D. Patin et al. / Biochimie 92 (2010) 1793e1800
1799
-ala-
Overexpressing individual Mur ligases genes in E. coli also did not
result in an increased rate of formation of peptidoglycan and cell-
wall thickening. This is likely also the case in S. aureus and other
Gram-positive bacteria. In the latter species, however, over-
production of some of the enzymes involved in earlier steps of the
pathway were shown to have an impact on the flow of metabolites
and cell peptidoglycan content. Recently for instance, over-
producing the PEP:UDP-GlcNAc enolpyruvyl transferase MurZ in
S. aureus was shown to increase by 20% the cell peptidoglycan
content [51]. Furthermore, it was earlier shown that S. aureus
mutant strains had developed low-level resistance to vancomycin
paralog amino acid invariants in the active site of the UDP-MurNAc-
nine: -glutamate ligase (MurD), Biochemistry 38 (1999) 12240e12247.
L
D
[
8] S.S. Eveland, D.L. Pompliano, M.S. Anderson, Conditionally lethal Escherichia
coli murein mutants contain point defects that map to regions conserved
among murein and folyl poly-g-glutamate ligases: identification of a ligase
superfamily, Biochemistry 36 (1997) 6223e6229.
9] A. Bouhss, S. Dementin, J. van Heijenoort, C. Parquet, D. Blanot, MurC and
MurD synthetases of peptidoglycan biosynthesis: borohydride trapping of
acyl-phosphate intermediates, Meth. Enzymol. 354 (2002) 189e196.
[
[
[
[
[
[
10] C.A. Smith, Structure, function and dynamics in the mur family of bacterial cell
wall ligases, J. Mol. Biol. 362 (2006) 640e655.
11] K.H. Schleifer, O. Kandler, Peptidoglycan types of bacterial cell walls and their
taxonomic implications, Bacteriol. Rev. 36 (1972) 407e477.
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J. Biol. Chem. 235 (1960) PC7ePC8.
(
VISA strains) by thickening the cell-wall peptidoglycan layer [52],
a finding which was then correlated to an increased expression of
the fructose-6-phosphate:glutamine amidotransferase GlmS
involved in early steps of synthesis of peptidoglycan precursor
UDP-GlcNAc [53]. No concomitant variation of the enzymatic
activities catalyzing subsequent steps in the pathway, such as the
Mur ligases, was reported in these cases, suggesting that these
intermediate activities were present in excess and adapted for
sustaining increased rates of peptidoglycan synthesis. The recent
demonstration that the MurC ligase from Corynebacterium gluta-
micum could be phosphorylated both in vitro and in vivo by the
PknA serine/threonine protein kinase, and that this modification
dramatically decreased the enzyme activity [54], suggests that Mur
ligase activities could potentially be negatively regulated by such
a mechanism, at least in some bacterial species.
In conclusion, the present work has allowed the purification and
enzymatic study of the four staphylococcal Mur ligases, enzymes
which participate in the synthesis of an essential cell-wall polymer
and therefore are potential targets for antibacterial agents. The
availability of these enzymes and acquired knowledge on their
enzymatic properties will be useful for the search for inhibitors,
a work that has already started in the case of MurESa [55e59].
14] E. Ito, J.L. Strominger, Enzymatic synthesis of the peptide in bacterial uridine
nucleotides. II. Enzymatic synthesis and addition of D-alanyl-D-alanine, J. Biol.
Chem. 237 (1962) 2696e2703.
[15] E. Ito, J.L. Strominger, Enzymatic synthesis of the peptide in bacterial uridine
nucleotides. I. Enzymatic addition of -alanine, -glutamic acid, and -lysine,
J. Biol. Chem. 237 (1962) 2689e2695.
16] E. Ito, J.L. Strominger, Enzymatic synthesis of the peptide in bacterial uridine
L
D
L
[
nucleotides. III. Purification and properties of
Chem. 239 (1964) 210e214.
L-lysine-adding enzyme, J. Biol.
