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Journal of Bacteriology, November 1998, p. 5792-5795, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
DdlN from Vancomycin-Producing Amycolatopsis
orientalis C329.2 Is a VanA Homologue with
D-Alanyl-D-Lactate Ligase Activity
C. Gary
Marshall and
Gerard D.
Wright*
Department of Biochemistry, McMaster
University, Hamilton, Ontario, Canada L8N 3Z5
Received 25 June 1998/Accepted 31 August 1998
 |
ABSTRACT |
Vancomycin-resistant enterococci acquire high-level resistance to
glycopeptide antibiotics through the synthesis of peptidoglycan terminating in D-alanyl-D-lactate. A key enzyme
in this process is a D-alanyl-D-alanine ligase
homologue, VanA or VanB, which preferentially catalyzes the synthesis
of the depsipeptide D-alanyl-D-lactate. We
report the overexpression, purification, and enzymatic characterization of DdlN, a VanA and VanB homologue encoded by a gene of the
vancomycin-producing organism Amycolatopsis orientalis
C329.2. Evaluation of kinetic parameters for the synthesis of peptides
and depsipeptides revealed a close relationship between VanA and
DdlN in that depsipeptide formation was kinetically preferred at
physiologic pH; however, the DdlN enzyme demonstrated a narrower
substrate specificity and commensurately increased affinity for
D-lactate in the C-terminal position over VanA. The
results of these functional experiments also reinforce the results of
previous studies that demonstrated that glycopeptide resistance enzymes
from glycopeptide-producing bacteria are potential sources of
resistance enzymes in clinically relevant bacteria.
| |
TEXT |
The origin of antibiotic resistance
determinants is of significant interest for several reasons, including
the prediction of the emergence and spread of resistance patterns, the
design of new antimicrobial agents, and the identification of potential reservoirs for resistance elements. Antibiotic resistance can occur either through spontaneous mutation in the target or by the
acquisition of external genetic elements such as plasmids or
transposons which carry resistance genes (7). The origins of
these acquired genes are varied, but it has long been recognized that
potential reservoirs are antibiotic-producing organisms which naturally
harbor antibiotic resistance genes to protect themselves from the
actions of toxic compounds (6).
High-level resistance to glycopeptide antibiotics such as
vancomycin and teicoplanin in vancomycin-resistant enterococci (VRE) is
conferred by the presence of three genes, vanH,
vanA (or vanB), and vanX, which, along
with auxiliary genes necessary for inducible gene expression, are found
on transposons integrated into plasmids or the bacterial genome
(1, 20). These three genes are essential to resistance and
serve to change the C-terminal peptide portion of the peptidoglycan
layer from D-alanyl-D-alanine
(D-Ala-D-Ala) to
D-alanyl-D-lactate
(D-Ala-D-Lac). This change results in the loss
of a critical hydrogen bond between vancomycin and the
D-Ala-D-Ala terminus and in a 1,000-fold
decrease in binding affinity between the antibiotic and the
peptidoglycan layer, which is the basis for the bactericidal action of
this class of compounds (5). The vanH gene
encodes a D-lactate dehydrogenase which provides the
requisite D-Lac (3, 5), while the
vanX gene encodes a highly specific DD-peptidase
which cleaves only D-Ala-D-Ala produced
endogenously while leaving D-Ala-D-Lac intact
(19, 21). The final gene, vanA or
vanB, encodes an ATP-dependent D-Ala-D-Lac ligase (4, 8, 10). This
enzyme has sequence homology with the chromosomal
D-Ala-D-Ala ligases, which are essential for
peptidoglycan synthesis but which generally lack the ability to
synthesize D-Ala-D-Lac (9).
We have recently cloned vanH, vanA, and
vanX homologues from two glycopeptide
antibiotic-synthesizing organisms: Amycolatopsis orientalis
C329.2, which produces vancomycin, and Streptomyces toyocaensis NRRL 15009, which produces A47934 (14). In
addition, the vanH-vanA-vanX gene cluster was identified in
several other glycopeptide producers. We have also
demonstrated that the VanA homologue from S. toyocaensis
NRRL 15009 can synthesize D-Ala-D-Lac in vitro
and in the glycopeptide-sensitive host Streptomyces
lividans (15, 16). We now report the expression of the
A. orientalis C329.2 VanA homologue DdlN in
Escherichia coli, its purification, and its enzymatic
characterization. These data reinforce the striking similarity between
vancomycin resistance elements in VRE and
glycopeptide-producing organisms and support the
possibility of a common origin for these enzymes.
Expression, purification, and specificity of DdlN.
DdlN was
overexpressed in E. coli under the control of the
bacteriophage T7 promoter. The construct gave good yields of highly purified enzyme following a four-step purification procedure (Table 1; Fig. 1).
Like other DD-ligases, DdlN behaved like a dimer in
solution (not shown).
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FIG. 1.
Purification of DdlN from E. coli BL21
(DE3)/pETDdlN. Proteins were separated on an SDS-11% polyacrylamide
gel and stained with Coomassie blue. Lane 1, molecular mass markers
(masses are noted at the left in kilodaltons); lane 2, whole-cell
lysate; lane 3, ammonium sulfate fraction (20 to 50% saturation);
lane 4, Sephacryl S200; lane 5, Q Sepharose; lane 6, phenyl Superose.
