Publications

• Structural complexes of human adenine phosphoribosyltransferase reveal novel features of the APRT catalytic mechanism.

  Silva C.H., Silva M., Iulek J., Thiemann O.H.

  Adenine phosphoribosyltransferase (APRT) is an important enzyme component of the purine recycling pathway. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and use the salvage pathway for the synthesis of purine bases rendering this biosynthetic pathway an at ... >> More

  Adenine phosphoribosyltransferase (APRT) is an important enzyme component of the purine recycling pathway. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and use the salvage pathway for the synthesis of purine bases rendering this biosynthetic pathway an attractive target for antiparasitic drug design. The recombinant human adenine phosphoribosyltransferase (hAPRT) structure was resolved in the presence of AMP in the active site to 1.76 A resolution and with the substrates PRPP and adenine simultaneously bound to the catalytic site to 1.83 A resolution. An additional structure was solved containing one subunit of the dimer in the apo-form to 2.10 A resolution. Comparisons of these three hAPRT structures with other 'type I' PRTases revealed several important features of this class of enzymes. Our data indicate that the flexible loop structure adopts an open conformation before and after binding of both substrates adenine and PRPP. Comparative analyses presented here provide structural evidence to propose the role of Glu104 as the residue that abstracts the proton of adenine N9 atom before its nucleophilic attack on the PRPP anomeric carbon. This work leads to new insights to the understanding of the APRT catalytic mechanism. << Less

  J Biomol Struct Dyn 25:589-597(2008) [PubMed] [EuropePMC]

• Structural analysis of adenine phosphoribosyltransferase from Saccharomyces cerevisiae.

  Shi W., Tanaka K.S.E., Crother T.R., Taylor M.W., Almo S.C., Schramm V.L.

  Adenine phosphoribosyltransferase (APRTase) is a widely distributed enzyme, and its deficiency in humans causes the accumulation of 2,8-dihydroxyadenine. It is the sole catalyst for adenine recycling in most eukaryotes. The most commonly expressed APRTase has subunits of approximately 187 amino ac ... >> More

  Adenine phosphoribosyltransferase (APRTase) is a widely distributed enzyme, and its deficiency in humans causes the accumulation of 2,8-dihydroxyadenine. It is the sole catalyst for adenine recycling in most eukaryotes. The most commonly expressed APRTase has subunits of approximately 187 amino acids, but the only crystal structure is from Leishmania donovani, which expresses a long form of the enzyme with 237 residues. Saccharomyces cerevisiae APRTase was selected as a representative of the short APRTases, and the structure of the apo-enzyme and sulfate bound forms were solved to 1.5 and 1.75 A, respectively. Yeast APRTase is a dimeric molecule, and each subunit is composed of a central five-stranded beta-sheet surrounded by five alpha-helices, a structural theme found in all known purine phosphoribosyltransferases. The structures reveal several important features of APRTase function: (i) sulfate ions bound at the 5'-phosphate and pyrophosphate binding sites; (ii) a nonproline cis peptide bond (Glu67-Ser68) at the pyrophosphate binding site in both apo-enzyme and sulfate-bound forms; and (iii) a catalytic loop that is open and ordered in the apo-enzyme but open and disordered in the sulfate-bound form. Alignment of conserved amino acids in short-APRTases from 33 species reveals 13 invariant and 15 highly conserved residues present in hinges, catalytic site loops, and the catalytic pocket. Mutagenesis of conserved residues in the catalytic loop, subunit interface, and phosphoribosylpyrophosphate binding site indicates critical roles for the tip of the catalytic loop (Glu106) and a catalytic site residue Arg69, respectively. Mutation of one loop residue (Tyr103Phe) increases k(cat) by 4-fold, implicating altered dynamics for the catalytic site loop. << Less

  Biochemistry 40:10800-10809(2001) [PubMed] [EuropePMC]

• The adenine phosphoribosyltransferase from Giardia lamblia has a unique reaction mechanism and unusual substrate binding properties.

  Sarver A.E., Wang C.C.

  Purine phosphoribosyltransferases catalyze the Mg2+ -dependent reaction that transforms a purine base into its corresponding nucleotide. They are present in a wide variety of organisms including plants, mammals, and parasitic protozoa. Giardia lamblia, the causative agent of giardiasis, lacks de n ... >> More

  Purine phosphoribosyltransferases catalyze the Mg2+ -dependent reaction that transforms a purine base into its corresponding nucleotide. They are present in a wide variety of organisms including plants, mammals, and parasitic protozoa. Giardia lamblia, the causative agent of giardiasis, lacks de novo purine biosynthesis and relies primarily on adenine and guanine phosphoribosyltransferases (APRTase and GPRTase) constituting two independent and essential purine salvage pathways. The APRTase from G. lamblia was cloned and expressed with a 6-His tag at its C terminus and purified to apparent homogeneity. Adenine and alpha-d-5-phosphoribosyl-1-pyrophosphate (PRPP) have K(m) values of 4.2 and 143 microm with a k(cat) of 2.8 s(-1) in the forward reaction, whereas AMP and PP(i) have K(m) values of 87 and 450 microm with a k(cat) of 9.5 x 10(-3) s(-1) in the reverse reaction. Product inhibition studies indicated that the forward reaction follows a random Bi Bi mechanism. Results from the kinetics of equilibrium isotope exchange further verified a random Bi Bi mechanism in the forward reaction. In a mutant enzyme, F25W, with kinetic constants similar to those of the wild type and a tryptophan residue at the adenine binding site, the addition of adenine or AMP to the free mutant enzyme resulted in fluorescence quenching, whereas PRPP caused fluorescence enhancement. The dissociation constants thus estimated are 16.5 microm for adenine, 14.3 microm for AMP, and 83.0 microm for PRPP. PP(i) exerted no detectable effect on the tryptophan fluorescence at all, suggesting a lack of PP(i) binding to the free enzyme. An ordered substrate binding in the reverse reaction with AMP bound first followed by PP(i) is thus postulated. << Less

