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Journal of Bacteriology, December 2000, p. 6827-6830, Vol. 182, No. 23
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Comparison of the
UDP-N-Acetylmuramate:L-Alanine Ligase Enzymes
from Mycobacterium tuberculosis and
Mycobacterium leprae
Sebabrata
Mahapatra,
Dean C.
Crick, and
Patrick J.
Brennan*
Department of Microbiology, Colorado State
University, Fort Collins, Colorado 80523-1677
Received 5 June 2000/Accepted 11 September 2000
 |
ABSTRACT |
In the peptidoglycan of Mycobacterium leprae,
L-alanine of the side chain is replaced by glycine. When
expressed in Escherichia coli, MurC
(UDP-N-acetyl-muramate:L-alanine ligase) of
M. leprae showed Km and
Vmax for L-alanine and glycine
similar to those of Mycobacterium tuberculosis MurC,
suggesting that another explanation should be sought for the presence
of glycine.
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TEXT |
Some chemical differences exist in
the peptidoglycan of Mycobacterium spp. compared to other
bacteria (3). Mycobacterial muramic acid is thought to be
glycolylated instead of acetylated (14). In the case of
Mycobacterium leprae, the first amino acid in the
tetrapeptide side chain of the peptidoglycan is Gly instead of
L-Ala (5), as found in Mycobacterium
tuberculosis and many other bacteria, implying that the M. leprae genome may encode a unique
UDP-N-acetylmuramate:L-Ala/Gly
(UDP-MurNAc:L-Ala/Gly) ligase (MurC) specific for the
addition of Gly to UDP-MurNAc. These special structural features of
mycobacterial peptidoglycan suggest the presence of unique enzymes that
could be exploited as drug targets.
The genes that encode MurC from several organisms have been sequenced
(1, 6, 8, 11, 13), and the Escherichia coli MurC
has been overexpressed and characterized (7, 11). However,
the mycobacterial counterparts have not been studied. The availability
of the genome sequences of M. tuberculosis (4) and M. leprae
(ftp://ftp.sanger.ac.uk/pub/pathogens/leprae/) provides an opportunity
to study the enzymes of these two pathogenic species, especially
important in the case of M. leprae, which is not accessible to direct enzymatic study.
murC genes of Mycobacterium.
The complete
sequence of the open reading frame of MLCB268.01c was revealed from the
assembled genome sequence of M. leprae; it corresponds to bp
1084518 to 1086003. The resulting protein contains 595 amino acid
residues with a theoretical molecular mass of 51 kDa, very similar to
M. tuberculosis MurC (about 79% identity) but with only
~34% identity to E. coli MurC. Both Rv2152c (M. tuberculosis) and MLCB268.01 (M. leprae) are found
within the mra cluster and contain eight of nine invariant
amino acids (2) that align perfectly with known MurCs from
other organisms (Fig. 1). However, the
MurC homologs found outside the mra clusters (Rv3712 and
MLCB2407.24c) have only ~22% identity with the putative MurCs found
within the clusters, and four of the nine invariant amino acids either
are absent or did not align.
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FIG. 1.
Alignment of amino acid residues of MurC from M. tuberculosis, M. leprae, and E. coli,
showing conserved residues (highlighted).
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Cloning, expression, and purification of
UDP-N-MurNAc:L-Ala ligase (MurC).
Rv2152c and Rv3712 were amplified from M. tuberculosis H37Rv
genomic DNA and cloned into the pET29a+ vector (Novagen, Madison, Wis.) (16), yielding pSM201 and pSM203, respectively.
The M. leprae MLCB268.01 and MLCB2407.24c genes were
amplified from M. leprae genomic DNA and cloned into pET28a+
and pET29a+, respectively, yielding pSM206 and pSM208, respectively
(16, 17). E. coli BL21(DE3) harboring plasmid
pSM201, pSM203, pSM206, or pSM208 was grown in Luria-Bertani broth
containing kanamycin, induced with
isopropyl-
-D-thiogalactopyranoside, lysed by sonication on ice, and centrifuged at 30,000 × g for 30 min
(17). The resulting supernatant containing the soluble
His-tagged fusion proteins were loaded on a nickel-nitrilotriacetic
acid (Ni-NTA) resin (Qiagen) column (18) which was washed
with 20 mM Tris-HCl (pH 8.0)-10 mM MgCl2-2 mM
-mercaptoethanol-30 mM imidazole (pH 8.0) and 0.5 M NaCl, and the
His-tagged proteins were eluted from the column with buffer containing
300 mM imidazole (pH 7.5) (18). Protein-containing fractions
were analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) (10) to show a high degree
of purification (Fig. 2).
