Catalysis by nucleoside hydrolases
Introduction
Nucleoside hydrolases (or nucleoside N-ribohydrolases; NHs) comprise a superfamily of structurally related metalloproteins with a unique β-sheet topology (Figure 1) 1., 2.. Functionally, NHs are glycosidases that hydrolyse the N-glycosidic bond (EC 3.2.2.-) of β-ribonucleosides, forming the free nucleic base and ribose (Figure 2a). All characterised members impose a stringent specificity for the ribose moiety, but exhibit variability in their preferences for the nature of the nucleic base. Sequence alignments highlight a recurring N-terminal DXDXXXDD motif as a hallmark of NH activity.
NHs are widely distributed in nature, and have been found in bacteria 3., 4., yeast [5], protozoa 6., 7., insects [8] and mesozoa [9]. Genes containing the characteristic NH fingerprint motif are also present in plants, amphibians and fish. Surprisingly, neither NH activity nor the encoding genes have ever been detected in mammals. The metabolic role of the NHs is well established only for parasitic protozoa (Trypanosoma, Leishmania, Giardia and so on). In these organisms, NHs are key enzymes of the salvage pathway that aims to scavenge purines from their environment 10., 11.. In this pathway, the NHs catalyse the hydrolysis of the assimilated nucleosides, allowing recycling of the purine bases and ribose. Parasitic protozoa rely on the purine salvage pathway for survival because — in contrast to most other living organisms — they lack a de novo biosynthetic pathway for purines. Considering this divergence in purine metabolism between parasite and host, the parasitic NHs have been studied extensively in recent years as potential targets for chemotherapeutic intervention.
Over the years, the protozoan NHs have been classified into three subgroups on the basis of their substrate specificity: the base-aspecific inosine-uridine preferring nucleoside hydrolases (IU-NHs) 12., 13., the purine-specific inosine-adenosine-guanosine preferring nucleoside hydrolases (IAG-NHs) 14.•, 15. and the 6-oxo-purine-specific inosine-guanosine preferring nucleoside hydrolases (IG-NHs) [16]. Recent data indicate that this classification is somewhat artificial and inadequate, and that an increasing number of NHs do not unequivocally fit in one of these three groups 3., 9., 17.. Moreover, there seems to be little correlation between the level of amino acid identity and the nucleobase specificity. As more NHs are characterised, the boundaries of the existing classification will probably fade.
This review will examine the current state of knowledge on the structural and functional similarities and differences among representatives of the superfamily of NHs.
Section snippets
Diverse quaternary structures built from a common fold
The structure of the archetypical IU-NH from the trypanosome parasite Crithidia fasciculata (CfNH) was solved by Schramm and co-workers as a free enzyme [18] and in complex with the inhibitor para-aminophenyliminoribitol (pAPIR) [19]. The same group also solved the crystal structure of the IU-NH from Leishmania major (LmNH) [13]. The crystal structures of the free IAG-specific enzyme from Trypanosoma vivax (TvNH) and its complex with the inhibitor 3-deaza-adenosine were reported in 2001 by our
The active site: specific for ribose but promiscuous for nucleobases
The NHs contain one deep narrow active site per subunit. A Ca2+ ion is tightly bound at the bottom of the active site (Figure 3) 14.•, 19.. This octacoordinated metal is chelated through a conserved network of interactions involving the sidechain oxygens of Asp10, Asp15 and Asp261, the mainchain carbonyl oxygen of Thr137 (TvNH numbering) and three water molecules. Upon substrate binding, the ribose moiety is fixed deep inside the active site cleft. In the complex, two Ca2+-bound water molecules
Oxocarbenium ion transition state
Analysis of a series of kinetic isotope effects by bond-energy bond-order vibrational analysis established a geometric model [22] and an electrostatic potential surface [23] of the transition state of the reaction catalysed by CfNH. These studies show that the enzyme-catalysed hydrolysis of inosine proceeds via an SN1-type mechanism with an oxocarbenium-ion-like transition state (Figure 2b).
In the transition state, the N-glycosidic bond is nearly cleaved with a length of almost 2 Å, while the
Nucleophile activation and oxocarbenium ion stabilisation
Solvent reactivity studies have shown that CfNH discriminates against methanol attack from solvent during steady-state catalysis, indicating a classical Koshland single-displacement mechanism, with an enzyme-bound water molecule as the nucleophile in the NH-catalysed SN1 reaction [12]. All crystal structures reveal a Ca2+-bound water molecule as the most likely candidate for this role 14.•, 19.. Asp10 is appropriately positioned to activate this water by abstracting a proton (Figure 3).
Leaving group activation
Kinetic isotope effects for the CfNH-catalysed hydrolysis of inosine imply that the hypoxanthine ring is protonated at N7 before reaching the transition state [22]. Protonation of the leaving group at N7 is a recurring theme in the mechanism of many other purine N-ribohydrolases and transferases, including purine nucleoside phosphorylases [29], AMP nucleosidases [30], phosphoribosyl transferases [31] and ribosome-inactivating proteins [32], and in the nonenzymatic acid-catalysed hydrolysis of
Conclusions
The crystal structures of NHs from three different organisms have revealed a common subunit structure, containing a unique β-sheet topology, for this superfamily of metalloenzymes. Many details concerning the catalytic mechanism have been inferred from kinetic data and site-directed mutagenesis experiments guided by the structures of IU-NHs and IAG-NHs in complex with (transition state) inhibitors and cryo-trapped substrates. The catalytic strategies employed are comparable to those of the
Update
The first pre-steady-state kinetic analysis of the conversion of a number of purine nucleosides by TvNH shows that this enzyme exhibits burst kinetics and behaves with half-of-the-sites reactivity [37]. The analysis suggests that this NH follows a complex multistep mechanism in which the release of the product ribose is rate limiting. A slow isomerisation step from a tight to a loose enzyme–ribose complex, rather than chemical turnover, determines the macroscopic turnover number of wild-type Tv
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
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of special interest
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of outstanding interest
Acknowledgements
We acknowledge the use of synchrotron beam time at the EMBL beamlines at the DORIS storage ring (Hamburg, Germany) and the European Synchrotron Radiation Facility (Grenoble, France).
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