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The amino acid substrate specificity of DdlN was assessed by incubation
of
14C-
D-Ala with all 20 common amino acids in
the
D configuration.
Purified DdlN catalyzed the synthesis
of
D-Ala-
D-Ala in addition
to that of
several other mixed dipeptides, including
D-Ala-
D-Met
and
D-Ala-
D-Phe (Fig.
2). Thus, DdlN exhibits a
substrate specificity
which is similar to that of VanA (
4),
with the capacity to
synthesize not only
D-Ala-
D-Ala but also mixed dipeptides with
bulky side chains in the C-terminal position.
Importantly, DdlN is a depsipeptide synthase with the ability to
synthesize
D-Ala-
D-Lac,
D-Ala-
D-hydroxybutyrate (Hbut), and
D-Ala-
D-hydroxyvalerate (Fig.
2). However, unlike VanA (
5),
D-hydroxycaproate and
D-phenyllactate are not
substrates (not
shown). Thus, DdlN is a broad-spectrum
D-Ala-
D-X ligase with depsipeptide
synthase
activity.
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FIG. 2.
Substrate specificity of DdlN. Autoradiogram from
thin-layer chromatography analysis of DdlN substrate specificity. All
reaction mixtures contained 2.5 mM D-Ala and 1 mM ATP, and
the radiolabel was 14C-D-Ala, except where
noted. Lane 1, D-Ala; lane 2, D-Lac with
14C-D-Lac label; lane 3, D,L-methionine; lane 4, DL-phenylalanine; lane
5, D-Hbut; lane 6, D-hydroxyvalerate. Letters
indicate the following: A, D-Ala-D-Lac; B,
D-Lac; C, D-Ala-D-Met; D,
D-Ala-D-Phe; E,
D-Ala-D-Hbut; F,
D-Ala-D-hydroxyvalerate.
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|
Characterization of D-Ala-D-X ligase
activity.
Following the initial assessment of the specificity of
the enzyme, several substrates were selected for quantitative analysis by evaluation of their steady-state kinetic parameters (Table 2). DdlN has two amino acid (or hydroxy
acid) Km values. Steady-state kinetic plots
indicated that, like other DD-ligases, the N-terminal Km (Km1) was significantly lower
(higher specificity) than the C-terminal Km
(Km2). Since the former value is expected to be independent of the C-terminal substrate, only
Km2 values were determined and are reported
here.
DdlN showed good
D-Ala-
D-Ala ligase activity
but with a very high and physiologically questionable
Km2 (21 mM). On the other
hand,
D-Ala-
D-Lac synthesis was excellent, with a
4-fold decrease
in
kcat, compared to
D-Ala-
D-Ala synthesis, which was offset by
a
52-fold drop in
Km that resulted in a >12-fold
increase in specificity
(
kcat/
Km2).
D-Hbut was also a good substrate, with a
kcat/
Km2
comparable to
that of
D-Ala.
Steady-state kinetic parameters for
D-Ala-
D-X
formation showed trends similar to those found with both VanA and DdlN.
For
example, the
kcat values between VanA and
DdlN were virtually
the same for most substrates. There were
significant differences,
however. For instance, while the
Km2 values for
D-Ala were very
high
for all three enzymes, DdlN does have greater affinity for
D-Ala, with a 1.8- and 7.9-fold lower
Km2 than those of VanA and
DdlM, respectively.
Additionally, the
Km2 for
D-Lac was
17.8-
and 2.7-fold lower than those for VanA and DdlM. Thus, DdlN has
a
more restrictive specificity for the C-terminal residue than
VanA,
which is compensated for by a higher affinity for the critical
substrate
D-Lac.
pH dependence of peptide versus that of depsipeptide synthesis
activity.
The partitioning of the syntheses of
D-Ala-D-Ala and
D-Ala-D-Hbut in VanA and other
depsipeptide-competent DD-ligases has been shown to be pH
dependent (17). Determination of the pH dependence of DdlN
in synthesizing peptide versus depsipeptide (Fig.
3) directly paralleled the results
obtained with VanA in similar experiments. At lower pHs (<7),
D-Ala-D-Hbut synthesis predominates and is
exclusive at a pH of <6 (Fig. 3). At pH 7.5, levels of synthesis of
D-Ala-D-Hbut and
D-Ala-D-Ala are relatively equal, while at a
pH greater than 8, the capacity to synthesize peptide overtakes
the capacity to synthesize depsipeptide, although the latter is never
abolished.
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FIG. 3.
pH dependence of partitioning of the syntheses of
peptide and depsipeptide by DdlN. (A) Autoradiogram of a thin-layer
chromatography separation of the products of reaction mixtures
containing 14C-D-Ala, unlabeled D-Ala, and
D-Hbut. (B) Quantification of reaction products following
phosphorimage analysis. Filled circles, D-Ala-D-Hbut; open
circles, D-Ala-D-Ala.
