Structural basis for the recognition of peptide RJPXD33 by acyltransferases in lipid A biosynthesis.

UDP-N-acetylglucosamine acyltransferase (LpxA) and UDP-3-O-(acyl)-glucosamine acyltransferase (LpxD) constitute the essential, early acyltransferases of lipid A biosynthesis. Recently, an antimicrobial peptide inhibitor, RJPXD33, was identified with dual affinity for LpxA and LpxD. To gain a fundamental understanding of the molecular basis of inhibitor binding, we determined the crystal structure of LpxA from Escherichia coli in complex with RJPXD33 at 1.9 Å resolutions. Our results suggest that the peptide binds in a unique modality that mimics (R)-ß-hydroxyacyl pantetheine binding to LpxA and displays how the peptide binds exclusive of the native substrate, acyl-acyl carrier protein. Acyltransferase binding studies with photo-labile RJPXD33 probes and truncations of RJPXD33 validated the structure and provided fundamental insights for future design of small molecule inhibitors. Overlay of the LpxA-RJPXD33 structure with E. coli LpxD identified a complementary peptide binding pocket within LpxD and serves as a model for further biochemical characterization of RJPXD33 binding to LpxD.

Extragenic suppressor mutations in ΔripA disrupt stability and function of LpxA.

BACKGROUND: Francisella tularensis is a Gram-negative bacterium that infects hundreds of species including humans, and has evolved to grow efficiently within a plethora of cell types. RipA is a conserved membrane protein of F. tularensis, which is required for growth inside host cells. As a means to determine RipA function we isolated and mapped independent extragenic suppressor mutants in ∆ripA that restored growth in host cells. Each suppressor mutation mapped to one of two essential genes, lpxA or glmU, which are involved in lipid A synthesis. We repaired the suppressor mutation in lpxA (S102, LpxA T36N) and the mutation in glmU (S103, GlmU E57D), and demonstrated that each mutation was responsible for the suppressor phenotype in their respective strains. We hypothesize that the mutation in S102 altered the stability of LpxA, which can provide a clue to RipA function. LpxA is an UDP-N-acetylglucosamine acyltransferase that catalyzes the transfer of an acyl chain from acyl carrier protein (ACP) to UDP-N-acetylglucosamine (UDP-GlcNAc) to begin lipid A synthesis. RESULTS: LpxA was more abundant in the presence of RipA. Induced expression of lpxA in the ΔripA strain stopped bacterial division. The LpxA T36N S102 protein was less stable and therefore less abundant than wild type LpxA protein. CONCLUSION: These data suggest RipA functions to modulate lipid A synthesis in F. tularensis as a way to adapt to the host cell environment by interacting with LpxA.

Dual targeting antibacterial peptide inhibitor of early lipid A biosynthesis.

UDP-3-O-(R-3-hydroxyacyl)GlcN N-acyltransferase (LpxD) has been shown to be essential to survival of lipid A producing Gram-negative bacteria. In this study, LpxD-binding peptides 12 amino acids in length were identified from a phage-bound random peptide library screen. Three peptides displayed antibacterial activity when expressed intracellularly, one of which (RJPXD33) represented 15% of the total hits. RJPXD33 binds to E. coli LpxD with a K(d) of 6 µM and is competitive with R-3-hydroxymyristoyl-ACP binding. RJPXD33 can be C-terminally fused in vivo with thioredoxin or N-terminally modified in vitro with ß-alanyl-fluorescein and maintain LpxD binding. The latter was used to develop an LpxD fluorescent binding assay used to evaluate unlabeled ligands and is amenable to small molecule library screening. Furthermore, RJPXD33 also binds to and inhibits E. coli UDP-N-acetylglucosamine acyltransferase (LpxA) with a K(d) of 20 µM, unearthing the possibility for the development of small molecule, dual-binding LpxA/LpxD inhibitors as novel antimicrobials.

A continuous fluorescent enzyme assay for early steps of lipid A biosynthesis.

UDP-N-acetylglucosamine acyltransferase (LpxA) and UDP-3-O-(R-3-hydroxyacyl)-glucosamine acyltransferase (LpxD) catalyze the first and third steps of lipid A biosynthesis, respectively. Both enzymes have been found to be essential for survival among gram-negative bacteria that synthesize lipopolysaccharide and are viable targets for antimicrobial development. Catalytically, both acyltransferases catalyze an acyl-acyl carrier protein (ACP)-dependent transfer of a fatty acyl moiety to a UDP-glucosamine core ring. Here, we exploited the single free thiol unveiled on holo-ACP after transfer of the fatty acyl group to the glucosamine ring using the thiol-specific labeling reagent, ThioGlo. The assay was continuously monitored as a change in fluorescence at λ(ex)=379 nm and λ(em)=513 nm using a microtiter plate reader. This assay marks the first continuous and nonradioactive assay for either acyltransferase.

Gradient elution isotachophoresis with direct ultraviolet absorption detection for sensitive amino acid analysis.

This work demonstrates coupling of the newly described electrophoretic enrichment technique of gradient elution isotachophoresis (GEITP) to a low-cost, conventional ultraviolet absorbance detector to realize sensitive measurements with a universal detector, eliminating the need for fluorescent analytes or derivatization. The effects of various parameters on enrichment were studied, including current density varied by leading electrolyte concentration, current density varied by applied electric field, and counter-flow acceleration across varying capillary inner diameters. Optimized parameters were applied to the enrichment and separation of the amino acids tryptophan (Trp) and tyrosine (Tyr). Limits of detection for Trp and Tyr were 51 and 215 nM, respectively, reflecting sensitivity enhancements of 860- and 1900-fold. Analysis times were less than 6 min, and peak height RSDs were less than 4%. A demonstration of enrichment and separation of these amino acids from artificial cerebrospinal fluid is additionally shown as a first step to realizing biochemical monitoring by GEITP.