Functional and Biochemical Analysis of the Chlamydia trachomatis Ligase MurE

Abstract

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 -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.