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 Å 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 ; 18 mM 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.Keywords: uridyltransferase; allosteric inhibitors; mechanism; UDP-N-acetylglucosamine N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU, also referred to as UDP-GlcNAc pyrophosphorylase) is an essential, bifunctional enzyme that catalyzes the production of UDP-N-acetylglucosamine (UDP-GlcNAc), an important precursor in bacterial peptidoglycan and lipopolysaccharide biosynthesis (Anderson and Raetz 1987). In the first step of the reaction, GlmU catalyzes the transfer of the acetyl group from acetyl-CoA to glucosamine-1-phosphate (GlcN-1-P) to produce N-acetylglucosamine-1-phosphate (GlcNAc-1-P):In the second reaction, uridyl monophosphate is transferred from UTP to GlcNAc-1-P to produce UDP-GlcNAc and pyrophosphate (PPi) (Mengin-Lecreulx and van Reprint requests to: Igor Mochalkin, Eastern Point Road, Groton, CT 06340, USA; e-mail: Igor.Mochalkin@pfizer.com; fax: (860) 715-3149.Abbreviations: GlmU, N-Acetylglucosamine-1-phosphate uridyltransferase; UDP-GlcNAc, uridine-diphospho-N-acetylglucosamine; GlcN-1-P, glucosamine-1-phosphate; GlcNAc-1-P, N-acetylglucosamine-1-phosphate; PPi, pyrophosphate; HTS, high-throughout screening; hiGlmU, GlmU from Haemophilus influenzae; ecGlmU, GlmU from Escherichia coli; saGlmU, GlmU from Staphylococcus aureus; PEG, polyethylene glycol.Article published online ahead of print. Article and publication date are at

In Vitro Validation of Acetyltransferase Activity of GlmU as an Antibacterial Target in Haemophilus influenzae

“…As synthesis of UDP-GlcNAc is essential for peptidoglycan formation and, in Gram-negative species, synthesis of lipopolysaccharides, inhibition of GlmU leads to a loss of cell wall integrity, resulting in cell death (5,6). Quinazolines and aminopiperidines are the first reported inhibitors of the uridyl transferase domain of Haemophilus influenzae GlmU (Protein Data Bank codes 2WOV and 2WOW) (7). Inhibitors of the acetyltransferase of Escherichia coli GlmU have also been identified (8).…”

PknB-Mediated Phosphorylation of a Novel Substrate, N-Acetylglucosamine-1-Phosphate Uridyltransferase, Modulates Its Acetyltransferase Activity

“…3b, which is also noted in the case of S. pneumoniae GlmU 19 and, to a lesser extent, in E. coli GlmU 18 and H. influenzae GlmU. 20 In addition, the low average B-values reveal that this domain is stabilized upon ligand binding. The interactions stabilizing UDP-GlcNAc in the active site are shown in Fig.…”

“…Crystal structures of GlmU from Escherichia coli, Haemophilus influenzae, and Streptococcus pneumoniae are available. [18][19][20] However, in GlmU from M. tuberculosis, we noted an interesting 30-amino-acid extension at the C-terminus that was absent in the aforementioned orthologs (Fig. 2a).…”

“…The structure of GlmU revealed the presence of two domains responsible for the two distinct biochemical activities that GlmU carries out (i.e., uridyltransfer and acetyltransfer). [18][19][20] Analysis of solvent-exposed residues suggested potential threonines that are likely to be phosphorylated by PknB (Fig. 2c).…”

High-throughput screen identifies small molecule inhibitors targeting acetyltransferase activity of Mycobacterium tuberculosis GlmU

“…It proves that GlmU is vital for the growth of the mycobacteria. Among the characterized bacterial GlmU enzymes are those from Escherichia coli [7], Streptococcus pneumonia [8], Haemophilus influenzae [9] and M. tuberculosis [10]. According to crystal structure of M. tuberculosis GlmU, it exits as homotrimer.…”