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FIG. 2.
SDS-PAGE gel showing the purification of Rv2152c (A) and
MLCB268.01c (B). Whole-cell lysate of uninduced E. coli
BL21(DE3) cells harboring pSM201 and pSM206 (lanes 1A and 1B,
respectively), whole-cell lysates of
isopropyl--D-thiogalactopyranoside-induced E. coli BL21(DE3) cells overproducing M. tuberculosis and
M. leprae MurC (Lanes 2A and 2B, respectively), and
clarified cell extracts (supernatant) obtained by centrifugation at
30,000 × g of the whole-cell lysate (lanes 3A and 3B).
Note that most of the overexpressed protein was insoluble and hence
gives the impression of lesser expression. Purified MurC proteins are
shown in lanes 4A and 4B. In each panel, the positions of molecular
size markers are shown on the left.
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Assay for UDP-MurNAc:L-Ala ligase (MurC).
Purified
fractions were pooled, and imidazole was removed by dialysis. The
UDP-MurNAc:L-Ala and UDP-MurNAc:Gly ligase activities were
assayed as described by Liger et al. (12). For this purpose, UDP-MurNAc was prepared by a two-step coupled enzymatic conversion of
UDP-GlcNAc to UDP-MurNAc according to Jin et al. (9) and identified through negative-ion fast atom bombardment-mass spectroscopy (FAB-MS) (the expected mass of 679 was observed) and nuclear magnetic resonance (NMR) (300 MHz). The following signals were clearly identified by 1H NMR spectroscopy in heavy water at 300 MHz: d7.94 (doublet, j = 8.1HZ, H-6; uracyl), d 5.96 (doublet, j = 4.5HZ, H-1; ribosyl), d 5.96 (doublet,
j = 8.1HZ, H-5; uracyl), d 5.60 broadened doublet (j = 4.2HZ, H-1; muramyl), d 2.03 (singlet, methyl;
N-acetyl muramyl), and d 1.32 (doublet, j = 6.6HZ, methyl; lactyl-muramyl). Reaction mixtures contained
100 mM Tris-HCl (pH 8.6), 25 mM
(NH4)2SO4, 20 mM MgCl2,
2 mM -mercaptoethanol, 1 mM UDP-MurNAc, 2 mM ATP, 50 µM
L-[14C]Ala (specific activity, 164 mCi/mmol)
(ICN Radiochemicals, Irvine, Calif.) or [14C]Gly (46.87 mCi/mmol) (NEN Life Science Products, Boston, Mass.), and a
predetermined amount of crude cell lysate or purified enzyme in a
25-µl reaction mix. Reactions were conducted under conditions in
which product formation was linear with respect to both time and
protein concentration. Reactions were stopped by the addition of 10 µl of glacial acetic acid and briefly centrifuged, and 3.5 µl of
the supernatant was applied to a silica gel thin-layer chromatography plate which was developed in isobutyric acid-1 M ammonium
hydroxide (5:3) to separate the reaction product from unreacted amino
acids. Radioactivity was measured using Bioscan Imaging Scanner System 200-IBM (Bioscan Inc., Washington, D.C.). The proportion of counts of
substrate and product compared to the total counts applied to the plate
was used to calculate enzyme activity.
The purified proteins arising from cloned Rv2152c and MLCB268.01c
showed good ligase activity using both
L-Ala and Gly as
substrates (Fig.
3 and
4). The
products of the ligase reactions
were also analyzed by MS;
UDP-MurNAc-
L-Ala gave the expected molecular
weight of 750, and the UDP-MurNAc-Gly gave the expected molecular
weight of 736. The
Km and
Vmax of both
Rv2152c and MLCB268.01c
were determined in the presence of either Gly
or
L-Ala (Fig.
3 and
4). Rv2152c showed an apparent
Km of 38 µM and a
Vmax
of 220
pmol/mg/min for Gly. When assayed with various amounts of
L-Ala,
this enzyme showed an apparent
Km of 14 µM and a
Vmax
of 1,200
pmol/mg/min. Even though the
Km values
for both of these substrates
were similar, the
Vmax for
L-Ala was found to be much
greater
than that seen for Gly, indicating better catalysis with
L-Ala
as the substrate. Similar results were obtained with
MLCB268.01c;
this
M. leprae enzyme had an apparent
Km of 25 µM and a
Vmax
of
76 pmol/mg/min for Gly, and, when assayed with various amounts
of
L-Ala, this enzyme showed an apparent
Km of 10 µM and a
Vmax of 460 pmol/mg/min.
