Functional and biochemical analysis of the Chlamydia trachomatis ligase MurE.
Chlamydiae are unusual obligately intracellular bacteria that do not synthesize detectable peptidoglycan. However, they possess genes that appear to encode products with peptidoglycan biosynthetic activity. Bioinformatic analysis predicts that chlamydial MurE possesses UDP-MurNAc-L-Ala-D-Glu:meso-diaminopimelic acid (UDP-MurNAc-L-Ala-D-Glu:meso-A(2)pm) ligase activity. Nevertheless, there are no experimental data to confirm this hypothesis. In this paper we demonstrate that the murE gene from Chlamydia trachomatis is capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-L-Ala-D-Glu:meso-A(2)pm ligase activity. Recombinant MurE from C. trachomatis (MurE(Ct)) was overproduced in and purified from E. coli in order to investigate its kinetic parameters in vitro. By use of UDP-MurNAc-L-Ala-D-Glu as the nucleotide substrate, MurE(Ct) demonstrated ATP-dependent meso-A(2)pm ligase activity with pH and magnesium ion optima of 8.6 and 30 mM, respectively. Other amino acids (meso-lanthionine, the ll and dd isomers of A(2)pm, D-lysine) were also recognized by MurE(Ct.) However, the activities for these amino acid substrates were weaker than that for meso-A(2)pm. The specificity of MurE(Ct) for three possible C. trachomatis peptidoglycan nucleotide substrates was also determined in order to deduce which amino acid might be present at the first position of the UDP-MurNAc-pentapeptide. Relative k(cat)/K(m) ratios for UDP-MurNAc-L-Ala-D-Glu, UDP-MurNAc-L-Ser-D-Glu, and UDP-MurNAc-Gly-D-Glu were 100, 115, and 27, respectively. Our results are consistent with the synthesis in chlamydiae of a UDP-MurNAc-pentapeptide in which the third amino acid is meso-A(2)pm. However, due to the lack of specificity of MurE(Ct) for nucleotide substrates in vitro, it is not obvious which amino acid is present at the first position of the pentapeptide.
Patin
Delphine
D
Université Paris-Sud, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, CNRS, Laboratoire des Enveloppes Bactériennes et Antibiotiques, UMR 8619, Orsay F-91405, France.
Bostock
Julieanne
J
Blanot
Didier
D
Mengin-Lecreulx
Dominique
D
Chopra
Ian
I
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002692:Chlamydia trachomatis / Q000201:enzymology*; D003067:Coenzymes / Q000494:pharmacology; D003960:Diaminopimelic Acid / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives / Q000378:metabolism
D001426:Bacterial Proteins; D003067:Coenzymes; D004151:Dipeptides; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C544599:uridine 5'-diphosphoryl N-acetylmuramoyl-alanyl-glutamate; D003960:Diaminopimelic Acid; D000255:Adenosine Triphosphate; D008025:Ligases; D008274:Magnesium