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-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, UDPMurNAc-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.

Structural characterization of muropeptides from Chlamydia trachomatis peptidoglycan by mass spectrometry resolves “chlamydial anomaly”

“…The chlamydial MurC enzyme, which ligates the first amino acid of the stem peptide to MurNAc, has been shown to lack selectivity for alanine over glycine or serine (23). Moreover, chlamydial MurE and MurF (which add subsequent amino acids to the growing stem peptide) are able to use glycine-containing peptides as substrate (10,24). These observations suggest that C. trachomatis PG may contain a mixture of alanine and glycine as the first amino acid in the stem peptide.…”

Biochemical characterisation of the chlamydial MurF ligase, and possible sequence of the chlamydial peptidoglycan pentapeptide stem

“…Such problems were already encountered for the puriWcation of MurE Ct (Patin et al 2009). To overcome them and increase the solubility of the protein, diVerent induction temperatures (15, 22, and 37°C), IPTG concentrations (0.2 and 0.75 mM) and E. coli host strains (BL21, C43, and Rosetta) were tested (data not shown).…”

“…The second amino acid of the pentapeptide in conventional peptidoglycan is always D-glutamic acid which after addition can be hydroxylated to generate threo-3-hydroxyglutamic acid, or amidated at the -carboxyl group to yield D-isoglutamine (Schleifer and Kandler 1972;Vollmer et al 2008). Although it has not proved possible to produce MurD Ct in an active form, MurE Ct does recognise UDP-MurNAcdipeptides terminating in D-Glu in vitro (Patin et al 2009). Therefore, it is plausible that the second amino acid in the chlamydial pentapeptide in vivo is indeed D-Glu.…”

“…Therefore, it is plausible that the second amino acid in the chlamydial pentapeptide in vivo is indeed D-Glu. MurE Ct demonstrates meso-diaminopimelic acid (meso-A 2 pm)-adding activity (Patin et al 2009). It exhibited a preference for meso-A 2 pm since other amino acids known to occur at position 3 in conventional peptidoglycan were less favoured substrates (Patin et al 2009).…”

“…MurE Ct demonstrates meso-diaminopimelic acid (meso-A 2 pm)-adding activity (Patin et al 2009). It exhibited a preference for meso-A 2 pm since other amino acids known to occur at position 3 in conventional peptidoglycan were less favoured substrates (Patin et al 2009). Consequently, the presence of meso-A 2 pm at the third position of the peptide in chlamydial peptidoglycan is likely.…”