Structures ofBacteroides fragilisuridine 5′-diphosphate-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (BfLpxA)

Abstract

Uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes a reversible reaction for adding an O-acyl group to the GlcNAc in UDP-GlcNAc in the first step of lipid A biosynthesis. Lipid A constitutes a major component of lipopolysaccharides, also referred to as endotoxins, which form the outer monolayer of the outer membrane of Gram-negative bacteria. Ligand-free and UDP-GlcNAc-bound crystal structures of LpxA from Bacteroides fragilis NCTC 9343, the most common pathogenic bacteria found in abdominal abscesses, have been determined and are presented here. The enzyme crystallizes in a cubic space group, with the crystallographic threefold axis generating the biological functional homotrimer and with each monomer forming a nine-rung left-handed β-helical (LβH) fold in the N-terminus followed by an α-helical motif in the C-terminus. The structure is highly similar to LpxA from other organisms. Yet, despite sharing a similar LβH structure with LpxAs from Escherichia coli and others, previously unseen calcium ions are observed on the threefold axis in B. fragilis LpxA to help stabilize the trimeric assembly.

Structures of Pseudomonas aeruginosa LpxA Reveal the Basis for Its Substrate Selectivity

“…coli and L. interrogans complex structures (backbone NH of residues Gly143 and Gly138, respectively) (Figure A,B) as well as in all other ortholog structures. ,,− However, this conformation is not observed in the P. aeruginosa LpxA structure (Figure ).…”

“…coli LpxA (PDB ID: 1LXA), there seems to be some variation at the C-terminal end of the third α-helix, involving different tilts compared to those of bacterial orthologs Acinetobacter baumannii (PDB ID: 4E6U), L. interrogans (PDB ID: 3HSQ), Campylobacter jejuni (PDB ID: 3R0S), Burkholderia thailandensis (PDB ID: 4EQY), Helicobacter pylori (PDB ID: 1J2Z), and Bacteroides fragilis (PDB ID: 4R36) and plant ortholog Arabidopsis thaliana (PDB ID: 3T57). …”

“…fragilis LpxA ortholog (PDB ID: 4R37). In the B. fragilis LpxA complex, the substrate shows a binding pose that is nearly identical to that of the P.…”

“…aeruginosa, a pathogenic Gram-negative bacterium with a wide range of antibiotic resistance mechanisms, has been causing increasing public health concern. , Due to its importance in LPS synthesis, LpxA is a valuable target in drug discovery against P. aeruginosa. , Even though eight ortholog LpxA structures have been previously solved, − only three were in complex with glucosamine ligands and the P. aeruginosa LpxA structure was still lacking.…”

“…LpxA is a soluble cytoplasmic protein that forms a functional homotrimer. , It shares sequence homology with LpxD, which also forms a homotrimer, unlike LpxC, which is a Zn 2+ -dependent monomeric enzyme that shares no sequence homology with other deacetylases . Previously solved crystal structures reveal that LpxA forms a left-handed parallel β-helix generated by ∼30 hexapeptide repeats. − Every turn in the β-helix is made up of a single hexapeptide . Conserved residues, identified previously through sequence alignments, assisted in the identification of the active site, which is located in a cleft formed by the interface of adjacent monomeric units of the trimer.…”