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-A₂pm) 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-A₂pm 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-A₂pm 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 A₂pm, d-lysine) were also recognized by MurE_Ct. However, the activities for these amino acid substrates were weaker than that for meso-A₂pm. 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-A₂pm. 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.Chlamydiae cause serious respiratory tract and genital infections in humans (9). They are obligately intracellular gram-negative bacteria, with a unique biphasic development cycle. Elementary bodies (EBs) are the infectious form of the organism and invade susceptible host cells. Once internalized, EBs differentiate into reticulate bodies (RBs), which have the capacity to divide (39, 40). The RBs are fragile and pleomorphic, whereas EBs are comparatively rigid and stable (19, 39). After repeated cycles of binary fission, the RBs differentiate into EBs, and the host cell lyses, releasing infectious EBs (1).In contrast to the vast majority of eubacteria, chlamydiae lack detectable amounts of peptidoglycan (PG), an essential polymer. PG is a giant macromolecule composed of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues cross-linked by short peptides. It determines the shape of bacteria, protects the cell from lysis due to internal osmotic pressure, and also plays a role in cell division. However, PG has not been detected in EBs (14, 20) or RBs (5).Although chlamydiae appear to lack PG, they contain penicillin-binding proteins and are sensitive to antibiotics that inhibit PG synthesis (5, 40). The Chlamydia trachomatis genome contains most of the genes coding for proteins involved in, or associated with, PG synthesis (54). Chlamydial MurA, MurC-Ddl, and the MurC domain of the latter fusion protein are active in vitro and complement Escherichia coli mutants deficient in the respective enzymes (23, 32, 33). Furthermore, proteomic analysis reveals that the murE gene product, which was assigned as UDP-MurNAc-l-Ala-d-Glu:meso-diaminopimelic acid ligase (UDP-MurNAc-l-Ala-d-Glu:meso-A₂pm ligase), is expressed in RBs (52).MurE ligases catalyze the addition of the third amino acid residue to the peptide chain of PG. This residue, generally a diamino acid, is usually meso-A₂pm for gram-negative bacteria and bacilli, and l-lysine for gram-positive bacteria, although other amino acids (for example, l-ornithine, meso-lanthionine, ll-A₂pm, l-diaminobutyric acid, or l-homoserine) occur in certain species (6, 50, 57). In many organisms, the third residue of the peptide chain participates in PG cross-linking; consequently, the MurE enzyme is highly specific for the relevant amino acid so as to avoid incorporation of incorrect amino acids into the macromolecule, which could result in deleterious morphological changes and cell lysis (35). Crystallization of MurE from E. coli (MurE_Ec) has permitted analysis of the structural basis for this high specificity (22). Sequence alignments of different MurE orthologues have also revealed the specific consensus sequences DNPR and D(D/N)P(N/A) located in the binding pockets for meso-A₂pm and l-Lys, respectively (11, 17). Chlamydia trachomatis MurE (MurE_Ct) possesses the DNPR motif, which suggests that it adds meso-A₂pm (17). However, there are no experimental data to confirm this prediction.In this paper we report for the first time the overproduction and purification of MurE_Ct, as well as a detailed investigation of its in vivo and in vitro biochemical properties. These studies contribute to our understanding of the nature and properties of the PG biosynthetic enzymes in chlamydiae and do indeed suggest that MurE_Ct has meso-A₂pm ligase activity.