A mechanism-inspired UDP-N-acetylglucosamine pyrophosphorylase inhibitor

UDP-N-acetylglucosamine pyrophosphorylase (UAP1) catalyses the last step in eukaryotic biosynthesis of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), converting UTP and GlcNAc-1P to the sugar nucleotide. Gene disruption studies have shown that this gene is essential in eukaryotes and a possible antifungal target, yet no inhibitors of fungal UAP1 have so far been reported. Here we describe the crystal structures of substrate/product complexes of UAP1 from Aspergillus fumigatus that together provide snapshots of catalysis. A structure with UDP-GlcNAc, pyrophosphate and Mg2+ provides the first Michaelis complex trapped for this class of enzyme, revealing the structural basis of the previously reported Mg2+ dependence and direct observation of pyrophosphorolysis. We also show that a highly conserved lysine mimics the role of a second metal observed in structures of bacterial orthologues. A mechanism-inspired UTP α,β-methylenebisphosphonate analogue (meUTP) was designed and synthesized and was shown to be a micromolar inhibitor of the enzyme. The mechanistic insights and inhibitor described here will facilitate future studies towards the discovery of small molecule inhibitors of this currently unexploited potential antifungal drug target.

1 Overall quality at a glance i O The following experimental techniques were used to determine the structure:

X-RAY DIFFRACTION
The reported resolution of this entry is 2.40 Å.
Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in the following graphic. The table shows the number of entries on which the scores are based.

Metric
Whole archive (#Entries) Similar resolution (#Entries, resolution range(Å)) The table below summarises the geometric issues observed across the polymeric chains and their t to the electron density. The red, orange, yellow and green segments on the lower bar indicate the fraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria. A grey segment represents the fraction of residues that are not modelled. The numeric value for each fraction is indicated below the corresponding segment, with a dot representing fractions <=5% The upper red bar (where present) indicates the fraction of residues that have poor t to the electron density. The numeric value is given above the bar.

Mol Chain Length
Quality of chain There are 3 unique types of molecules in this entry. The entry contains 7507 atoms, of which 0 are hydrogens and 0 are deuteriums.
In the tables below, the ZeroOcc column contains the number of atoms modelled with zero occupancy, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.
Molecule 1 is a protein called UDP-N-acetylglucosamine pyrophosphorylase.
Mol Chain Residues Atoms ZeroOcc AltConf Trace  3  A  50  Total O  50  50  0  0   3  B  70  Total O  70  70  0  0 Page 5 Full wwPDB X-ray Structure Validation Report 6G9W 3 Residue-property plots i O These plots are drawn for all protein, RNA and DNA chains in the entry. The rst graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic. The second graphic shows the sequence view annotated by issues in geometryand electron density. Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more. A red dot above a residue indicates a poor t to the electron density (RSRZ > 2). Stretches of 2 or more consecutive residues without any outlier are shown as a green connector. Residues present in the sample, but not in the model, are shown in grey.
• Molecule 1: UDP-N-acetylglucosamine pyrophosphorylase Chain A:  There are no bond length outliers.
There are no bond angle outliers.
There are no chirality outliers.
There are no planarity outliers. The all-atom clashscore is dened as the number of clashes found per 1000 atoms (including hydrogen atoms). The all-atom clashscore for this structure is 5.
All (77)  There are no Ramachandran outliers to report.

Protein sidechains i O
In the following table, the Percentiles column shows the percent sidechain outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the sidechain conformation was analysed, and the total number of residues. In the following table, the Counts columns list the number of bonds (or angles) for which Mogul statistics could be retrieved, the number of bonds (or angles) that are observed in the model and the number of bonds (or angles) that are dened in the Chemical Component Dictionary. The Link column lists molecule types, if any, to which the group is linked. The Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value. A bond length (or angle) with |Z| > 2 is considered an outlier worth inspection. RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles).  (and percentage) of RSRZ outliers, followed by percent RSRZ outliers for the chain as percentile scores relative to all X-ray entries and entries of similar resolution. The OWAB column contains the minimum, median, 95 th percentile and maximum values of the occupancy-weighted average B-factor per residue. The column labelled`Q< 0.9' lists the number of (and percentage) of residues with an average occupancy less than 0.9.