Binding of Uridine 5‘-Diphosphate in the “Basic Patch” of the Zinc Deacetylase LpxC and Implications for Substrate Binding^,

Abstract

LpxC is a zinc metalloenzyme that catalyzes the first committed step in the biosynthesis of lipid A, a vital component of the outer membrane of Gram-negative bacteria. Accordingly, the inhibition of LpxC is an attractive strategy for the treatment of Gram-negative bacterial infections. Here, we report the 2.7 Å resolution X-ray crystal structure of LpxC from Aquifex aeolicus complexed with uridine 5′-diphosphate (UDP), and the 3.1 Å resolution structure of LpxC complexed with pyrophosphate. The X-ray crystal structure of the LpxC-UDP complex provides the first view of interactions likely to be exploited by the substrate UDP group in the "basic patch" of the active site. The diphosphate group of UDP makes hydrogen bond interactions with strictly conserved residue K239 as well as solvent molecules. The ribose moiety of UDP interacts with partially conserved residue E197. The UDP uracil group hydrogen bonds with both the backbone NH group and the backbone carbonyl group of E160, and with the backbone NH group of K162 through an intervening water molecule. Finally, the α-phosphate and uracil groups of UDP interact with R143 and R262 through intervening water molecules. The structure of LpxC complexed with pyrophosphate reveals generally similar intermolecular interactions in the basic patch. Unexpectedly, diphosphate binding in both complexes is accompanied by coordination to an additional zinc ion, resulting in the identification of a new metal-binding site termed the E-site. The structures of the LpxC-UDP and LpxC-pyrophosphate complexes provide new insights regarding substrate recognition in the basic patch and metal ion coordination in the active site of LpxC.The zinc metalloenzyme UDP -{3-O-[(R)-3-hydroxymyristoyl]}-N-acetylglucosamine deacetylase (LpxC) catalyzes the first irreversible step in the biosynthesis of lipid A ( Figure 1) (1-4), which is ultimately incorporated into the outer leaflet of the outer membrane of Gramnegative bacteria as the hydrophobic anchor of lipopolysaccharide (LPS) (5-9). 1 The exterior sheath of LPS serves as a protective barrier that resists penetration by many common antibiotics such as erythromycin (8)(9)(10)(11)(12)(13)(14). Importantly, lipid A is the toxic component of LPS that triggers a severe immune response to Gram-negative bacterial infections, which can lead to septic shock accompanied by acute hypotension, multiple organ failure, and death (15-18). Accordingly, inhibitors of LpxC and lipid A biosynthesis represent a potential treatment option for Gramnegative sepsis. † This work was supported by the National Institutes of Health grant GM49758. ‡ The atomic coordinates of LpxC complexed with uridine 5′-diphosphate and pyrophosphate have been deposited in the Protein Data Bank, www.rcsb.org, with accession codes 2IER and 2IES, respectively. *To whom correspondence should be addressed: Tel: 215-898-5714. Fax: 215-573-2201. E-mail: chris@sas.upenn.edu.. 1 Abbreviations: LpxC, UDP -{3-O-[(R)-3-hydroxymyristoyl]}-N-acetylglucosamine deacetylase; LPS, lipopolysaccha...

Structure of the Bacterial Deacetylase LpxC Bound to the Nucleotide Reaction Product Reveals Mechanisms of Oxyanion Stabilization and Proton Transfer

“…Structural superposition revealed that interactions to UDP observed in the A. aeolicus LpxC structure (25) are significantly different from those observed here in the product-bound E. coli LpxC structure. Although the ␤-phosphates are similarly positioned, the ␣-phosphate and ribose groups do not superimpose (Fig.…”

“…UDP and TU-514), the structure of LpxC bound to an intact reaction product precisely defines the ligand interactions required for LpxC function. Importantly, the structure reveals conformations of the GlcN and UDP moieties that are distinct from those inferred based on previous crystal structures (18,25). These differences are likely due to truncation of the ligand and possibly sequence divergence between A. aeolicus and E. coli LpxC.…”

“…2, A and B). In contrast to previous serendipitous observations of co-purified (25), Y. enterocolitica (green, PDB code 3nzk) (29), E. coli (red, PDB code 3p3g) (30), and P. aeruginosa (gray, PDB code 3uhm) (28). D, surface representation of E. coli LpxC with myr-UDP-GlcN.…”

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

“…First, the CHIR-090 threonyl group is located adjacent to, but does not occupy, the UDP-binding pocket, which is common to all LpxC orthologs (Fig. 3E) (16). Analysis of the UDP pocket reveals conserved hydrogen bond donors and acceptors, as well as hydrophobic surfaces that might be readily accessed by a CHIR-090 analog.…”

“…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.…”