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.

Biology Centre CAS Institute of Parasitology České Budějovice Czech Republic

Faculty of Science University of South Bohemia České Budějovice Czech Republic

The Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague 6 Czech Republic

The School of Chemistry and Molecular Biosciences The University of Queensland Brisbane QLD Australia

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Simarro PP, Diarra A, Ruiz Postigo JA, Franco JR & Jannin JG (2011) The human African trypanosomiasis control and surveillance programme of the World Health Organization 2000-2009: the way forward. PLoS Negl Trop Dis 5, e1007.

Simarro PP, Cecchi G, Franco JR, Paone M, Diarra A, Ruiz-Postigo JA, Fevre EM, Mattioli RC & Jannin JG (2012) Estimating and mapping the population at risk of sleeping sickness. PLoS Negl Trop Dis 6, e1859.

Who WHO (2013) Control and surveillance of human African trypanosomiasis. World Health Organ Tech Rep Ser 1-237.

Wright MH, Paape D, Price HP, Smith DF & Tate EW (2016) Global profiling and inhibition of protein lipidation in vector and host stages of the sleeping sickness parasite Trypanosoma brucei. ACS Infect Dis 2, 427-441.

Nagle AS, Khare S, Kumar AB, Supek F, Buchynskyy A, Mathison CJ, Chennamaneni NK, Pendem N, Buckner FS, Gelb MH et al. (2014) Recent developments in drug discovery for leishmaniasis and human African trypanosomiasis. Chem Rev 114, 11305-11347.

Munday JC, Tagoe DN, Eze AA, Krezdorn JA, Rojas Lopez KE, Alkhaldi AA, McDonald F, Still J, Alzahrani KJ, Settimo L et al. (2015) Functional analysis of drug resistance-associated mutations in the Trypanosoma brucei adenosine transporter 1 (TbAT1) and the proposal of a structural model for the protein. Mol Microbiol 96, 887-900.

Graf FE, Ludin P, Arquint C, Schmidt RS, Schaub N, Kunz Renggli C, Munday JC, Krezdorn J, Baker N, Horn D et al. (2016) Comparative genomics of drug resistance in Trypanosoma brucei rhodesiense. Cell Mol Life Sci 73, 3387-3400.

Wenzler T, Schumann Burkard G, Schmidt RS, Maser P, Bergner A, Roditi I & Brun R (2016) A new approach to chemotherapy: drug-induced differentiation kills African trypanosomes. Sci Rep 6, 22451.

Scotti L, Mendonca FJ, da Silva MS & Scotti MT (2016) Enzymatic targets in Trypanosoma brucei. Curr Protein Pept Sci 17, 243-259.

Van Rompay AR, Johansson M & Karlsson A (2000) Phosphorylation of nucleosides and nucleoside analogs by mammalian nucleoside monophosphate kinases. Pharmacol Ther 87, 189-198.

Allen TE & Ullman B (1993) Cloning and expression of the hypoxanthine-guanine phosphoribosyltransferase gene from Trypanosoma brucei. Nucleic Acids Res 21, 5431-5438.

Boitz JM, Ullman B, Jardim A & Carter NS (2012) Purine salvage in Leishmania: complex or simple by design? Trends Parasitol 28, 345-352.

Eakin AE, Nievesalicea R, Tosadoacevedo R, Chin MS, Wang CC & Craig SP (1995) Comparative complement selection in bacteria enables screening for lead compounds targeted to a purine salvage enzyme of parasites. Antimicrob Agents Chemother 39, 620-625.

de Jersey J, Holy A, Hockova D, Naesens L, Keough DT & Guddat LW (2011) 6-oxopurine phosphoribosyltransferase: a target for the development of antimalarial drugs. Curr Top Med Chem 11, 2085-2102.

Jardim A, Bergeson SE, Shih S, Carter N, Lucas RW, Merlin G, Myler PJ, Stuart K & Ullman B (1999) Xanthine phosphoribosyltransferase from Leishmania donovani. Molecular cloning, biochemical characterization, and genetic analysis. J Biol Chem 274, 34403-34410.