[
17] S.G. Nathenson, J.L. Strominger, E. Ito, Enzymatic synthesis of the peptide in
bacterial uridine nucleotides. IV. Purification and properties of the
acid-adding enzyme, J. Biol. Chem. 239 (1964) 1773e1776.
D-glutamic
[18] Y. Mizuno, M. Yaegashi, E. Ito, Purification and properties of uridine diphos-
phate N-acetylmuramate: -alanine ligase, J. Biochem. 74 (1973) 525e538.
19] K. Kurokawa, S. Nishida, M. Ishibashi, H. Mizumura, K. Ueno, T. Yutsudo,
L
[
H. Maki, K. Murakami, K. Sekimizu, Staphylococcus aureus MurC participates in
L-alanine recognition via histidine 343, a conserved motif in the shallow
hydrophobic pocket, J. Biochem. 143 (2008) 417e424.
[
20] A.W. Walsh, P.J. Falk, J. Thanassi, L. Discotto, M.J. Pucci, H.-T. Ho, Comparison
of the D-glutamate-adding enzymes from selected Gram-positive and Gram-
negative bacteria, J. Bacteriol. 181 (1999) 5395e5401.
[
21] A. Babi ꢀc , D. Patin, A. Boniface, M. Hervé, D. Mengin-Lecreulx, S. Pe ꢀc ar, S. Gobec,
D. Blanot, Chemoenzymatic synthesis of the nucleotide substrates of the Mur
ligases, in: D. Kikelj (Ed.), 5th Joint Meeting on Medicinal Chemistry, June 17-
21, Portoro ꢀz , Slovenia, Medimond Srl, Bologna, Italy, 2007, pp. 1e4.
[22] A. Boniface, A. Bouhss, D. Mengin-Lecreulx, D. Blanot, The MurE synthetase
from Thermotoga maritima is endowed with an unusual
activity, J. Biol. Chem. 281 (2006) 15680e15686.
D-lysine adding
Acknowledgments
[23] D. Mengin-Lecreulx, C. Michaud, C. Richaud, D. Blanot, J. van Heijenoort,
Incorporation of LL-diaminopimelic acid into peptidoglycan of Escherichia coli
mutants lacking diaminopimelate epimerase encoded by dapF, J. Bacteriol.
70 (1988) 2031e2039.
[24] C. Michaud, D. Blanot, B. Flouret, J. van Heijenoort, Partial purification and
specificity studies of the -glutamate-adding and -alanyl- -alanine-adding
enzymes from Escherichia coli K12, Eur. J. Biochem. 166 (1987) 631e637.
25] K.E. Amrein, B. Takacs, M. Stieger, J. Molnos, N.A. Flint, P. Burn, Purification and
characterization of recombinant human p50csk protein-tyrosine kinase from
an Escherichia coli expression system overproducing the bacterial chaperones
GroES and GroEL, Proc. Natl. Acad. Sci. U S A 92 (1995) 1048e1052.
This work was supported by the European Commission through
the EUR-INTAFAR project (LSHM-CT-2004-512138), the Centre
National de la Recherche Scientifique (PICS 3729), the Ministère de
l’Education Nationale, de la Recherche et de la Technologie
1
D
D
D
[
[
[
(
(
scholarship to S.D.), the Délégation Générale pour l’Armement
Contrats Jeune Chercheur 036000104 and 056000030 to A.B.), and
the Franco-Slovene Proteus programme.
26] A. Caravano, D. Mengin-Lecreulx, J.-M. Brondello, S.P. Vincent, P. Sinaÿ,
Synthesis and inhibition properties of conformation probes for the mutase-
Appendix. Supplementary data
catalyzed UDP-galactopyranose/furanose interconversion, Chem. Eur. J.
2003) 5888e5898.
9
(
27] F. Pompeo, J. van Heijenoort, D. Mengin-Lecreulx, Probing the role of cysteine
residues in glucosamine-1-phosphate acetyltransferase activity of the
bifunctional GlmU protein from Escherichia coli: site-directed mutagenesis
and characterization of the mutant enzymes, J. Bacteriol. 180 (1998)
4
799e4803.
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