Functional and Biochemical Analysis of theChlamydia trachomatisLigase MurE

Abstract

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-A₂pm) ligase activity. Nevertheless, there are no experimental data to confirm this hypothesis. In this paper we demonstrate that themurEgene fromChlamydia trachomatisis capable of complementing a conditionalEscherichia colimutant impaired in UDP-MurNAc-l-Ala-d-Glu:meso-A₂pm ligase activity. Recombinant MurE fromC. trachomatis(MurE_Ct) was overproduced in and purified fromE. coliin order to investigate its kinetic parameters in vitro. By use of UDP-MurNAc-l-Ala-d-Glu as the nucleotide substrate, MurE_Ctdemonstrated ATP-dependentmeso-A₂pm ligase activity with pH and magnesium ion optima of 8.6 and 30 mM, respectively. Other amino acids (meso-lanthionine, thellandddisomers of A₂pm,d-lysine) were also recognized by MurE_Ct.However, the activities for these amino acid substrates were weaker than that formeso-A₂pm. The specificity of MurE_Ctfor three possibleC. trachomatispeptidoglycan nucleotide substrates was also determined in order to deduce which amino acid might be present at the first position of the UDP-MurNAc-pentapeptide. Relativek_cat/K_mratios 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 ismeso-A₂pm. However, due to the lack of specificity of MurE_Ctfor nucleotide substrates in vitro, it is not obvious which amino acid is present at the first position of the pentapeptide.