Hwang HY & Ullman B (1997) Genetic analysis of purine metabolism in Leishmania donovani. J Biol Chem 272, 19488-19496.

Berg M, Van der Veken P, Goeminne A, Haemers A & Augustyns K (2010) Inhibitors of the purine salvage pathway: a valuable approach for antiprotozoal chemotherapy? Curr Med Chem 17, 2456-2481.

Berg M, Kohl L, Van der Veken P, Joossens J, Al-Salabi MI, Castagna V, Giannese F, Cos P, Versees W, Steyaert J et al. (2010) Evaluation of nucleoside hydrolase inhibitors for treatment of African trypanosomiasis. Antimicrob Agents Chemother 54, 1900-1908.

Dolezelova E, Teran D, Gahura O, Kotrbova Z, Prochazkova M, Keough D, Spacek P, Hockova D, Guddat L & Zikova A (2018) Evaluation of the Trypanosoma brucei 6-oxopurine salvage pathway as a potential target for drug discovery. PLoS Negl Trop Dis 12, e0006301.

Heroux A, White EL, Ross LJ, Davis RL & Borhani DW (1999) Crystal structure of Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase with XMP, pyrophosphate, and two Mg2+ ions bound: insights into the catalytic mechanism. Biochemistry 38, 14495-14506.

Heroux A, White EL, Ross LJ, Kuzin AP & Borhani DW (2000) Substrate deformation in a hypoxanthine-guanine phosphoribosyltransferase ternary complex: the structural basis for catalysis. Structure 8, 1309-1318.

Somoza JR, Chin MS, Focia PJ, Wang CC & Fletterick RJ (1996) Crystal structure of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from the protozoan parasite Tritrichomonas foetus. Biochemistry 35, 7032-7040.

Munagala NR & Wang CC (1998) Altering the purine specificity of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus by structure-based point mutations in the enzyme protein. Biochemistry 37, 16612-16619.

Teran D, Hockova D, Cesnek M, Zikova A, Naesens L, Keough DT & Guddat LW (2016) Crystal structures and inhibition of Trypanosoma brucei hypoxanthine-guanine phosphoribosyltransferase. Sci Rep 6, https://doi.org/10.1038/srep35894

Munagala NR, Chin MS & Wang CC (1998) Steady-state kinetics of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus: the role of threonine-47. Biochemistry 37, 4045-4051.

Keough DT, Ng A, Winzor DJ, Emmerson BT & de Jersey J (1999) Purification and characterization of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase and comparison with the human enzyme. Mol Biochem Parasitol 98, 29-41.

Moynie L, Giraud MF, Breton A, Boissier F, Daignan-Fornier B & Dautant A (2012) Functional significance of four successive glycine residues in the pyrophosphate binding loop of fungal 6-oxopurine phosphoribosyltransferases. Protein Sci 21, 1185-1196.

Shi W, Li CM, Tyler PC, Furneaux RH, Grubmeyer C, Schramm VL & Almo SC (1999) The 2.0 Å structure of human hypoxanthine-guanine phosphoribosyltransferase in complex with a transition-state analog inhibitor. Nat Struct Biol 6, 588-593.

Valsecchi WM, Cousido-Siah A, Defelipe LA, Mitschler A, Podjarny A, Santos J & Delfino JM (2016) The role of the C-terminal region on the oligomeric state and enzymatic activity of Trypanosoma cruzi hypoxanthine phosphoribosyl transferase. Biochim Biophys Acta 1864, 655-666.

Gasteiger E, Hoogland C, Gattiker A, Wilkins MR, Appel RD & Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In The Proteomics Protocols Handbook (Walker JM, eds), pp. 571-607. Springer, Totowa, NJ.

Krissinel E (2015) Stock-based detection of protein oligomeric states in jsPISA. Nucleic Acids Res 43, W314-W319.

