydrogenases. Samples of D-lactate dehydrogenases from L. mesenteroides and L. leichmanii (Sigma) were transferred by electroblotting from a 9% SDS-PAGE gel to PVDF membrane, and N-terminal sequences were determined by Dr. W. Lane, Harvard Microchemistry Facility. The N-terminal sequences obtained are shown in Figure 1. Assays of 2-Hydroxy Acids as Substrates for VanA. k,,, values were determined by coupled spectrophotometric assay (Daub et al., 1988), in the presence of 10 mM D-alanine and 50 mM DL-2-hydroxyacid, in VanA assay buffer [ 100 mM Tris (pH 8.6), 10 mM MgCI,, 10 mM KCl]. K, values were determined by thin layer chromatographic separation of [ 14C]~-Ala-Xproduct from [ I4C]~-AIa-~-Ala at varying concentrations of X, followed by scintillation counting. Assays (25 pL) contained 100 mM Tris (pH 8.6), 10 mM MgCI2, 10 mM KCl, 0.2 mM [U-14C]~-Ala(5 nmol, 40 pCi/pmol), 6 mM ATP, VanA (3.2 pg), and ~ ~ - 2 - h y d r o xacid y (0.1-80 mM). Reactions were incubated for 1 h at 25 OC, and 10 pL of each reaction was spotted onto a cellulose thin layer chromatography plate (Kodak), eluted for 3 h in a n-butanol/acetic acid/water (12:3:5), dried, and exposed to film overnight. Spots visualized by autoradiography corresponding to D-Ala-X product were cut out and scintillation counted. K, values were calculated by plots of l/(D-Ala-X product formation) against 1/ [XI. A K , value of 7.1 mM was measured for D-lactate by measurement of the incorporation of radioactivity into DAla-['4C]~-Lacat varying concentrations (2-40 mM) of [I4C]~-lactate,generated from [14C]pyruvateby L. mesenteroides D-lactate dehydrogenase, and a fixed concentration of D-alanine (1 mM). Preparation of Crude Extract of BM4147 and BM4147-I. Cultures of E. faecium strains BM4147 and BM4147-1 (500 mL) were grown in brain heart infusion media at 37 OC for 8 h, BM4147 in the presence of 10 pg/mL vancomycin. Cells were harvested by centrifugation at 8OOOg for 10 min, resuspended in 10 mL of HEPES lysis buffer [ 100 mM HEPES buffer (pH 7.2) containing 300 mM NaCl, 5 mM MgCl,, 1 mM EDTA, and 5 mM DTT]. The suspensions were then passed twice through a French press at 1000 psi, and cell debris was pelleted by centrifugation at 25000g for 30 min.
Powdered ammonium sulfate was gradually added to the supernatant to a final concentration of 70% saturation, and the solution was stirred for 1 h. The precipitate was collected by centrifugation at 12000g for 30 min and stored at 4 OC. When required for assays, aliquots of ammonium sulfate pellets were resuspended in HEPES lysis buffer. Synthesis of Peptides and Depsipeptides. NAc-DAla-D-Ala was prepared by acetylation of commerical D-Ala-D-Ala with freshly distilled acetic anhydride (Nieto & Perkins, 1971). Dipeptides were prepared from NAc-D-Ala and appropriately protected amino acids by conventional methodologies. Benzyl esters of D-lactate and DL-2-hydroxybutyrate were prepared by the general method of Gisin et al. (1969). Depsipeptides were prepared from Cbz-DAla and hydroxy acid benzyl esters using isoprenyl chloroformate as coupling agent in the presence of dimethylaminopyridine and triethylamine in dichloromethane (Zeggaf et al., 1989). Debenzylation was carried out in ethanol under H, atmosphere with 10% Pd over charcoal for 3-4 h at 23 OC. After filtration through Celite and concentration at room temperature, the crude compounds were applied to a Sephadex G-10 column (35 X 1 cm) and eluted with water. Depsipeptide containing fractions were lyophilized and further purified by RP-HPLC (Bio-Rad HI Pore 318, 250 X 21.5 mm) using 25 mM NH4HC03as the mobile phase and collecting fractions at 0 OC followed by lyophilization. The free depsipeptides were stored at -80 OC. Diastereomers of ~-Ala-~~-2-hydroxybutyrate were obtained as separate peaks by RP-HPLC. ~-Ala-~-2-hydroxybutyrate was identified by the retention time of the I4C-labeled compound prepared by incubation of [14C]~-Alawith Van A and DL-2-hydroxybutyrate. D-Ala-D-lactate. 'H NMR (D,O) 6: 4.82 (quart., J = 7 Hz, lH, OCH), 4.09 (quart., J = 7 Hz, l H , NCH), 1.53 (d, J = 7 Hz, 3H, CH,), 1.48 ( d , J = 7 Hz, 3H, CH,); FAB mass spectrum: m / z 162 ( M + 1); calculated 162.0762, found 162.0760. ~-Ala-~-2-hydroxybutyrate. 'H NMR (D,O) 6: 4.7 (t, J = 7 Hz, lH, OCH), 4.07 (quart., J = 7 Hz, lH, NCH), 1.75 (quintet, J = 7 Hz, 2H, CH,CH,), 1.48 (d, J = 7 Hz, 3H, CH,), 0.86 (t, J = 7 Hz, 3H, CH,CH,); FAB mass spectrum: m / z 176 ( M + 1); calculated 176.0918, found 176.0899. The N-acetylated depsipeptides were prepared by acetylation of the free depsipeptides with acetic anhydride (1.1 equiv) in ethanol in the presence of diisopropylethyl amine (1.1 equiv) or by coupling of N-Ac-D-alanine with the appropriate hydroxy acid benzyl ester. The N-acylated depsipeptides were purified by chromatography over Sephadex G-10 followed by RPHPLC using 0.1% TFA as the mobile phase and eluting with a linear gradient of 0-25% CH3CN (N-Ac-D-Ala-D-lactate) or 0-30% (N-Ac-~-Ala-~~-2-hydroxybutyrate). NAc-D-Ala-D-lactate. 'H NMR (D,O) 6: 4.55 (quart., J = 7 Hz, l H , OCH), 4.42 (quart., J = 7 Hz, lH, NCH), 2.01 (s, 3H, CH,CO), 1.53 (d, J = 7 Hz, 3H, CH,), 1.47 (d, J = 7 Hz, 3H, CH,); FAB mass spectrum: m / z 204 ( M + 1); calculated 204.0867, found 204.0873. NAc-~-Ala-~~-2-hydroxybutyrate. 'H NMR (D,O) 6: 4.95 (m, l H , OCH), 4.42 (quint., J = 4 Hz, lH, NCH), 2.02 (s, 3H, CH,CO), 1.9 (m, 2H, CH,), 1.46 [d, J = 7 Hz, 3H,
Biochemistry, Vol. 30, No. 43, 1991
Vancomycin Resistance Mechanism
1041 1
Table 11: Kinetic Data for VanH and D-Lactate Dehydrogenases" Leuconostoc mesenteroides D-lactate DH
VanH substrate pyruvate a-ketobutyrate a-ketovalerate a-ketoisovalerate a-ketocaproate a-ketoisocaproate phenyl pyruvate
k,, (s-I) 40 35 35 5.2 2.4 5.2
