Enzyme-substrate interactions in the purine-specific nucleoside hydrolase from Trypanosoma vivax

Nucleoside hydrolases are key enzymes in the purine salvage pathway of Trypanosomatidae and are considered as targets for drug design. We previously reported the first x-ray structure of an inosine-adenosine-guanosine preferring nucleoside hydrolase (IAG-NH) from Trypanosoma vivax (). Here we report the 2.0-A crystal structure of the slow D10A mutant in complex with the inhibitor 3-deaza-adenosine and the 1.6-A crystal structure of the same enzyme in complex with a genuine substrate inosine. The enzyme-substrate complex shows the substrate bound to the enzyme in a different conformation from 3-deaza-adenosine and provides a snapshot along the reaction coordinate of the enzyme-catalyzed reaction. The chemical groups on the substrate important for binding and catalysis are mapped. The 2'-OH, 3'-OH, and 5'-OH contribute 4.6, 7.5, and 5.4 kcal/mol to k(cat)/K(m), respectively. Specific interactions with the exocyclic groups on the purine ring are not required for catalysis. Site-directed mutagenesis indicates that the purine specificity of the IAG-NHs is imposed by a parallel aromatic stacking interaction involving Trp(83) and Trp(260). The pH profiles of k(cat) and k(cat)/K(m) indicate the existence of one or more proton donors, possibly involved in leaving group activation. However, mutagenesis of the active site residues around the nucleoside base and an alanine scan of a flexible loop near the active site fail to identify this general acid. The parallel aromatic stacking seems to provide the most likely alternative mechanism for leaving group activation.     

Possible roles of beta-elimination and delta-elimination reactions in the repair of DNA containing AP (apurinic/apyrimidinic) sites in mammalian cells.

Histones and polyamines nick the phosphodiester bond 3' to AP (apurinic/apyrimidinic) sites in DNA by inducing a beta-elimination reaction, which can be followed by delta-elimination. These beta- and delta-elimination reactions might be important for the repair of AP sites in chromatin DNA in either of two ways. In one pathway, after the phosphodiester bond 5' to the AP site has been hydrolysed with an AP endonuclease, the 5'-terminal base-free sugar 5'-phosphate is released by beta-elimination. The one-nucleotide gap limited by 3'-OH and 5'-phosphate ends is then closed by DNA polymerase-beta and DNA ligase. We have shown in vitro that such a repair is possible. In the other pathway, the nicking 3' to the AP site by beta-elimination occurs first. We have shown that the 3'-terminal base-free sugar so produced cannot be released by the chromatin AP endonuclease from rat liver. But it can be released by delta-elimination, leaving a gap limited by 3'-phosphate and 5'-phosphate. After conversion of the 3'-phosphate into a 3'-OH group by the chromatin 3'-phosphatase, there will be the same one-nucleotide gap, limited by 3'-OH and 5'-phosphate, as that formed by the successive actions of the AP endonuclease and the beta-elimination catalyst in the first pathway.     

Energetic mapping of transition state analogue interactions with human and Plasmodium falciparum purine nucleoside phosphorylases

Human purine nucleoside phosphorylase (huPNP) is essential for human T-cell division by removing deoxyguanosine and preventing dGTP imbalance. Plasmodium falciparum expresses a distinct PNP (PfPNP) with a unique substrate specificity that includes 5'-methylthioinosine. The PfPNP functions both in purine salvage and in recycling purine groups from the polyamine synthetic pathway. Immucillin-H is an inhibitor of both huPNP and PfPNPs. It kills activated human T-cells and induces purine-less death in P. falciparum. Immucillin-H is a transition state analogue designed to mimic the early transition state of bovine PNP. The DADMe-Immucillins are second generation transition state analogues designed to match the fully dissociated transition states of huPNP and PfPNP. Immucillins, DADMe-Immucillins and related analogues are compared for their energetic interactions with human and P. falciparum PNPs. Immucillin-H and DADMe-Immucillin-H are 860 and 500 pM inhibitors against P. falciparum PNP but bind human PNP 15-35 times more tightly. This common pattern is a result of kcat for huPNP being 18-fold greater than kcat for PfPNP. This energetic binding difference between huPNP and PfPNP supports the k(chem)/kcat binding argument for transition state analogues. Preferential PfPNP inhibition is gained in the Immucillins by 5'-methylthio substitution which exploits the unique substrate specificity of PfPNP. Human PNP achieves part of its catalytic potential from 5'-OH neighboring group participation. When PfPNP acts on 5'-methylthioinosine, this interaction is not possible. Compensation for the 5'-OH effect in the P. falciparum enzyme is provided by improved leaving group interactions with Asp206 as a general acid compared with Asn at this position in huPNP. Specific atomic modifications in the transition state analogues cause disproportionate binding differences between huPNP and PfPNPs and pinpoint energetic binding differences despite similar transition states.     

