Journal of Molecular Biology
Volume 307, Issue 5, 13 April 2001, Pages 1363-1379
Journal home page for Journal of Molecular Biology

Regular article
Structure and function of a novel purine specific nucleoside hydrolase from Trypanosoma vivax1

https://doi.org/10.1006/jmbi.2001.4548Get rights and content

Abstract

The purine salvage pathway of parasitic protozoa is currently considered as a target for drug development because these organisms cannot synthesize purines de novo. Insight into the structure and mechanism of the involved enzymes can aid in the development of potent inhibitors, leading to new curative drugs. Nucleoside hydrolases are key enzymes in the purine salvage pathway of Trypanosomatidae, and they are especially attractive because they have no equivalent in mammalian cells. We cloned, expressed and purified a nucleoside hydrolase from Trypanosoma vivax. The substrate activity profile establishes the enzyme to be a member of the inosine-adenosine-guanosine-preferring nucleoside hydrolases (IAG-NH). We solved the crystal structure of the enzyme at 1.6 Å resolution using MAD techniques. The complex of the enzyme with the substrate analogue 3-deaza-adenosine is presented. These are the first structures of an IAG-NH reported in the literature. The T. vivax IAG-NH is a homodimer, with each subunit consisting of ten β-strands, 12 α-helices and three small 310-helices. Six of the eight strands of the central β-sheet form a motif resembling the Rossmann fold. Superposition of the active sites of this IAG-NH and the inosine-uridine-preferring nucleoside hydrolase (IU-NH) of Crithidia fasciculata shows the molecular basis of the different substrate specificity distinguishing these two classes of nucleoside hydrolases. An “aromatic stacking network” in the active site of the IAG-NH, absent from the IU-NH, imposes the purine specificity. Asp10 is the proposed general base in the reaction mechanism, abstracting a proton from a nucleophilic water molecule. Asp40 (replaced by Asn39 in the IU-NH) is positioned appropriately to act as a general acid and to protonate the purine leaving group. The second general acid, needed for full enzymatic activity, is probably part of a flexible loop located in the vicinity of the active site.

Introduction

Parasitic diseases remain a huge health care problem, especially in developing countries. Trypanosomiasis, or sleeping sickness, is caused by a unicellular protozoon of the genus Trypanosoma. Annually, about 25,000 to 50,000 new cases of sleeping sickness are reported in the sub-Sahara region.1, 2, 3 Trypanosomiasis is, moreover, one of the most important illnesses of domestic livestock in Africa. Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense are the causative agents of sleeping sickness in human beings, while Trypanosoma vivax, Trypanosoma congolense and Trypanosoma brucei brucei cause the disease in domestic animals and wildlife. It is clear that there is an urgent need for new efficient drugs to treat trypanosomiasis, especially since the drugs used at present show very high host toxicity. Moreover, the wide-spreading drug resistance makes treatment very difficult in large areas of Africa.4, 5 In this regard, a number of trypanosomal pathways are being investigated as possible targets for drug development.6, 7

One of these pathways is the purine salvage pathway.8 All parasitic protozoa (e.g. Plasmodium, Toxoplasma, Leishmania, Trypanosoma) lack the ability to synthesize purine nucleotides de novo. Instead, they use salvage enzymes to obtain purine bases and nucleosides from their hosts and convert them to the corresponding nucleotides.9, 10, 11 After their uptake by specific transporters,12, 13 the purine nucleosides are first hydrolyzed to ribose and the base by nucleoside hydrolases. The bases are then converted to the concomitant nucleotides by phophoribosyltransferases (PRTases). Interfering with purine salvage by blocking the key enzymes could be an effective way of killing these organisms. The PRTases have already been studied extensively as targets for specific inhibitor design.14, 15, 16 However, due to the many parallel routes that exist in the trypanosomal purine salvage pathways, blocking of this enzyme alone probably will not be enough to kill the parasite.7 The nucleoside hydrolases form another important and ubiquitous class of purine salvage enzymes. Considering drug development, they have the advantage that, in contrast to the PRTases, no nucleoside hydrolase activity has been reported thus far in mammalian cells.10

