(Received for publication, October 31, 1996, and in revised form, January 24, 1997)
From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
The Gram-positive bacterium Leuconostoc
mesenteroides, ATCC 8293, is intrinsically resistant to the
antibiotic vancomycin. This phenotype correlates with substitution of
D-Ala-D-lactate (D-Ala-D-Lac) termini for
D-Ala-D-Ala termini in peptidoglycan intermediates in which the depsipeptide has much lower affinity than
the dipeptide for vancomycin binding. Overproduction of the L. mesenteroides D-Ala-D-Ala ligase (LmDdl)
2 in E. coli and its purification to ~90% homogeneity
allow demonstration that the LmDdl2 does have both depsipeptide and
dipeptide ligase activity. Recently, we reported that mutation of an
active site tyrosine (Tyr), Tyr216, to phenylalanine (Phe)
in the E. coli DdlB leads to gain of D-Ala-D-Lac depsipeptide ligase activity in
that enzyme. The vancomycin-resistant LmDdl2 has a Phe at the
equivalent site, Phe261. To test the prediction that a Tyr
residue predicts dipeptide ligase while an Phe residue predicts both
depsipeptide and dipeptide ligase activity, the F261Y mutant protein of
LmDdl2 was constructed and purified to ~90% purity. F216Y LmDdl2
showed complete loss of the ability to couple D-Lac but
retained D-Ala-D-Ala dipeptide ligase activity.
The TyrPhe substitution on the active site omega-loop in
D-Ala-D-Ala ligases is thus a molecular
indicator of both the ability to make
D-Ala-D-Lac and intrinsic resistance to the
vancomycin class of glycopeptide antibiotics.
The vancomycin class of glycopeptide antibiotics binds with high affinity to the N-acylated D-Ala-D-Ala termini of intermediates in assembly and cross-linking of peptidoglycan (PG)1 strands in bacterial cell wall biosynthesis (1-4) at the external face of the cell membrane. The vancomycin·PG·D-Ala-D-Ala complex is blocked for subsequent transpeptidations and transglycosylations, by which new chains are added and existing PG chains are cross-linked. The net reduction in covalent connectivity of PG translates into reduced tensile strength and increased susceptibility to osmotic lysis and bacterial death.
Clinically significant vancomycin resistance has been detected in pathogenic enterococci in three phenotypic forms, designated VanA, VanB, and VanC type resistance (2). The VanA phenotype has been best studied and found to require the expression of five genes where all five encoded proteins VanR, -S, -H, -A, -X have enzymatic activities (2, 4). VanS and -R act as partners in a two-component regulatory system, VanS as a transmembrane sensor kinase (5, 6) and VanR as a response regulator (6, 7), which account for inducible transcriptional activation of VanH, -A, and -X. VanH encodes a D-Lac dehydrogenase, functioning as a D-specific pyruvate reductase (8) to provide D-Lac for VanA, which is a D-Ala-D-Ala ligase homolog that has gained D-Ala-D-Lac depsipeptide ligase activity (8, 9). In a cell producing both D-Ala-D-Ala and D-Ala-D-Lac (Scheme 1, a and b), VanX acts selectively as a D-Ala-D-Ala dipeptidase. Thus, D-Ala-D-Lac accumulates (10) and serves as a substrate for the MurF enzyme that normally adds D-Ala-D-Ala as a unit to a UDP-N-acetylmuramic acid (MurNAc) tripeptide. Instead of the normal UDP-MurNAc pentapeptide terminating in D-Ala-D-Ala, a UDP-MurNAc tetrapeptide ester terminating in D-Ala-D-Lac is produced in such a vancomycin-resistant Enterococcus sp. Vancomycin binds with three orders of magnitude lower affinity to the D-Ala-D-Lac terminus versus the D-Ala-D-Ala terminus, accounting quantitatively for observed resistance levels (8).
[View Larger Version of this Image (6K GIF file)]Scheme 1.