Lanthanide Catalyzed Cyclization of Uridine 3'-p-Nitrophenyl Phosphate

Steady state kinetics and (15)N isotope effects have been used to study the cyclization reaction of uridine 3'-p-nitrophenyl phosphate. The cyclization reaction is catalyzed by transition metal ions and lanthanides, as are substitution reactions of many phosphate esters. Kinetic analysis reveals that the erbium-catalyzed cyclization reaction involves the concerted deprotonation of the 2'-OH group and departure of the leaving group. The transition state is very late, with a very large degree of bond cleavage to the leaving group, which could be due to a large degree of polarization of the P&bond;O bonds by erbium. Copyright 2000 Academic Press.     

T4 DNA ligase can seal a nick in double-stranded DNA limited by a 5''-phosphorylated base-free deoxyribose residue.

The 5' AP endodeoxyribonucleases hydrolyze the phosphodiester bond 5' to AP (apurinic or apyrimidinic) sites in double-stranded DNA leaving 3'-OH and 5'-phosphate ends. These nicks are sealed by T4 DNA ligase although the 5'-phosphate end belongs to a base-free deoxyribose.     

Leaving group stabilization by metal ion coordination and hydrogen bond donation is an evolutionarily conserved feature of group I introns.

To understand the behavior of group I introns on a biologically fundamental level, we must distinguish those traits that arise as the products of natural selection (selected traits) from those that arise as the products of neutral drift (non-selected traits). In practice, this distinction relies on comparing the similarities and differences among widely divergent introns to identify conserved traits. Here we address whether the strategies used by the eukaryotic group I intron from the Tetrahymena ciliate to stabilize the leaving group during splicing are maintained in the group I intron from the widely divergent Azoarcus bacterium. A substrate analogue containing a 3'-phosphorothiolate linkage, in which a sulfur atom replaces the bridging 3'-oxygen atom of the scissile phosphate, reacts 20-fold slower in the Azoarcus reaction than the corresponding unmodified substrate in the presence of Mg(II) as the only divalent cation. However, Mn(II) relieves this negative effect such that the 3'-S-P bond cleaves 21-fold faster than does the 3'O-P bond. Other thiophilic divalent metal ions such as Co(II), Cd(II), and Zn(II) similarly support cleavage of the S-P bond. These results indicate that a metal ion directly coordinates to the leaving group in the transition state of the Azoarcus ribozyme reaction. Additionally, the 3'-sulfur substitution eliminates the approximately 10(3)-fold contribution of the adjacent 2'-OH to transition state stabilization. Considering that sulfur accepts hydrogen bonds weakly compared to oxygen, this result suggests that the 2'-OH contributes to catalysis by donating a hydrogen bond to the 3'-oxygen leaving group in the transition state, presumably acting in conjunction with the metal ion to stabilize the developing negative charge. These same catalytic strategies of metal ion coordination and hydrogen bond donation operate in the Tetrahymena ribozyme reaction, suggesting that these features of catalysis have been conserved during evolution and thus extend to all group I introns. The two ribozymes also exhibit quantitative differences in their response to 3'-sulfur substitution. The Azoarcus ribozyme binds and cleaves the phosphorothiolate substrate more efficiently relative to the natural substrate than the Tetrahymena ribozyme under the same conditions, suggesting that the Azoarcus ribozyme better accommodates the phosphorothiolate at the active site both in the ground state and in the transition state. These differences may reflect either a less tightly knit Azoarcus structure and/or spatial deviations between backbone atoms in the two ribozymes that arise during divergent evolution, analogous to the well-documented relationship between protein sequence and structure.     

Introduction of a benzyl group onto the 2'-OH of 6-chloropurine 3'-O-benzoylriboside

A new method to introduce a benzyl group onto the 2'-OH of purine ribonucleoside is described. Thus, 6-chloropurine 3'-O-benzoylriboside and its 5'-O-trityl congener were condensed with benzyl alcohol using the Mitsunobu reaction to give the 2'-O-benzyl derivative. The yields were varied from 4.6 to 62.9% depending on the solvent. The product was converted to adenosine, indicating that the stereochemistry at C-2' is retained.     

Phosphorus-31 Fourier transform nuclear magnetic resonance study of mononucleotides and dinucleotides. 1. Chemical shifts.