The nucleoside hydrolases catalyze the hydrolysis of the N-glycosidic bond between the anomeric carbon atom of ribose and the purine or pyrimidine base. A nucleoside hydrolase activity has been detected in crude lysates of different species of the Trypanosomatidae.11, 17, 18, 19, 20 Only four nucleoside hydrolases (two from Crithidia fasciculata.21, 22 one from T. brucei brucei23 and one from Leishmania major24) have thus far been purified and characterized. These have been divided into three subclasses according to their substrate specificity. The first class is the base-aspecific inosine-uridine-preferring nucleoside hydrolases (IU-NH) found in C. fasciculata21 and L. major.24 The other two classes, an inosine-adenosine-guanosine-preferring nucleoside hydrolase (IAG-NH) from T. brucei brucei23 and an inosine-guanosine-preferring nucleoside hydrolase (IG-NH) from C. fasciculata,22 show specificity toward purine nucleosides.

The IU-NHs have been studied in depth. Using kinetic isotope effects, it was shown that the enzyme-catalyzed reaction occurs via an SN1 mechanism with an oxocarbenium ion transition state.25, 26 The IU-NHs obtain most of their catalytic power via stabilization of this intrinsically very unstable oxocarbenium ion. Only minor interactions occur with the purine or pyrimidine leaving group.27 Three IU-NH crystal structures (up to a resolution of 2.3 Å) have been reported.24, 28, 29 The structural properties of the purine-specific nucleoside hydrolases (IAG-NH and IG-NH) have not been characterized. However, the substrate specificity and the pH-dependence of the kinetic constants indicate that the catalytic mechanisms and the transition state structures are fundamentally different for the IU and IAG isozymes.23 In comparison with the IU-NHs, the IAG-NHs rely much more on leaving group activation for catalysis.30

Here, we report the cloning of a nucleoside hydrolase from Trypanosoma vivax, the causative agent of the Souma disease in African cattle.31 The protein is expressed in Escherichia coli as a His-tagged fusion protein that allows easy purification. Determination of the substrate specificity establishes the enzyme to be an IAG-NH. Kinetic data obtained with a substrate analogue confirm the importance of leaving group activation in the catalytic mechanism. A 1.6 Å resolution X-ray structure of the unliganded protein and a 2.1 Å resolution X-ray structure of the protein complexed with the substrate analogue 3-deaza-adenosine are reported. These structures reveal insights into the differences between IU and IAG-NHs and are an indispensable tool for inhibitor design.

Section snippets

Cloning and sequence comparison

The cloned cDNA encoding the T. vivax IAG-NH is 1245 nt long and contains a mini-exon or spliced leader sequence from bases 1 to 39. The nucleoside hydrolase ORF codes for a protein of 327 amino acid residues, including the N-terminal methionine residue. For cloning and purification purposes, the start codon was removed and replaced by a sequence coding for 13 amino acid residues, including the six histidine residues from the His-tag. Figure 1(a) compares the amino acid sequence of the

Conclusions

T. vivax is a causative agent of trypanosomiasis in African cattle. The crystal structures of a T. vivax IAG-NH reported here provide the first structural description of a purine-specific nucleoside hydrolase. The structure in complex with the substrate analogue 3-deaza-adenosine sheds light on the mechanism of the enzyme-catalysed reaction. All hydroxyl groups of the ribose are involved in interactions with (mainly acidic) residues of the enzyme and with a calcium ion in the bottom of the

Cloning of the complete IAG-NH cDNA

The T. vivax IAG-NH homologue was cloned by PCR amplification of ss-cDNA generated from total poly(A)+ RNA of the bloodstream form of T. vivax, IL2160. The primers were designed from T. brucei brucei and T. congolense conserved amino acid sequences ADCFVE (forward primer 5′-GCG-GAT-TGC-TTC-GTT-GA-3′) and KVEEC (reverse primer 5′-GCA-TTC-CTC-CAC-CTT-3′). The forward primer was used with an oligo(dT) primer to generate the 3′ fragment. The reverse primer was used with the mini-exon-derived

Acknowledgements

This work was supported by the Vlaams Interuniversitair Instituut voor Biotechnologie and by the Nationaal Fonds voor Wetenschappelijk Onderzoek - Vlaanderen. W.V. is a recipient of a grant from the FWO-Vlaanderen. The authors thank Maia De Kerpel for excellent technical assistance. We are also grateful to Yves Geunes for computer support. The substrate analogue p-nitrophenyl β-d-ribofuranoside was a generous gift from Dr Vern Schramm from the Albert Einstein College of Medicine, New York. We

References (69)

  • D.W. Parkin et al.

