Crystal structure of LpxC, a zinc-dependent deacetylase essential for endotoxin biosynthesis

199

389

Streptococcus pneumoniae peptidoglycan GlcNAc deacetylase (SpPgdA) protects the Gram-positive bacterial cell wall from host lysozymes by deacetylating peptidoglycan GlcNAc residues. Deletion of the pgda gene has been shown to result in hypersensitivity to lysozyme and reduction of infectivity in a mouse model. SpPgdA is a member of the family 4 carbohydrate esterases, for which little structural information exists, and no catalytic mechanism has yet been defined. Here we describe the native crystal structure and product complexes of SpPgdA biochemical characterization and mutagenesis. The structural data show that SpPgdA is an elongated three-domain protein in the crystal. The structure, in combination with mutagenesis, shows that SpPgdA is a metalloenzyme using a His-His-Asp zinc-binding triad with a nearby aspartic acid and histidine acting as the catalytic base and acid, respectively, somewhat similar to other zinc deacetylases such as LpxC. The enzyme is able to accept GlcNAc 3 as a substrate (Km ‫؍‬ 3.8 mM, kcat ‫؍‬ 0.55 s ؊1 ), with the N-acetyl of the middle sugar being removed by the enzyme. The data described here show that SpPgdA and the other family 4 carbohydrate esterases are metalloenzymes and present a step toward identification of mechanismbased inhibitors for this important class of enzymes.crystal structure ͉ metalloenzyme

Section: Resultsmentioning

confidence: 99%

Structure and metal-dependent mechanism of peptidoglycan deacetylase, a streptococcal virulence factor

Blair

Schüttelkopf

MacRae

et al. 2005

199

389

“…Extensive structural studies show that LpxC displays a novel ''␤-␣-␣-␤ sandwich'' fold, formed by two domains with similar topologies (13)(14)(15)(16)(17)(18)(19)(20). Each domain consists of a layer of two helices packing against a ␤-sheet containing mixed parallel and antiparallel strands, and the overall structure is characterized by the ␣-helices of each domain sandwiched between the ␤-sheets.…”

mentioning

confidence: 99%

“…LpxC binds the myristoyl chain of the hydroxamate-containing substrate-analog TU-514 within a hydrophobic passage that is largely formed by Insert II and a loop (␤1-␤2 loop) from Domain I (14,15,17). The lining of this passage mostly consists of conserved hydrophobic residues, providing high-affinity binding for the acyl chain of the substrate, and potentially, for the diverse range of potent LpxC inhibitors (15,20).…”

mentioning

confidence: 99%

Structure of the deacetylase LpxC bound to the antibiotic CHIR-090: Time-dependent inhibition and specificity in ligand binding

Barb

Jiang

Raetz

et al. 2007

125

The UDP-3- O -( R -3-hydroxyacyl)- N -acetylglucosamine deacetylase LpxC is an essential enzyme of lipid A biosynthesis in Gram-negative bacteria and a promising antibiotic target. CHIR-090, the most potent LpxC inhibitor discovered to date, displays two-step time-dependent inhibition and kills a wide range of Gram-negative pathogens as effectively as ciprofloxacin or tobramycin. In this study, we report the solution structure of the LpxC–CHIR-090 complex. CHIR-090 exploits conserved features of LpxC that are critical for catalysis, including the hydrophobic passage and essential active-site residues. CHIR-090 is adjacent to, but does not occupy, the UDP-binding pocket of LpxC, suggesting that a fragment-based approach may facilitate further optimization of LpxC inhibitors. Additionally, we identified key residues in the Insert II hydrophobic passage that modulate time-dependent inhibition and CHIR-090 resistance. CHIR-090 shares a similar, although previously unrecognized, chemical scaffold with other small-molecule antibiotics such as L-161,240 targeting LpxC, and provides a template for understanding the binding mode of these inhibitors. Consistent with this model, we provide evidence that L-161,240 also occupies the hydrophobic passage.