A phosphorus-31 nuclear magnetic resonance (NMR) study of adenine, uracil, and thymine mononucleotides, their cyclic analogues, and the corresponding dinucleotides is reported. From the pH dependence of phosphate chemical shifts, pKa values of 6.25-6.30 are found for all 5'-mononucleotides secondary phosphate ionization, independently from the nature of the base and the presence of a hydroxyl group at the 2' position. Conversely, substitution of a hydrogen atom for a 2'-OH lowers the pKa of 3'-monoribonucleotides from 6.25 down to 5.71-5.85. This indication of a strong influence of the 2'-hydroxyl group on the 3'-phosphate is confirmed by the existence of a 0.4 to 0.5 ppm downfield shift induced by the 2'-OH on the phosphate resonance of 3'-monoribonucleotides, and 3',5'-cyclic nucleotides and dinucleotides with respect to the deoxyribosyl analogues. Phosphate chemical shifts and titration curves are affected by the ionization and the type of the base. Typically, deviations from the theoretical Henderson-Hasselbalch plots are observed upon base titration. In addition, purine displays a more deshielding influence than pyrimidine on the phosphate groups of most of the mononucleotides (0.10 to 0.25 ppm downfield shift) with a reverse situation for dinucleotides. These effects together with the importance of stereochemical arrangement (furanose ring pucker, furanose-phosphate backbone conformation, O-P-O bond angle) on the phosphate chemical shifts are discussed.     

A new method for the synthesis of 2'-O-benzyladenosine using Mitsunobu reaction

A new method to introduce a benzyl group onto the 2'-OH of purine ribonucleoside is described. Thus, 6-chloropurine 3'-O-benzoylriboside and its 5'-O-trityl congener were condensed with benzyl alcohol using the Mitsunobu reaction to give the 2'-O-benzyl derivative. The yields were varied from 4.6 to 62.9% depending on the solvent used. The product was converted to adenosine, indicating that the stereochemistry at C-2' is retained.     

Catalysis by nucleoside hydrolases

Nucleoside hydrolases cleave the N-glycosidic bond of ribonucleosides. Because of their vital role in the protozoan purine salvage pathway, nucleoside hydrolases from parasitic protozoa in particular have been studied extensively by X-ray crystallography, kinetic methods and site-directed mutagenesis. An elaborate network of conserved interactions between the metalloenzyme and the ribose enables steric and electrostatic stabilisation of the oxocarbenium-ion-like transition state. Activation of the leaving group by protonation before the formation of the transition state is a recurring catalytic strategy of enzymes that cleave N-glycosidic bonds. However, the mechanisms underlying leaving group activation are still the subject of debate for the nucleoside hydrolases.     

Specificity and mode of cleavage of the pH 4.0 endonuclease from adenovirus type 2 - infected KB cells.

Adenovirus type 2 or lambda DNA was digested with the pH 4.0 endonuclease, purified from adenovirus 2-infected KB cells. The enzyme produces a limit digest of approximate size in the range of 140-210 base pairs long. The termini of the DNA fragments generated by the endonuclease digestion had 3'-P and 5'-OH groups. The 3' and 5' end groups of the products were analyzed. Our data indicate that 3' end group was a purine (68-76%), dA occuring about twice the frequency of dG. The 5' end group was either dG or dC with equal frequency. Data obtained by treatment of the 5' labeled endonuclease product of lambda DNA with single-strand specific S1 nuclease from Asperigillus oryzae or exonuclease VII from Escherichia coli indicated that the majority of the products had a short 5' protruding ends. The mode of cleavage of this endonuclease seems to be through initial formation of several single-strand breaks with some base specificity. If these breaks are at close proximity on opposite strands, double-stranded fragments with protruding ends are generated.     

Structural and biochemical characterization of the nucleoside hydrolase from C. elegans reveals the role of two active site cysteine residues in catalysis

Nucleoside hydrolases (NHs) catalyze the hydrolysis of the N‐glycoside bond in ribonucleosides and are found in all three domains of life. Although in parasitic protozoa a role in purine salvage has been well established, their precise function in bacteria and higher eukaryotes is still largely unknown. NHs have been classified into three homology groups based on the conservation of active site residues. While many structures are available of representatives of group I and II, structural information for group III NHs is lacking. Here, we report the first crystal structure of a purine‐specific nucleoside hydrolase belonging to homology group III from the nematode Caenorhabditis elegans (CeNH) to 1.65Å resolution. In contrast to dimeric purine‐specific NHs from group II, CeNH is a homotetramer. A cysteine residue that characterizes group III NHs (Cys253) structurally aligns with the catalytic histidine and tryptophan residues of group I and group II enzymes, respectively. Moreover, a second cysteine (Cys42) points into the active site of CeNH. Substrate docking shows that both cysteine residues are appropriately positioned to interact with the purine ring. Site‐directed mutagenesis and kinetic analysis proposes a catalytic role for both cysteines residues, with Cys253 playing the most prominent role in leaving group activation.     