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The partitioning of the formation of peptide versus depsipeptide as a
function of pH by DdlM is comparable to that by VanA
and
depsipeptide-competent mutants of DdlB (
17), which
show
essentially exclusively depsipeptide formation at lower pHs and
increasing peptide formation as the pH increases. This implies
a
potential role for the protonated ammonium group of
D-Ala2
in
second-substrate recognition and suggests a mechanism for the
discrimination between
D-Ala and
D-Lac at
physiologic pH. The
structural basis for this distinction remains
obscure for DdlB
and VanA or DdlN.
Concluding remarks.
Resistance to vancomycin and other
glycopeptides is mediated through the synthesis of a
peptidoglycan which does not terminate with the canonical
D-Ala-D-Ala dipeptide. Thus, enterococci which exhibit the VanC phenotype, which consists of low-level, noninducible resistance to vancomycin only, have peptidoglycan terminating in
D-Ala-D-Ser (19). On the other hand,
bacteria which are constitutively resistant to high concentrations of
glycopeptides, such as lactic acid bacteria and VRE
exhibiting the VanA or VanB phenotype (high-level inducible resistance
to vancomycin), incorporate the depsipeptide D-Ala-D-Lac into their cell walls (2, 12,
13). The enzymes responsible for the intracellular synthesis of
D-Ala-D-Lac not surprisingly have significant
amino acid sequence similarity with D-Ala-D-Ala
ligases, which are responsible for
D-Ala-D-Ala synthesis in all bacteria with a
cell wall (9).
The
D-Ala-
D-Lac synthases can be
subdivided into two groups based on sequence homology: those
found in the constitutively
resistant lactic acid bacteria and those
found in glycopeptide-producing
organisms and VanA or VanB
VRE (
9,
14). The former have more
similarity with exclusive
D-Ala-
D-Ala ligases. Indeed, single
point
mutations in
D-Ala-
D-Ala ligases which yield
sequences more
similar to those of lactic acid bacterium
D-Ala-
D-Lac ligases
are sufficient to induce
significant depsipeptide synthase activity
in these enzymes
(
17). Similarly, mutational studies of the
D-Ala-
D-Lac ligase from
Leuconostoc
mesenteroides have demonstrated
that the converse also holds
(
18). On the other hand, the molecular
basis for
depsipeptide synthesis by the VanA or VanB ligases is
unknown, in large
part due to the lack of protein structural information
on which to base
mutational studies, unlike the situation with
D-Ala-
D-Ala ligases, where the
E. coli DdlB structure serves as
a template for mechanistic research
(
11).
Significantly, a major difference in the VanA or VanB ligases and other
DD-ligases lies in the amino acid sequence of the
-loop
region, which closes off the active site of DdlB (
11)
and
has been shown to contribute amino acid residues with the
capacity
to control the syntheses of
D-Ala-
D-Ala and
D-Ala-
D-Lac,
notably, Tyr216 (
17,
18). Until recently, the VanA and VanB
ligases were
exceptional in amino acid structure and had no known
homologues. The sequencing of resistance genes from
glycopeptide-producing
bacteria has uncovered enzymes with
>60% homology to VanA or VanB
and which are virtually superimposable
in the critical
-loop
region (
14,
15). One of these, DdlM
from
S. toyocaensis NRRL
15009, has been shown to have
D-Ala-
D-Lac ligase ability (
15,
16),
although no rigorous analysis of this activity has been
performed. The
results presented here demonstrate that DdlN from
the vancomycin
producer
A. orientalis C329.2 not only is a
D-Ala-
D-Lac
ligase but also has significant
functional homology with VanA.
It is not known at present if, like
S. toyocaensis NRRL 15009
(
16),
A. orientalis C329.2 also possess a
D-Ala-
D-Ala-exclusive
ligase, though the
presence of a
vanX gene (
14) suggests that
it
may.
These studies demonstrate that DdlN cloned from a vancomycin-producing
bacterium is a
D-Ala-
D-Lac ligase which has not
only
amino acid sequence homology with the
DD-ligases from
VRE but
also functional homology. Thus, VanA, VanB, DdlN, and DdlM have
likely evolved from similar origins. The fact that a
vanH-vanA-vanX gene cluster can be found in other
glycopeptide producers as well
(
14) suggests
that the genes now found in VRE may have originated
in
glycopeptide-producing bacteria. Our finding that
overexpressed,
purified, DdlN shows many enzymatic
characteristics similar (though
not identical) to those of VanA
suggests that the genes from glycopeptide-producing
bacteria can be important in elucidating biochemical and protein
structural aspects of the VRE proteins.
| |
ACKNOWLEDGMENTS |
This research was supported by an operating grant from the Natural
Sciences and Engineering Research Council of Canada (NSERC), by a
Medical Research Council of Canada (MRC) graduate studentship to
C.G.M., and by an MRC Scholar Award to G.D.W.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, McMaster University, 1200 Main St. W., Hamilton, Ontario, Canada L8N 3Z5. Phone: (905) 525-9140, ext. 22943. Fax: (905) 522-9033. E-mail: wrightge{at}fhs.csu.mcmaster.ca.
| |
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Journal of Bacteriology, November 1998, p. 5792-5795, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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