The molecular analysis of vancomycin resistance has led to the
similarities and differences between the dipeptide forming D-Ala-D-Ala ligases and the ~28% identical
38 kDa D-Ala-D-Ala ligase homolog VanA, whose
gain of depsipeptide ligase activity is crucial for phenotypic
resistance (8, 9). The x-ray structure of the DdlB isoform of
Escherichia coli in complex with a phosphinophosphate analog
of a dipeptidyl reaction intermediate has allowed definition of the
ligase active site (11) and predicted functions for several residues
that were validated by mutagenesis (12). Most intriguingly, mutations
at Tyr216 (Y216F) or Ser150 (S150A) in the
E. coli DdlB convert the dipeptide ligase to an enzyme that
has now gained substantial depsipeptide ligase activity (13) that is
the hallmark of a VanA and -B type dipeptide/depsipeptide ligase. An
x-ray structure of E. coli Y216F DdlB has been obtained (14). Both Tyr216, on a mobile omega-loop, and
Ser150 participate, with Glu15, in wild-type
E. coli DdlB in a hydrogen bonding array that fixes the
omega-loop to cover the substrates and intermediates in the active site
and to hydrogen bond (Glu15) to the amino group of
D-Ala1 to orient this electrophilic substrate (Fig. 1). It was this hydrogen bonding array that
suggested Tyr216 as a potentially important residue.
In addition to studying the vanR, -S, -H, -A, and -X operon function, the structure of PG intermediates in Gram-positive bacteria with intrinsic vancomycin resistance such as Lactobacillus, Pediococcus, and Leuconostoc species has been analyzed (16-19). In these cases, PG intermediates terminating in D-Ala-D-Lac were also detected (20, 21), suggesting a common evolutionary mechanism and a possible origin for at least the VanH and -A genes. Polymerase chain reaction (PCR) analysis has been used to identify and sequence fragments of the ddl genes in such organisms (22), and there is a correlation of Tyr/Phe in the D-Ala-D-Ala ligases at the position corresponding to Tyr216 in E. coli DdlB with sensitivity/resistance phenotypes (13). To test the prediction that a phenylalanine in the omega-loop region does indeed predict a D-Ala-D-Ala ligase with depsipeptide ligase activity, D-Ala-D-Ala ligase from vancomycin-resistant Leuconostoc mesenteroides ATCC 8293 was overproduced, purified from E. coli extract, and characterized for D-Ala-D-Ala and D-Ala-D-Lac ligase activities. When Phe261 was then mutated to Tyr, the resultant enzyme shows retention of D-Ala-D-Ala dipeptide ligase activity but loss of D-Ala-D-Lac depsipeptide ligase activity.
L. mesenteroides ATCC 8293 was from
American type culture collection (ATCC). Oligonucleotides were
purchased from Integrated DNA Technologies, Inc (Coralville, IA), and
restriction enzymes and polymerases were from U. S. Biochemical, Corp.
(Cleveland, OH). ATP, D-Ala,
DL--hydroxybutyrate (Hbut), D-Lac,
phosphoenolpyruvate, and HEPES were purchased from
Sigma. NADH, L-Lac dehydrogenase, and
pyruvate kinase were from Boehringer Mannheim.
D-[14C]-Ala and
D-[14C]-Lac were from American Radiolabeled
Chemicals Inc. (St. Louis, MO), and thin layer chromatography (TLC)
cellulose sheets were from Kodak (Rochester, NY).
Cloning
was carried out by amplification of a DNA fragment by using two rounds
of PCR of genomic DNA purified from L. mesenteroides ATCC
8293. Three PCR oligonucleotides (oligomer 1, CATAC AAGGT GAGGA CGGAA
AGATG; oligomer 2, GCGGA TCCTT AGTTA AACTT CCCTA TCTTT TCTTC TCCAA
GTGAC; and oligomer 3, GCACA TTCTA GAAGG AGACG GACAT ATGAC TAAAA AAAGA
GTAGC) used in this reaction were designed on the basis of the
published L. mesenteroides CIP 16407 sequences (22). The
first round of PCR with oligomers 1 and 2 was for the amplification of
the corresponding genomic DNA fragment. Oligomer 1 was designed to
hybridize to the upstream region of the lmddl gene, and
oligomer 2 contains the 3 terminal 37 bases of the gene and additional
sequences to introduce a BamHI restriction site. Three
separate clonings resulted in the same DNA sequence. In the sequence,
some variations were detected, as noted below, compared with the
sequence previously reported for a different subtype (ATCC 8293 versus CIP 16407, see Ref. 22). Therefore, this gene is
designated lmddl2. The differences in the DNA sequences and
their corresponding effects on the encoded peptide sequence (CIP
versus ATCC strains) are Ala130 to Gly
(Asp35 to Gly), Thr236 to Cys (silent),
Ala258 Gly259 to Gly-Cys (Ser78 to
Ala), Gly317 to Cys (Leu97 to Phe), and
Cys686 to Thr (silent), resulting in three amino acid
changes between LmDdl and LmDdl2. The subsequent round of PCR where the
above PCR product was template with oligomers 2 and 3, was performed to
produce an XbaI restriction enzyme site and Shine-Dalgano
sequences at the upstream site of the gene as described earlier (23). The DNA fragment was subcloned into the
XbaI-BamHI site of pET22b (Novagen, Madison, WI)
and transformed into E. coli DH5
and subsequently into
E. coli BL21(DE3). Site-directed mutagenesis at the
Phe261 site was carried out by using two-round megaprimer
PCR (24). The oligonucleotides used were the above two oligmers 2 and 3 and a mutated sequence oligomer (GGAAT TATCA ACGTA CTTAT TATTA TAATC
ATACC). After mutagenesis the sequences were confimed by DNA
sequencing.