Structure-function relationship in Escherichia coli initiation factors. Biochemical and biophysical characterization of the interaction between IF-2 and guanosine nucleotides

Equilibrium dialysis and protection from heat inactivation and proteolysis show that initiation factor 2 (IF-2) interacts not only with GTP but also with GDP and that its conformation is changed upon binding of either nucleotide. The apparent Ka (at 25 degrees C) for the IF-2 X GDP and IF-2 X GTP complexes was 8.0 X 10(4) and 7.0 X 10(3) M(-1), respectively. The lower affinity for GTP is associated with a more negative delta S0. The interaction, monitored by 1HNMR spectroscopy, is characterized by fast exchange and results in line broadening and downfield shift of the purine C-8 and ribose C-1' protons of GTP as well as of the beta, gamma-methylene protons of (beta-gamma-methylene)guanosine 5'-triphosphate. The interaction of guanosine nucleotides with IF-2 requires an H bond donor (or acceptor) group at position C-2 of the purine and involves the beta- and/or gamma-phosphate of the nucleotide while the ribose 2'-OH group or the integrity of the furan ring are less critical. IF-2 binds to ribosomal particles with decreasing affinity: 30 S greater than 70 S greater than 50 S. GTP and GDP have no effect on the binding to 70 S. GTP stimulates the binding to the 30 S and depresses somewhat the binding to the 50 S subunits; GDP has the opposite effect. These results seem to rule out that the release of IF 2 from 70 S is due to a "GDP-conformation" of the factor incompatible with its permanence on the ribosome. The rate and the extent of 30 S initiation complex formation are approximately 2-fold higher with IF-2 X GTP than with IF-2 alone. At low concentrations of IF-2 and 30 S subunits, GDP inhibits this reaction, acting as a strong competitive inhibitor of GTP (Ki = 1.25 X 10(-5)m) and preventing IF-2 from binding to the ribosomal subunit.     

Determination of the activation energy for pseudorotation of the furanose ring in nucleosides by 13-C nuclear-magnetic-resonance relaxation.

The activation energies for the pseudorotation of the furanose ring in adenosine, guanosine, inosine and xanthosine dissolved in liquid deuteroammonia have been determined by analysis of the longitudinal relaxation rates of the single tertiary carbons between +40 degrees C and minus 60 degrees C. For the purine ribosides the average activation energy was found to be 4.7 plus or minus 0.5 kcal x mol-1 (20 plus or minus 2 kJ x mol-1). For the pyrimidine nucleosides cytidine and uridine the respective activation energy should be higher since it could not be determined by 13-C relaxation measurements. This result can be explained by the formation of a hydrogen bond between the 5'-hydroxymethyl group and the base. In adenosine, guanosine, inosine and xanthosine the relaxation rates of C(5') are smaller than all others thus excluding the formation of a hydrogen bond between the purine base and the 5'-hydroxymethyl group of a strength comparable to the one suggested for cytidine and uridine.     

Hydration of [d(CGC)r(aaa)d(TTTGCG)](2)

We have studied the hydration and dynamics of RNA C2'-OH in a DNA. RNA hybrid chimeric duplex [d(CGC)r(aaa)d(TTTGCG)](2). Long-lived water molecules with correlation time tau(c) larger than 0.3 ns were found close to the RNA adenine H2 and H1' protons in the hybrid segment. A possible long-lived water molecule was also detected close to the methyl group of 7T in the RNA-DNA junction but not to the other two thymine bases (8T and 9T). This result correlates with the structural studies that only DNA residue 7T in the RNA-DNA junction adopts an O4'-endo sugar conformation (intermediate between B-form and A-form), while the other DNA residues including 3C in the DNA-RNA junction, adopt C1'-exo or C2'-endo conformations (in the B-form domain). Based on the NOE cross-peak patterns, we have found that RNA C2'-OH tends to orient toward the O3' direction, forming a possible hydrogen bond with the 3'-phosphate group. The exchange rates for RNA C2'-OH were found to be around 5-20 s(-1), compared to 26.7(+/-13.8) s(-1) reported previously for the other DNA.RNA hybrid duplex. This slow exchange rate may be due to the narrow minor groove width of [d(CGC)r(aaa)d(TTTGCG)](2), which may trap the water molecules and restrict the dynamic motion of hydroxyl protons. The distinct hydration patterns of the RNA adenine H2 and H1' protons and the DNA 7T methyl group in the hybrid segment, as well as the orientation and dynamics of the RNA C2'-OH protons, may provide a molecular basis for further understanding the structure and recognition of DNA.RNA hybrid and chimeric duplexes.     

