Garry D. Dotson

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 β-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.

Structural Basis for the Recognition of Peptide RJPXD33 by Acyltransferases in Lipid A Biosynthesis

Background: Peptide RJPXD33 binds to and inhibits both LpxA and LpxD acyltransferases. Results: The crystal structure of the antibacterial peptide RJPXD33 complexed to E. coli LpxA was determined. Conclusion: RJPXD33 binds to E. coli LpxA in a unique modality that mimics the (R)-β-hydroxyacyl pantetheine moiety of substrate acyl-ACP. Significance: Bioactive, dual binding LpxA/LpxD peptides raise the possibility of designing less resistance-prone peptidomimetics and/or small molecule antibacterials. 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)-β-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

BackgroundFrancisella 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.ResultsLpxA 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.ConclusionThese 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.

Kinetic characterization of human phosphopantothenoylcysteine synthetase

Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the formation of phosphopantothenoylcysteine from (R)-phosphopantothenate and L-cysteine with the concomitant consumption of a nucleotide triphosphate. Herein, the human coaB gene encoding PPCS is cloned into pET23a and overexpressed in E. coli BL21(DE3), to yield 10mg of purified enzyme per liter of culture. Detailed kinetic studies found that this PPCS follows a similar Bi Uni Uni Bi Ping Pong mechanism as previously described for the E. faecalis PPCS, except that the human enzyme can use both ATP and CTP with similar affinity. One significant difference for human PPCS catalysis with respect to ATP and CTP is that the enzyme shows cooperative binding of ATP, measured as a Hill constant of 1.7. PPCS catalysis under CTP conditions displayed Michaelis constants of 265 microM, 57 microM, and 16 microM for CTP, PPA, and cysteine, respectively, with a kcat of 0.53+/-0.01 s(-1) for the reaction. Taking into account the cooperativity under ATP condition, PPCS exhibited Michaelis constants of 269 microM, 13 microM, and 14 microM for ATP, PPA, and cysteine, respectively, with a kcat of 0.56 s(-1) for the reaction. Oxygen transfer studies found that 18O from [carboxyl-18O] phosphopantothenate is incorporated into the AMP or CMP produced during PPCS catalysis, consistent with the formation of a phosphopantothenoyl cytidylate or phosphopantothenoyl adenylate intermediate, supporting similar catalytic mechanisms under both CTP and ATP conditions. Inhibition studies with GTP and UTP as well as product inhibition studies with CMP and AMP suggest that human PPCS lacks strong nucleotide selectivity.

Conversion of the covalent intermediate 3-fluoro-2-phospho-lactyl-EPTase to 3-fluoro-2-phospholactyl-UDP-GlcNAc

The 3-fluoro-2-phospholactyl-UDP-GlcNAc enolpyruvyl transferase (EPTase) intermediate is prepared by the incubation of 3-fluorophosphoenolpyruvate (3-FPEP) and 3-deoxy-UDP-GlcNAc with “PEPfree” UDP-GlcNAc enolpyruvyl transferase (EPTase). This intermediate is converted directly into the 3-fluoro-2-phospholactyl-UDP-GlcNAc intermediate by incubation with UDP-GlcNAc. Utilization of UDP-GalNAc as substrate results in the formation of two fluoro-containing tetrahedral intermediates but in a ratio different from that seen with UDP-GlcNAc.