Phosphatidic acid is the central intermediate in membrane phospholipid synthesis and is generated by two acyltransferases in a pathway conserved in all life forms. The second step in this pathway is catalyzed by 1-acyl-sn-glycero-3-phosphate acyltransferase, called PlsC in bacteria. The crystal structure of PlsC from Thermotoga maritima reveals an unusual hydrophobic/aromatic N-terminal two-helix motif linked to an acyltransferase αβ domain that contains the catalytic HX4D motif. PlsC dictates the acyl chain composition of the 2-position of phospholipids, and the acyl chain selectivity ‘ruler’ is an appropriately placed and closed hydrophobic tunnel. This was confirmed by site-directed mutagenesis and membrane composition analysis of Escherichia coli cells expressing the mutated proteins. MD simulations reveal that the two-helix motif represents a novel substructure that firmly anchors the protein to one leaflet of the membrane. This binding mode allows the PlsC active site to acylate lysophospholipids within the membrane bilayer using soluble acyl donors.
Background: Chlamydia trachomatis has a phospholipid composition that resembles its eukaryotic host, but it contains branched-chain fatty acids of chlamydial origin. Results: The inhibition of the enoyl-acyl carrier protein reductase (FabI) in chlamydial fatty acid synthesis blocks C. trachomatis replication. Conclusion: Bacterial FASII is required for C. trachomatis proliferation. Significance: FabI is a therapeutic target against C. trachomatis.
The human-restricted pathogen Neisseria gonorrhoeae encodes a single N-acetylmuramyl-L-alanine amidase involved in cell separation (AmiC), as compared with three largely redundant cell separation amidases found in Escherichia coli (AmiA, AmiB, and AmiC). Deletion of amiC from N. gonorrhoeae results in severely impaired cell separation and altered peptidoglycan (PG) fragment release, but little else is known about how AmiC functions in gonococci. Here, we demonstrated that gonococcal AmiC can act on macromolecular PG to liberate cross-linked and non-cross-linked peptides indicative of amidase activity, and we provided the first evidence that a cell separation amidase can utilize a small synthetic PG fragment as substrate (GlcNAc-MurNAc(pentapeptide)-GlcNAc-MurNAc (pentapeptide)). An investigation of two residues in the active site of AmiC revealed that Glu-229 is critical for both normal cell separation and the release of PG fragments by gonococci during growth. In contrast, Gln-316 has an autoinhibitory role, and its mutation to lysine resulted in an AmiC with increased enzymatic activity on macromolecular PG and on the synthetic PG derivative. Curiously, the same Q316K mutation that increased AmiC activity also resulted in cell separation and PG fragment release defects, indicating that activation state is not the only factor determining normal AmiC activity. In addition to displaying high basal activity on PG, gonococcal AmiC can utilize metal ions other than the zinc cofactor typically used by cell separation amidases, potentially protecting its ability to function in zinc-limiting environments. Thus gonococcal AmiC has distinct differences from related enzymes, and these studies revealed parameters for how AmiC functions in cell separation and PG fragment release.
Peptidoglycan (PG)2 is a critical structural macromolecule that makes up the cell wall surrounding the cytoplasmic membrane of nearly all bacteria. This cage-like structure is made up of long polysaccharide strands that are composed of repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) with short peptide stems (3-5 amino acids) covalently attached to the MurNAc moiety (1). Depending on the organism, some proportion of peptide stems are crosslinked to peptides on adjacent strands, whereas others remain non-cross-linked. Cross-linking provides structural integrity for the macromolecule and contributes to the determination of bacterial shape.Neisseria gonorrhoeae (the gonococcus or GC) is a Gramnegative bacterium, obligate human pathogen, and etiologic agent of the sexually transmitted infection gonorrhea. Gonorrhea is the second most common reportable bacterial infection in the United States with an estimated 820,000 cases annually (2). A robust inflammatory response is the cause of much of the damage observed during GC infection and is triggered by the release of lipo-oligosaccharide, porin proteins, and fragments of PG (3-5). In particular, N. gonorrhoeae release a greater abundance of PG fragments than Escherichia co...
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