Structure of a small-molecule inhibitor complexed with GlmU from Haemophilus influenzae reveals an allosteric binding site

Abstract

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential enzyme in aminosugars metabolism and an attractive target for antibiotic drug discovery. GlmU catalyzes the formation of uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc), an important precursor in the peptidoglycan and lipopolisaccharide biosynthesis in both Gram-negative and Gram-positive bacteria. Here we disclose a 1.9 A resolution crystal structure of a synthetic small-molecule inhibitor of GlmU from Haemophilus influenzae (hiGlmU). The compound was identified through a high-throughput screening (HTS) configured to detect inhibitors that target the uridyltransferase active site of hiGlmU. The original HTS hit exhibited a modest micromolar potency (IC(50) approximately 18 microM in a racemic mixture) against hiGlmU and no activity against Staphylococcus aureus GlmU (saGlmU). The determined crystal structure indicated that the inhibitor occupies an allosteric site adjacent to the GlcNAc-1-P substrate-binding region. Analysis of the mechanistic model of the uridyltransferase reaction suggests that the binding of this allosteric inhibitor prevents structural rearrangements that are required for the enzymatic reaction, thus providing a basis for structure-guided design of a new class of mechanism-based inhibitors of GlmU.

Scheme 1.

Figure 1.

hiGlmU in complex with allosteric inhibitor 1. (A) View of the electrostatic surface potential of hiGlmU at the uridyltransferase active site and the allosteric region. Positive electrostatic potential is colored blue. Negative potential is colored red. Inhibitor 1 bound at the allosteric site is colored in the following atom colors: carbon, green; nitrogen, blue; oxygen, red; chlorine, orange. UDP-GlcNAc (PDB code 2v0i) shown at the substrate-binding site is colored in the following atom colors: carbon, yellow; nitrogen, blue; oxygen, red. (B) View of an OMIT electron density map contoured at 3σ level. (C) A schematic representation of inhibitor 1/hiGlmU interactions drawn using the MOE program (Chemical Computing Group, CCG). Hydrophobic residues are colored with a green interior; polar residues are colored in light purple. Basic residues are further annotated by a blue interior ring, and acidic residues with a red ring. The hydrogen-bonding interaction between the E224 side chain of hiGlmU and inhibitor 1 is drawn with a green arrowhead. (D) View of the superimposed hiGlmU coordinates from the complex with inhibitor 1 (colored in magenta), the product UDP-GlcNAc (colored in blue; PDB code 2v0i) and the apo form (colored in gold; PDB code 2v0h). In the UDP-GlcNAc and inhibitor 1 bound structures, the active site undergoes a substantial structural rearrangement, including the movement of residues N146, K156, and A160 by 5.5 Å, 5.9 Å, and 6.9 Å, respectively (distance measures between Cα pairs). Binding of inhibitor 1 prevents the structural rearrangement from open (apo-like) to closed (product-bound) conformations.

Scheme 2.

Scheme 3.