  J Biol Chem 277:39973-39980(2002) [PubMed] [EuropePMC]

• Crystal structures of APRT from Francisella tularensis - an N-H...N hydrogen bond imparts adenine specificity in adenine phosporibosyltransferases.

  Pavithra G.C., Ramagopal U.A.

  Francisella tularensisis, the causative agent of tularemia has been classified as a category A bioterrorism agent. Here, we present the crystal structure of apo and adenine bound form of the adenine phosphoribosyltransferase (APRT) from Francisella tularensis. APRT is an enzyme involved in the sal ... >> More

  Francisella tularensisis, the causative agent of tularemia has been classified as a category A bioterrorism agent. Here, we present the crystal structure of apo and adenine bound form of the adenine phosphoribosyltransferase (APRT) from Francisella tularensis. APRT is an enzyme involved in the salvage of adenine (a 6-aminopurine), converting it to AMP. The purine salvage pathway relies on two essential and distinct enzymes to convert 6-aminopurine and 6-oxopurines into corresponding nucleotides. The mechanism by which these enzymes differentiate different purines is not clearly understood. Analysis of the structures of apo and adenine-bound APRT from F. tularensis, together with all other available structures of APRTs, suggests that (a) the base-binding loop is stabilized by a cluster of aromatic and conformation-restricting proline residues, and (b) an N-H···N hydrogen bond between the base-binding loop and the N1 atom of adenine is the key interaction that differentiates adenine from 6-oxopurines. These observations were corroborated by bioinformatics analysis of ~ 4000 sequences of APRTs (with 80% identity cutoff), which confirmed that the residues conferring rigidity to the base-binding loop are highly conserved. Furthermore, an F23A mutation on the base-binding loop severely affects the efficiency of the enzyme. We extended our analysis to the structure and sequences of APRTs from the Trypanosomatidae family with a destabilizing insertion on the base-binding loop and propose the mechanism by which these evolutionarily divergent enzymes achieve base specificity. Our results suggest that the base-binding loop not only confers appropriate affinity but also provides defined specificity for adenine.<h4>Enzyme</h4>EC 2.4.2.7 DATABASE: Structural data are available in Protein Data Bank (PDB) under the accession numbers 5YW2 and 5YW5. << Less

  FEBS J 285:2306-2318(2018) [PubMed] [EuropePMC]

• Structural basis for substrate selectivity and nucleophilic substitution mechanisms in human adenine phosphoribosyltransferase catalyzed reaction.

  Ozeir M., Huyet J., Burgevin M.C., Pinson B., Chesney F., Remy J.M., Siddiqi A.R., Lupoli R., Pinon G., Saint-Marc C., Gibert J.F., Morales R., Ceballos-Picot I., Barouki R., Daignan-Fornier B., Olivier-Bandini A., Auge F., Nioche P.

  The reversible adenine phosphoribosyltransferase enzyme (APRT) is essential for purine homeostasis in prokaryotes and eukaryotes. In humans, APRT (hAPRT) is the only enzyme known to produce AMP in cells from dietary adenine. APRT can also process adenine analogs, which are involved in plant develo ... >> More

  The reversible adenine phosphoribosyltransferase enzyme (APRT) is essential for purine homeostasis in prokaryotes and eukaryotes. In humans, APRT (hAPRT) is the only enzyme known to produce AMP in cells from dietary adenine. APRT can also process adenine analogs, which are involved in plant development or neuronal homeostasis. However, the molecular mechanism underlying substrate specificity of APRT and catalysis in both directions of the reaction remains poorly understood. Here we present the crystal structures of hAPRT complexed to three cellular nucleotide analogs (hypoxanthine, IMP, and GMP) that we compare with the phosphate-bound enzyme. We established that binding to hAPRT is substrate shape-specific in the forward reaction, whereas it is base-specific in the reverse reaction. Furthermore, a quantum mechanics/molecular mechanics (QM/MM) analysis suggests that the forward reaction is mainly a nucleophilic substitution of type 2 (S_N2) with a mix of S_N1-type molecular mechanism. Based on our structural analysis, a magnesium-assisted S_N2-type mechanism would be involved in the reverse reaction. These results provide a framework for understanding the molecular mechanism and substrate discrimination in both directions by APRTs. This knowledge can play an instrumental role in the design of inhibitors, such as antiparasitic agents, or adenine-based substrates. << Less

  J Biol Chem 294:11980-11991(2019) [PubMed] [EuropePMC]