Cell culture and
purification of DdlB, Y216F, and VanA were performed as described
previously (12). Those of wild type and F261Y LmDdl2 are essentially
the same as the above proteins with minor modifications. Briefly,
Luria-Bertani medium was inoculated by 1/40 volume of the overnight
culture of the corresponding strain and incubated at 30 °C to
A595 0.6. At this time, 0.4 mM
(final concentration) isopropyl-thio--D-galactoside was
added to induce lmddl2 gene expression, and the culture was
further incubated for ~3 h. The harvested cells in buffer P
containing 50 mM Hepes buffer, pH 7.2, and 10 mM MgCl2 were disrupted by French press (18,000 psi/in2), and the supernatant was obtained after 30 min of
centrifugation of the resulting cell extract at 100,000 × g. Most of the LmDdl2 protein was recovered in the
supernatant. Purification of the protein was followed by ammonium
sulfate fractionation (25~50% fraction), ACA54 gel (BioSepra,
Marlborough, MA) filtration, and Q-Sepharose chromatography (column
size, 80 ml; elution, 400 ml of buffer P; gradient, 0-1 M
KCl). In the gel filtration chromatography, the LmDdl2 protein (~42
kDa) was recovered in similar fractions as DdlB, which is a dimer of a
32 kDa polypeptide. Thus, LmDdl2 also appears to be a dimer. The
purities of both wild-type and F216Y mutant proteins were measured to
be around 90% by gel scanning densitometry of an SDS gel.
The assay mixture included 0.2 mM D-[1-14C]-Ala (0.1 mCi/ml, 55 µCi/µmol) or 0.2 mM D-[1-14C]-Lac (0.1 mCi/ml, 55 µCi/µmol), 100 mM HEPES, pH 7.5, 10 mM KCl, 10 mM MgCl2, 10 mM ATP, and additional unlabeled D-Ala, D-Lac, or DL-Hbut with enzyme (~10 µM), which was incubated at room temperature for 3 h. Three µl of each sample was analyzed on TLC cellulose plate as described previously (8, 9).
Enzyme Assay by Coupled ADP ReleaseThe TLC assay was not suitable for measurement of kinetic parameters of LmDdl2 because it was necessary to incubate the reaction mixture for more than 30 min to detect any turnover, and it was difficult to find the linear region of enzyme activity. For these reasons, the ADP release-coupled assay (25) was routinely used for evaluating kinetic parameters. The reaction mixture was composed of 100 mM HEPES, pH 7.5, 10 mM KCl, 10 mM MgCl2, 10 mM ATP, 2.5 mM phosphoenolpyruvate, 0.15-0.2 mM NADH, 50 units/ml L-Lac dehydrogenase, 50 units/ml pyruvate kinase, D-Ala and D-Lac. The assay was initiated by adding enzymes at 30 °C, and reaction progress was monitored at 340 nm.
Kinetic AnalysisThe kinetic analysis was carried out as described previously (13, 26-29). The basic equation for D-Ala-D-Ala ligase (Equation 2) was derived based on the steady-state kinetics for two identical substrate molecules of D-Ala (Equation 1).
