Binding of uridine 5'-diphosphate in the "basic patch" of the zinc deacetylase LpxC and implications for substrate binding.

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 Gram-negative 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 (8-14). 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 Gram-negative sepsis. Figure 1 Proposed catalytic mechanism of LpxC. The structure of LpxC from Aquifex aeolicus was first determined by X-ray crystallography at 2.0 A resolution and reveals a ~20 A-deep active site located at the interface of two topologically similar α + β domains (19). Enzyme crystals were prepared in the presence of excess Zn2+, resulting in the formation of a binuclear zinc cluster composed of a catalytic zinc ion (Zn2+A, coordinated by H79, H238, D242, and a solvent molecule with tetrahedral geometry) and an inhibitory zinc ion (Zn2+B, coordinated by E78, H265, a solvent molecule, and a fatty acid with tetrahedral geometry) (19).22 The aliphatic tail of the fatty acid was bound in a ~15 A-long hydrophobic tunnel leading out of the active site cleft and was hypothesized to represent the binding mode of the 3-O-(R-3-hydroxymyristoyl) group of the substrate (19). Consistent with this hypothesis, the subsequently determined NMR structure (20-21) and the X-ray crystal structure (22) of LpxC complexed with the substrate analogue inhibitor TU-514 (Figure 2) revealed the binding of the corresponding fatty acid substituent in the hydrophobic tunnel. Figure 2 (A) LpxC substrate, UDP –{3-O-[(R)-3-hydroxymyristoyl]}-N-acetylglucosamine. (B) Substrate analogue inhibitor, TU-514 (Ki ~ 650 nM (27)). (C) uridine 5′-diphosphate (UDP). Experimental results acquired to date suggest that LpxC catalyzes substrate deacetylation through a mechanism involving a general acid-base catalyst pair (Figure 1) (23). In the first step of catalysis, E78 likely serves as a general base by abstracting a proton from a zinc-bound water molecule, thereby promoting nucleophilic attack at the substrate and formation of a tetrahedral oxyanion intermediate (19). Site directed mutagenesis studies (21, 23, 25) and X-ray crystal structures of LpxC-cacodylate and LpxC-palmitate complexes (23) are consistent with the general base function proposed for E78. The X-ray crystal structure of LpxC complexed with the tetrahedral cacodylate anion further implicates T191 and H265 in the stabilization of the tetrahedral intermediate and its flanking transition states (23). Protonation of the leaving amino group by general acid H265 (pKa ~ 8 (21)) facilitates the collapse of the tetrahedral intermediate to yield the final products (21, 23, 25). The NMR (20-21) and X-ray crystal (22) structures of the LpxC–TU-514 complex yield generally similar inferences about substrate binding in the active site of LpxC despite significant differences in the zinc coordination polyhedron and intermolecular interactions of the hexose ring. The X-ray crystal structure reveals that the TU-514 hydroxamate moiety chelates Zn2+A to form a pentacoordinate zinc complex with square pyramidal coordination geometry (22). Hydrogen bond interactions between the hydroxamate OH group and D242 and H265, and the hydroxamate C=O group and T191, also stabilize the zinc complex. Hexose ring substituents hydrogen bond with E197 and H58, and K239 through a water-mediated hydrogen bond interaction. Although TU-514 is similar in structure to the actual LpxC substrate (Figure 2), it lacks the 3-hydroxy group of the 3-O myristoyl substituent and a UDP group (26, 27). Therefore, the structures of LpxC–TU-514 complexes (20-22) provide no information on the binding of the UDP portion of an intact substrate. Based on analysis of the refined NMR structure of the LpxC–TU-514 complex (21), the negatively charged diphosphate group of UDP is hypothesized to interact with residues in the so-called “basic patch” defined by K239, R143, R262, and H265. The binding of a sulfate ion in the basic patch of LpxC complexed with cacodylate (PDB entry 1YHC) (23) supports this hypothesis (21). In order to explicitly define the role of the basic patch in substrate binding, we now report the crystal structure of the LpxC-UDP complex at 2.7 A resolution. We also report the structure of LpxC complexed with pyrophosphate at 3.1 A resolution. Surprisingly, both structures reveal that diphosphate binding in the basic patch is accommodated by a new metal ion in addition to an array of hydrogen bond interactions. Together, these structures provide key inferences on substrate recognition in the basic patch and changes in metal ion coordination that may accompany catalysis.