Three-dimensional structure of ribonuclease T1 complexed with an isosteric phosphonate substrate analogue of GpU: alternate substrate binding modes and catalysis

The X-ray crystal structure of a complex between ribonuclease T1 and guanylyl(3'-6')-6'-deoxyhomouridine (GpcU) has been determined at 2. 0 A resolution. This ligand is an isosteric analogue of the minimal RNA substrate, guanylyl(3'-5')uridine (GpU), where a methylene is substituted for the uridine 5'-oxygen atom. Two protein molecules are part of the asymmetric unit and both have a GpcU bound at the active site in the same manner. The protein-protein interface reveals an extended aromatic stack involving both guanines and three enzyme phenolic groups. A third GpcU has its guanine moiety stacked on His92 at the active site on enzyme molecule A and interacts with GpcU on molecule B in a neighboring unit via hydrogen bonding between uridine ribose 2'- and 3'-OH groups. None of the uridine moieties of the three GpcU molecules in the asymmetric unit interacts directly with the protein. GpcU-active-site interactions involve extensive hydrogen bonding of the guanine moiety at the primary recognition site and of the guanosine 2'-hydroxyl group with His40 and Glu58. On the other hand, the phosphonate group is weakly bound only by a single hydrogen bond with Tyr38, unlike ligand phosphate groups of other substrate analogues and 3'-GMP, which hydrogen-bonded with three additional active-site residues. Hydrogen bonding of the guanylyl 2'-OH group and the phosphonate moiety is essentially the same as that recently observed for a novel structure of a RNase T1-3'-GMP complex obtained immediately after in situ hydrolysis of exo-(Sp)-guanosine 2',3'-cyclophosphorothioate [Zegers et al. (1998) Nature Struct. Biol. 5, 280-283]. It is likely that GpcU at the active site represents a nonproductive binding mode for GpU [Steyaert, J., and Engleborghs (1995) Eur. J. Biochem. 233, 140-144]. The results suggest that the active site of ribonuclease T1 is adapted for optimal tight binding of both the guanylyl 2'-OH and phosphate groups (of GpU) only in the transition state for catalytic transesterification, which is stabilized by adjacent binding of the leaving nucleoside (U) group.     

Structural requirements for the activation of Escherichia coli CTP synthase by the allosteric effector GTP are stringent, but requirements for inhibition are lax

Cytidine 5'-triphosphate synthase catalyzes the ATP-dependent formation of CTP from UTP using either NH(3) or l-glutamine (Gln) as the source of nitrogen. GTP acts as an allosteric effector promoting Gln hydrolysis but inhibiting Gln-dependent CTP formation at concentrations of >0.15 mM and NH(3)-dependent CTP formation at all concentrations. A structure-activity study using a variety of GTP and guanosine analogues revealed that only a few GTP analogues were capable of activating Gln-dependent CTP formation to varying degrees: GTP approximately 6-thio-GTP > ITP approximately guanosine 5'-tetraphosphate > O(6)-methyl-GTP > 2'-deoxy-GTP. No activation was observed with guanosine, GMP, GDP, 2',3'-dideoxy-GTP, acycloguanosine, and acycloguanosine monophosphate, indicating that the 5'-triphosphate, 2'-OH, and 3'-OH are required for full activation. The 2-NH(2) group plays an important role in binding recognition, whereas substituents at the 6-position play an important role in activation. The presence of a 6-NH(2) group obviates activation, consistent with the inability of ATP to substitute for GTP. Nucleotide and nucleoside analogues of GTP and guanosine, respectively, all inhibited NH(3)- and Gln-dependent CTP formation (often in a cooperative manner) to a similar extent (IC(50) approximately 0.2-0.5 mM). This inhibition appeared to be due solely to the purine base and was relatively insensitive to the identity of the purine with the exception of inosine, ITP, and adenosine (IC(50) approximately 4-12 mM). 8-Oxoguanosine was the best inhibitor identified (IC(50) = 80 microM). Our findings suggest that modifying 2-aminopurine or 2-aminopurine riboside may serve as an effective strategy for developing cytidine 5'-triphosphate synthase inhibitors.