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
![]() |
(Eq. 3) |
![]() |
(Eq. 4) |
![]() |
(Eq. 5) |
![]() |
(Eq. 6) |
D-Ala-D-Lac ligase activities were measured as described previously (13). Briefly, Equation 8 was used based on Equation 7, where S1 and S2 are D-Ala and D-Lac, respectively. Those activities were measured by the ADP release-coupled assay. Because this method cannot discriminate D-Ala-D-Lac ligase activity from D-Ala-D-Ala ligase activity (paths a and b in Scheme 1), the velocity values were corrected. The value obtained from the observed activity minus D-Ala-D-Ala ligase activity in the absence of D-Lac was regarded as D-Ala-D-Lac ligase activity because, in the conditions used (for D-Ala-D-Lac ligase activity of LmDdl2 wild-type), the D-Ala-D-Ala ligase activity is less than 5% of the maximum observed activity.
![]() |
(Eq. 7) |
![]() |
(Eq. 8) |
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The gene encoding
the putative D-Ala-D-Ala ligase from the
vancomycin-resistant L. mesenteroides ATCC 8293 (reported MIC 1012 µg/ml) (20, 21) was subcloned into plasmid pET22b
behind the T7 promoter as noted under "Experimental Procedures,"
expressed in E. coli BL21(DE3), and purified as summarized
in Fig. 2. Overproduction of the ~42 kDa LmDdl2 was
obtained in soluble form and was readily purified by gel filtration and
ion exchange chromatography. A yield of 30 mg of enzyme from ~5 g
(wet weight) of E. coli was obtained. Fig.
3A shows the profile of products from pure LmDdl2 by TLC
analysis in which radiolabeled D-[14C]-Ala
was the tracer substrate and
D-[14C]-Ala-D-[14C]-Ala
and D-[14C]-Ala-D-Hbut products
were detected. D-Hbut was utilized as a surrogate substrate
for D-Lac in these assays as in earlier studies (8, 9, 13)
since D-Ala-D-Lac comigrates with
D-Ala-D-Ala on TLC while
D-Ala-D-Hbut depsipeptide migrates with higher
mobility. The enzyme LmDdl2 makes
D-[14C]-Ala-D-Hbut as does VanA
and, albeit more slowly, E. coli Y216F DdlB, shown as
positive controls for depsipeptide ligase activity, whereas wild-type
E. coli DdlB makes only dipeptide. In Fig. 3B, cognate incubations with D-[14C]-Lac as
tracer show the depsipeptide ligase capacity with D-Lac as
a nucleophilic hydroxy acid cosubstrate for LmDdl2.
Table I summarizes steady-state kinetic data for LmDdl2
for D-Ala1, D-Ala2, and
D-Lac, using a continuous coupled assay for ADP production
as previously reported (25). The affinity for D-Ala1 and D-Ala2 could
not be readily determined in contrast to several of the other enzyme
forms in the table. It is clear that Km2 for
D-Ala2 is very high and most probably
non-physiological. The kcat values for
D-Ala-D-Lac ligase activity of LmDdl2 (23 min1) and of the E. coli Y216F DdlB mutant (42 min
1) approximate those previously reported for VanA (45 min
1) (13). Typically, these kcat
values are less than those for D-Ala-D-Ala
dipeptide synthesis, perhaps reflecting the weaker nucleophilicity of
D-Lac versus D-Ala2 in
capturing the D-Ala1-PO3 intermediate. The best determined steady-state parameter for wild-type LmDdl2 was kcat/Km2 of 0.35 min
1 mM
1 for
D-Ala2 as the nucleophile cosubstrate and 1.2 for D-Lac as the nucleophile substrate for a catalytic
efficiency ratio of ~3/1 in favor of depsipeptide. By comparison, the
corresponding (kcat/KD-Lac)/(kcat/KD-Ala2)
for VanA is ~30/1. The E. coli DdlB Y216F mutant that has
gained D-Ala-D-Lac depsipeptide ligase activity has a corresponding ratio of 0.04. Thus, the LmDdl2 is intermediate in
its preferential catalytic efficiency to make
D-Ala-D-Lac instead of
D-Ala-D-Ala. Given the vancomycin resistance
phenotype and the exclusive detection of
D-Ala-D-Lac termini in UDP-MurNAc peptide intermediate (20, 21), it may be that LmDdl2 functions in depsipeptide
ligase mode in vivo.
In the LmDdl2 F261Y mutant (see below) in which no residual activity to
synthesize D-Ala-D-Lac is detectable, the
kcat/KD-Ala2 value has
increased three-fold (to 1.1 mM1
min
1), as shown by the data of Fig.
4C.