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FIG. 3.
Effect of amino acid concentration on the rate of
UDP-MurNAc-[14C]Gly (A) or
UDP-MurNAc-L-[14C]Ala (B) biosynthesis by
purified Rv2152c from M. tuberculosis. The apparent
Km and Vmax values were
derived from a double-reciprocal plot of these data (inset).
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FIG. 4.
Effect of amino acid concentration on the rate of
UDP-MurNAc:[14C]Gly (A) or
UDP-MurNAc:L-[14C]Ala (B) biosynthesis by
purified MLCB268.01c from M. leprae. The apparent
Km and Vmax values were
derived from a double-reciprocal plot of these data (inset).
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Nonactive MurC homologs outside the mra cluster.
The MurC homologues Rv3712 and MLCB2407.24c were also expressed in
E. coli as C-terminally His-tagged proteins using the T7 expression system, resulting in soluble proteins capable of being purified by Ni-NTA column chromatography. However, the purified proteins as well as the crude cell lysate from the overproducing E. coli cells showed no significant ligase activity when tested.
Molecular organization of the M. leprae, M. tuberculosis, and E. coli mra clusters.
In
general, the basic genetic organization of the mra gene
cluster responsible for peptidoglycan biosynthesis (19) of
M. tuberculosis, M. leprae, and E. coli is similar except for four additional open reading frames in
the M. tuberculosis mra cluster between pbpB and
murE (Fig. 5). Clearly,
Rv2152c from M. tuberculosis and MLCB268.01c from M. leprae encode the MurC enzymes of their respective species, in
that the E. coli-overexpressed enzymes were enzymatically
active, indicating that they underwent proper folding even when
expressed in a nonhomologous system. The properties of these two
ligases are very similar. The calculated apparent Km for Gly of both ligases was found to be much
lower than the reported Km value of ~2.5 to 10 mM for Gly of E. coli MurC (7, 12). The
mycobacterial MurCs also had similar Km values
for L-Ala, and in both cases, this value was slightly lower
than the Km for Gly. However, the apparent
Vmax for L-Ala is much higher than
that for Gly in both cases, suggesting better catalysis of L-Ala ligase activity.
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FIG. 5.
Comparison of the chromosomal organization of the
mra clusters in M. tuberculosis, M. leprae, and E. coli.
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The other two open reading frames (Rv3712 and MLCB2407.24c) that show
homology to
E. coli murC do not appear to encode any
ligase
activity, probably due to the absence of four of the nine
invariant
amino acids found in bona fide members of the MurC enzyme
family.
Therefore it can be concluded that
M. tuberculosis and
M. leprae, like other bacteria, have only one such ligase.
Thus,
the presence of a Gly-specific ligase can apparently be ruled
out
as the reason for the specific occurrence of Gly instead of
L-Ala in the
M. leprae peptidoglycan.
M. leprae is always derived
from host tissue because it is not
possible to cultivate it in
vitro, which may be due to the unusual
peptidoglycan structure
in this species. When
E. coli and
Salmonella cells are grown in
human epithelial cells,
changes in the chemical composition of
the peptidoglycan are observed
(
15). From the data presented
here, it can be hypothesized
that, in
M. leprae, the incorporation
of Gly into
peptidoglycan is due to a combination of the substrate
specificity of
the MurC and the nature of the intracellular
environment.
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ACKNOWLEDGMENTS |
We thank Philip Draper for his helpful discussions.
M. tuberculosis genomic DNA was obtained from J. T. Belisle through NIH, NIAID contract NO1 AI-75320. M. leprae
genomic DNA was obtained through the resources of NIH, NIAID contract
NO1 AI-55262. This work was supported by grant NIH, NIAID 18357 and contract NIH, NIAID NO1 AI-55262.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Colorado State University, Fort Collins, CO 80523-1677. Phone: (970) 491-6700. Fax: (970) 491-1815. E-mail:
pbrennan{at}cvmbs.colostate.edu.
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Journal of Bacteriology, December 2000, p. 6827-6830, Vol. 182, No. 23
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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