Crystal structures of Trypanosoma brucei hypoxanthine – guanine – xanthine phosphoribosyltransferase in complex with IMP, GMP and XMP

Abstract

The 6‐oxopurine phosphoribosyltransferases (PRTs) are drug targets for the treatment of parasitic diseases. This is due to the fact that parasites are auxotrophic for the 6‐oxopurine bases relying on salvage enzymes for the synthesis of their 6‐oxopurine nucleoside monophosphates. In Trypanosoma brucei, the parasite that is the aetiological agent for sleeping sickness, there are three 6‐oxopurine PRT isoforms. Two are specific for hypoxanthine and guanine, whilst the third, characterized here, uses all three naturally occurring bases with similar efficiency. Here, we have determined crystal structures for TbrHGXPRT in complex with GMP, XMP and IMP to investigate the structural basis for substrate specificity. The results show that Y201 and E208, not commonly observed within the purine binding pocket of 6‐oxopurine PRTs, contribute to the versatility of this enzyme. The structures further show that a nearby water can act as an adaptor to facilitate the binding of XMP and GMP. When GMP binds, a water can accept a proton from the 2‐amino group but when XMP binds, the equivalent water can donate its proton to the 2‐oxo group. However, when IMP is bound, no water molecule is observed at that location. Database Coordinates and structure factors were submitted to the Protein Data Bank and have accession codes of 6MXB, 6MXC, 6MXD and 6MXG for the TbrHGXPRT.XMP complex, TbrHGXPRT.GMP complex, TbrHGXPRT.IMP complex, and TbrHGPRT.XMP complex, respectively.

Acyclic nucleoside phosphonates with adenine nucleobase inhibit Trypanosoma brucei adenine phosphoribosyltransferase in vitro

“…This dependency represents a potentially promising ground for the discovery of anti-parasitic compounds with a purine-based scaffold. Indeed, we showed that potent 6-oxopurine acyclic nucleoside phosphonates (ANPs) inhibit 6-oxopurine phosphoribosyl transferases in vitro as well as possess strong cytotoxic effects on T. brucei bloodstream form cells 6 , 17 , 18 . Using our experience with this system, we designed new selective adenine-based ANPs and evaluated their activity in vitro and in T. brucei culture.…”

“…In our previous work, we determined the crystal structures of two T. brucei PSP enzymes, 6-oxopurine PRTases, in the complex with several ANPs, and showed that the prodrugs of the selected inhibitors possess strong anti-trypanosomal activity in the cell-based assay 6 , 17 , 18 . Inspired by these results, here we focused on the 6-aminopurine salvage route, which is linked to the 6-oxopurine pathway via an AMP deaminase that converts AMP to IMP (Fig.…”

Stereo-Defined Acyclic Nucleoside Phosphonates are Selective and Potent Inhibitors of Parasite 6-Oxopurine Phosphoribosyltransferases

“…The structure was solved by molecular replacement using the program PHASER within PHENIX 1.18.2 . For human HGPRT, the starting model was PDB code: 5HIA; for Tbr HGPRT1, PDB code: 5JV5; and for Tbr HGXPRT, PDB code: 6AQO . Subsequent refinement and model building were done with PHENIX 1.18.2 and COOT 0.8.9.2 .…”

“…18.2. 40 For human HGPRT, the starting model was PDB code: 5HIA; 18 for TbrHGPRT1, PDB code: 5JV5; 41 and for TbrHGXPRT, PDB code: 6AQO. 9 Subsequent refinement and model building were done with PHENIX 1.…”

Kinetic and Structural Characterization of Trypanosoma cruzi Hypoxanthine–Guanine–Xanthine Phosphoribosyltransferases and Repurposing of Transition-State Analogue Inhibitors

“…The authors proposed the role of the conserved water molecule as an adapter to facilitate binding of both XMP and GMP to HGXPRT. 69 This water plays no role in the binding of Hx, as its C2-carbon cannot form hydrogen bonds but instead makes hydrophobic interactions with Phe204 (a Leu in TcHGPRT, the last of the three substitutions observed between the T. cruzi isoforms). Binding of XMP, GMP, and IMP to the active sites of TbbHGXPRT is highly conserved.…”