To test the proposition that Phe261 in LmDdl2, analogous to the Y216F mutant of E. coli DdlB (13), is involved in the gain of function depsipeptide ligase activity of wild-type LmDdl2, the LmDdl2 F261Y mutant was constructed, and the enzyme was purified (to ~90% homogeneity) with the prediction that it should retain dipeptide ligase activity but be selectively ablated for depsipeptide ligase activity. This enzyme has the activity shown in Fig. 4B as a function of added D-Lac. While wild-type LmDdl2 (Phe261) has both D-Ala-D-Ala ligase activity and D-Ala-D-Lac ligase activity, the F261Y enzyme has no detectable ability to utilize D-Lac (at up to 100 mM concentration) either at 3 or 10 mM concentrations of cosubstrate D-Ala. Wild-type LmDdl2 saturates at about 50 mM D-Lac. While the F261Y LmDdl2 is inactive with D-Lac, it actually has a more robust D-Ala-D-Ala ligase activity (Fig. 3C).
This work describes the purification and initial kinetic characterization of a D-Ala-D-Ala ligase for the first time from a Gram-positive bacterium, L. mesenteroides, known to possess intrinsic chromosomally mediated resistance to the antibiotic vancomycin (20, 21). As previously demonstrated for the enterococcal VanA enzyme (8, 9), LmDdl2 does indeed possess both dipeptide ligase (D-Ala-D-Ala) and depsipeptide ligase activity (D-Ala-D-Lac, D-Ala-D-Hbut), consistent with both the vancomycin-resistance phenotype and the detection of cell wall PG intermediates terminating in D-Ala-D-Lac2 (20, 21). These activities pinpoint this chromosomal D-Ala-D-Ala ligase as a key molecular determinant in the antibiotic resistance phenotype. The gain of depsipeptide ligase activity of LmDdl2 generalizes previous observations on VanA and VanB-containing drug-resistant enterococci to the naturally resistant Gram-positive soil bacterium and increases the probability that this will be the immunity mechanism for vancomycin producing steptomyces.
It has been noted that a striking correlation exists for the occurrence
of Tyr/Phe at position 216 with the vancomycin sensitivity/resistance phenotype (13). In the comparison of the Tyr216 or
equivalent residue (Fig. 5), it is conceivable that
there are three classes. The first class, with Tyr in that position, includes 11 proteins. Among them, eight proteins are known or proposed
to be D-Ala-D-Ala ligases (8, 9, 25, 29),
whereas VanC1, 2, 3 were predicted to be
D-Ala-D-Ser ligases since
D-Ala-D-Ser terminating UDP-MurNAc was detected
in enterococci containing these proteins (37). The lower affinity for
D-Ala-D-Ser (amide) versus
D-Ala-D-Ala (amide) is ascribed to steric clash
of the hydroxymethyl side chain of D-Ser in the complex
with vancomycin.
The second group includes four proteins from Gram-positive bacteria (Lc, Lm, Lp, Ls D-Ala-D-Ala ligases in Fig. 5) that contain Phe instead of Tyr. The Tyr/Phe7 replacement has previously been tested in one direction, by converting wild-type E. coli DdlB to the Y216F mutant (13). As summarized in Table II, this results in specific gain of function of depsipeptide ligase activity. Wild-type LmDdl2 with the Phe residue similarly has D-Ala-D-Lac ligase activity. The reverse mutation F261Y yields mutant LmDdl2 that retains an increasingly efficient D-Ala-D-Ala ligase but, indeed, has lost all detectable depsipeptide ligase activity. X-ray structures are available for wild-type E. coli DdlB (11) and now for the E. coli Y216F DdlB (14), but the altered ability to activate D-Lac C2-OH as a nucleophile is not yet clear. Crystal structures for the wild-type and mutant pair of LmDdls may also be needed to decipher the amine versus hydroxyl specificity in D-Ala2 versus D-Lac.
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In VanA and B isoforms, which may comprise the third group, the homology suggests a slightly altered loop region and no obviously discernible aromatic residue that is isofunctional to Phe216 or Phe261 of E. coli DdlB or LmDdls. This may indicate a microscopically different structural solution for a loop in VanA and VanB, permitting D-Lac to function as a nucleophilic cosubstrate. D-Ala-D-Lac has 800-1000-fold lower affinity for vancomycin compared with D-Ala-D-Ala because of the ester for amide substitution (8, 9).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U75444[GenBank].
We thank Roeger Flugel for assistance in using computer programs, Dr. Ivan Lessard for help in PCR, and the laboratory members for discussions.