    Nucleoside hydrolase from Crithidia fasciculata. Metabolic role, purification, specificity, and kinetic mechanism

    J. Biol. Chem.

    (1991)
  • B. Estupiñán et al.

    Guanosine-Inosine-preferring nucleoside N-glycohydrolase from Crithidia fasciculata

    J. Biol. Chem.

    (1994)
  • D.W. Parkin

    Purine-specific nucleoside N-ribohydrolase from Trypanosoma brucei brucei. Purification, specificity, and kinetic mechanism

    J. Biol. Chem.

    (1996)
  • W. Shi et al.

    Nucleoside hydrolase from Leishmania major. Cloning, expression, catalytic properties, transition state inhibitors, and the 2.5-Å crystal structure

    J. Biol. Chem.

    (1999)
  • R. Pellé et al.

    Molecular cloning and expression of a purine-specific N-ribohydrolase from Trypanosoma brucei brucei. Sequence, expression, and molecular analysis

    J. Biol. Chem.

    (1998)
  • A.E.V. Haschemeyer et al.

    Nucleoside conformationan analysis of steric barriers to rotation about the glycosidic bond

    J. Mol. Biol.

    (1967)
  • H. Berthod et al.

    Complementary studies on the rigidity-flexibility of nucleotides

    FEBS Letters

    (1973)
  • S.K. Burley et al.

    Weakly polar interactions in proteins

    Advan. Protein Chem.

    (1988)
  • G.B. McGaughey et al.

    π-Stacking interactions. Alive and well in proteins

    J. Biol. Chem.

    (1998)
  • R. Wolfenden

    Conformational aspects of inhibitor designenzyme-substrate interactions in the transition state

    Bioorg. Med. Chem.

    (1999)
  • W.E.J. DeWolf et al.

    The catalytic site of AMP nucleosidase. Substrate specificity and pH effects with AMP and formycin 5′-PO4

    J. Biol. Chem.

    (1979)
  • V.L. Schramm et al.

    Transition state analysis and inhibitor design for enzymatic reactions

    J. Biol. Chem.

    (1994)
  • S.N. Ho et al.

    Site-directed mutagenesis by overlap extension using the polymerase chain reaction

    Gene

    (1989)
  • Z. Otwinowski et al.

    Processing of X-ray diffraction data collected in oscillation mode

  • D.H. Molyneux

    Current public health status of the trypanosomiases and leishmaniases

  • D.H. Smith et al.

    Human African trypanosomiasisan emerging public health crisis

    Brit. Med. Bull.

    (1998)
  • C.A. Ross et al.

    Drug resistance in trypanosomatids

  • A.H. Fairlamb

    Novel biochemical pathways in parasitic protozoa

    Parasitology

    (1989)
  • P.O.J. Ogbunude et al.

    Adenosine cycle in African trypanosomes

    Ann. Trop. Med. Parasitol.

    (1985)
  • M.J. Davies et al.

    The enzymes of purine salvage in Trypanosoma cruzi, Trypanosoma brucei and Leishmania mexicana

    Parasitology

    (1983)
  • D.M. James et al.

    Uptake of purine bases and nucleosides in African trypanosomes

    Parasitology

    (1980)
  • C.M. Li et al.

    Transition-state analogs as inhibitors of human and malarial hypoxanthine-guanine phosphoribosyltransferases

    Nature Struct. Biol.

    (1999)
  • A.M. Aronov et al.

    Rational design of selective submicromolar inhibitors of Trichichomonas foetus hypoxanthine-guanine-xanthine phosphoribosyltransferase

    Biochemistry

    (2000)
  • G. Schmidt et al.

    A purine nucleoside hydrolase from Trypanosoma gambiense, purification and properties

    Tropenmed. Parasitol.

    (1975)
  • Cited by (90)

    • Novel nucleoside-based antimalarial compounds

      2016, Bioorganic and Medicinal Chemistry Letters
    View all citing articles on Scopus
    1

    Edited by R. Huber

    View full text