Krissinel E & Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372, 774-797.

Eads JC, Scapin G, Xu Y, Grubmeyer C & Sacchettini JC (1994) The crystal structure of human hypoxanthine-guanine phosphoribosyltransferase with bound GMP. Cell 78, 325-334.

Craig SP & Eakin AE (2000) Purine phosphoribosyltransferases. J Biol Chem 275, 20231-20234.

Sinha SC & Smith JL (2001) The PRT protein family. Curr Opin Struct Biol 11, 733-739.

Vos S, Parry RJ, Burns MR, de Jersey J & Martin JL (1998) Structures of free and complexed forms of Escherichia coli xanthine-guanine phosphoribosyltransferase. J Mol Biol 282, 875-889.

Vos S, de Jersey J & Martin JL (1997) Crystal structure of Escherichia coli xanthine phosphoribosyltransferase. Biochemistry 36, 4125-4134.

Holm L & Rosenstrom P (2010) Dali server: conservation mapping in 3D. Nucleic Acids Res 38, W545-W549.

Eng WS, Hockova D, Spacek P, Janeba Z, West NP, Woods K, Naesens LM, Keough DT & Guddat LW (2015) First crystal structures of Mycobacterium tuberculosis 6-oxopurine phosphoribosyltransferase: complexes with GMP and pyrophosphate and with acyclic nucleoside phosphonates whose prodrugs have antituberculosis activity. J Med Chem 58, 4822-4838.

Kulikowska E, Kierdaszuk B & Shugar D (2004) Xanthine, xanthosine and its nucleotides: solution structures of neutral and ionic forms, and relevance to substrate properties in various enzyme systems and metabolic pathways. Acta Biochim Pol 51, 493-531.

Guddat LW, Vos S, Martin JL, Keough DT & de Jersey J (2002) Crystal structures of free, IMP-, and GMP-bound Escherichia coli hypoxanthine phosphoribosyltransferase. Prot Sci 11, 1626-1638.

Monzani PS, Trapani S, Thiemann OH & Oliva G (2007) Crystal structure of Leishmania tarentolae hypoxanthine-guanine phosphoribosyltransferase. BMC Struct Biol 7, 59.

Keough DT, Brereton IM, de Jersey J & Guddat LW (2005) The crystal structure of free human hypoxanthine-guanine phosphoribosyltransferase reveals extensive conformational plasticity throughout the catalytic cycle. J Mol Biol 351, 170-181.

Shi W, Li CM, Tyler PC, Furneaux RH, Cahill SM, Girvin ME, Grubmeyer C, Schramm VL & Almo SC (1999) The 2.0 Å structure of malarial purine phosphoribosyltransferase in complex with a transition-state analogue inhibitor. Biochemistry 38, 9872-9880.

Wen J, Arakawa T & Philo JS (1996) Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions. Anal Biochem 240, 155-166.

Ullah MO, Valkov E, Ve T, Williams S, Mas C, Mansell A & Kobe B (2015) Recombinant production of functional full-length and truncated human TRAM/TICAM-2 adaptor protein involved in Toll-like receptor and interferon signaling. Protein Expr Purif 106, 31-40.

McPhillips TM, McPhillips SE, Chiu HJ, Cohen AE, Deacon AM, Ellis PJ, Garman E, Gonzalez A, Sauter NK, Phizackerley RP et al. (2002) Blu-ice and the distributed control system: software for data acquisition and instrument control at macromolecular crystallography beamlines. J Synchrotron Radiat 9, 401-406.

Kabsch WX (2010) Xds. Acta Crystallogr D 66, 125-132.

McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC & Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40, 658-674.

Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW et al. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D 66, 213-221.

Emsley P, Lohkamp B, Scott WG & Cowtan K (2010) Features and development of Coot. Acta Crystallogr D 66, 486-501.

Battye TG, Kontogiannis L, Johnson O, Powell HR & Leslie AG (2011) iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D 67, 271-281.

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