“…Many pathogens, such as strains of Pseudomonas aeruginosa in cystic fibrosis patients (29,30), now are resistant to commercially available antibiotics. Therefore it is necessary to develop new antibacterial agents that target previously unexploited systems, such as the enzymes that assemble the lipid A component of lipopolysaccharide (2,8,9,(31)(32)(33)(34)(35). The cytosolic acyltransferase LpxA catalyzes the first step of the lipid A pathway ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Structure of UDP-N-acetylglucosamine acyltransferase with a bound antibacterial pentadecapeptide

Williams

Immormino

Gewirth

et al. 2006

UDP-GlcNAc acyltransferase (LpxA) catalyzes the first step of lipid A biosynthesis, the transfer of the R-3-hydroxyacyl chain from R-3-hydroxyacyl acyl carrier protein (ACP) to the glucosamine 3-OH group of UDP-GlcNAc. LpxA is essential for the growth of Escherichia coli and related Gram-negative bacteria. The crystal structure of the E. coli LpxA homotrimer, determined previously at 2.6 Å in the absence of substrates or inhibitors, revealed that LpxA contains an unusual, left-handed parallel ␤-helix fold. We now present the crystal structure at 1.8 Å resolution of E. coli LpxA in a complex with a pentadecapeptide, peptide 920. Three peptides, each of which adopts a ␤-hairpin conformation, are bound per LpxA trimer. The peptides are located at the interfaces of adjacent subunits in the vicinity of the three active sites. Each peptide interacts with residues from both adjacent subunits. Peptide 920 is a potent inhibitor of E. coli LpxA (Ki ‫؍‬ 50 nM). It is competitive with respect to acyl-ACP but not UDP-GlcNAc. The compact ␤-turn structure of peptide 920 bound to LpxA may open previously uncharacterized approaches to the rational design of LpxA inhibitors with antibiotic activity.antibiotic ͉ crystal structure ͉ Escherichia coli ͉ inhibitor ͉ outer membrane L ipopolysaccharide constitutes the outer monolayer of the outer membrane in Gram-negative bacteria (1-3). Lipopolysaccharide maintains the integrity of the outer membrane, which functions as a barrier to molecules Ͼ500 Da (4). Lipid A (endotoxin) is the hydrophobic moiety that anchors lipopolysaccharide into the outer membrane (1, 2). It is a potent activator of the human innate immune system via Toll-like receptor 4 (TLR4) (5-7). The minimal lipopolysaccharide required for growth usually consists of two 3-deoxy-D-manno-octulosonic acid (Kdo) residues attached to lipid A ( Fig. 1; ref. 2). Because lipid A biosynthetic enzymes have no mammalian counterparts, they are attractive targets for the design of new antibiotics (10, 11).Kdo 2 -lipid A is synthesized by a conserved pathway consisting of nine enzymes (1, 2). LpxA catalyzes the first step (Fig. 1), the thermodynamically unfavorable 3-O-acylation of UDP-GlcNAc (K eq ϭ 0.01) (12-14). The crystal structure of Escherichia coli LpxA, previously determined at 2.6 Å (15), revealed that the enzyme is a homotrimer. It adopts a distinctive, left-handed parallel ␤-helix fold ( Fig. 2 A and B), which is specified by the presence of 24 complete and six partial hexad repeats (15). Three contiguous hexad repeats (18-aa residues) fold into one coil of the ␤-helix (Fig. 2 A and B). Many other bacterial acyl and acetyl transferases later were shown to possess this fold (17)(18)(19)(20)(21)(22).Structures of LpxA have not been solved in the presence of substrates or inhibitors. Mutagenesis suggests that the active site is located in a large cleft between adjacent subunits (8, 23). This region contains several conserved basic residues (K76, H122, H125, H144, H160, and R204) (23). H125 (Fig. 2 C and D) may be the catalytic ...