The crystal structures of Thermus thermophilus CMP kinase complexed with a phosphoryl group acceptor and donor.

Nucleoside monophosphate kinases play crucial roles in biosynthesis and regeneration of nucleotides. These are bi-substrate enzymes that catalyze reversible transfers of a phosphoryl group between ATP and nucleoside monophosphate. These enzymes are comprised of the CORE domain, the NMP-binding domain, and the LID domain. Large conformational rearrangement of the three domains occurs during the catalytic cycle. Although many structures of CMP kinase have been determined, only limited structural information has been available on the conformational changes along the reaction pathway. We determined five crystal structures of CMP kinase of Thermus thermophilus HB8 in ligand-free form and the CMP "open", CMP "closed", ADP-CDP-Gd3+-, and CDP-bound forms at resolutions of 1.7, 2.2, 1.5, 1.6, and 1.7 Å, respectively. The ligand-free form was in an open conformation, whereas the structures of the CMP "closed", ADP-CDP-Gd3+-, and CDP-bound forms were in a closed conformation, in which the shift of the NMP-binding domain and LID domain caused closure of the substrate-binding cleft. Interestingly, the CMP "open" form was in an open conformation even with CMP bound, implying intrinsic conformational fluctuation. The structure of the ADP-CDP complex is the first structure of CMP kinase with a phosphoryl group donor and an acceptor. Upon simultaneous binding of ADP and CDP, the side chains of several residues in the LID domain moved toward the nucleotides without global open-closed conformational changes compared to those in the CMP "closed" and CDP complexes. These global and local conformational changes may be crucial for the substrate recognition and catalysis. The terminal phosphate groups of ADP and CDP had similar geometry to those of two ADP in AMP kinase, suggesting common catalytic mechanisms to other nucleoside monophosphate kinases. Our findings are expected to contribute to detailed understanding of the reaction mechanism of CMP kinase.

Structural characterization of human O-phosphoethanolamine phospho-lyase.

Human O-phosphoethanolamine phospho-lyase (hEtnppl; EC 4.2.3.2) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the degradation of O-phosphoethanolamine (PEA) into acetaldehyde, phosphate and ammonia. Physiologically, the enzyme is involved in phospholipid metabolism, as PEA is the precursor of phosphatidylethanolamine in the CDP-ethanolamine (Kennedy) pathway. Here, the crystal structure of hEtnppl in complex with pyridoxamine 5'-phosphate was determined at 2.05 Šresolution by molecular replacement using the structure of A1RDF1 from Arthrobacter aurescens TC1 (PDB entry 5g4i) as the search model. Structural analysis reveals that the two proteins share the same general fold and a similar arrangement of active-site residues. These results provide novel and useful information for the complete characterization of the human enzyme.

Structural and functional characterization of CMP-N-acetylneuraminate synthetase from Vibrio cholerae.

Several pathogenic bacteria utilize sialic acid, including host-derived N-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, including Neisseria meningitidis, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, from Vibrio cholerae are reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg2+ in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure of V. cholerae CMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process.

Probing specific ligand-protein interactions by native-denatured exchange mass spectrometry.

Probing ligand-target protein interactions provides essential information for deep understanding of biochemical machinery and design of drug screening assays. Native electrospray ionization-mass spectrometry (ESI-MS) is promising for direct analysis of ligand-protein complexes. However, it lacks the ability to distinguish between specific and non-specific ligand-protein interactions, and to further recognize the specifically bound proteins as drug target candidates, which remains as a major challenge in the field of drug developments by far. Herein we report a native-denatured exchange (NDX) mass spectrometry (MS) acquisition approach using a liquid sample-desorption electrospray ionization (LS-DESI) setup, and demonstrate its capability in enabling a change from native detection of noncovalent ligand-protein complexes to denatured analysis using three model ligand-protein complexes including myoglobin, CDP-ribonuclease and N,N',N″-triacetylchitotriose (NAG3)-lysozyme. Notably, we found the NDX-MS approach can readily discriminate specific ligand-protein interactions from nonspecific ones, as revealed by their distinct dynamic profiles of Kd as a function of the DESI spraying flow rate. Consequently, this NDX-MS approach holds promise for future applications to discovering specific protein targets for ligands of interest, and to screening compounds with high specificity to drug targets and thus eliminates off-target effects.

Influence of copper(II) ions on the noncovalent interactions between cytidine-5'-diphosphate or cytidine-5'-triphosphate and biogenic amines putrescine or spermidine.

Potentiometric and NMR spectroscopic studies of the nucleotide (NucP)/polyamine (PA) system (where NucP = CDP, CTP, PA = putrescine or spermidine) revealed the formation of molecular complexes (NucP)(Hx+y)(PA) (where Hx+y = number of protons; x - from NucP and y - from PA). Their thermodynamic parameters were determined and the modes of their interactions were proposed. The main reaction centers were found to be the protonated amine groups of polyamine (positive centers) and phosphate groups of nucleotide (negative centers). The pH ranges in which the complex occurs correspond to those of amine protonation and -PO3x- group deprotonation, which unambiguously confirms the dipole-dipole type of interaction. In the pH range of total deprotonation of NHx+ groups from the polyamine, the molecular complexes disappear. The equilibrium and spectroscopic studies of the ternary systems Cu(II)/NucP/PA evidenced the formation of Cu(NucP)Hx+y(PA) type coordination compounds and Cu(NucP)⋯(PA)(Hx) type molecular complexes with polyamine in the outer coordination sphere. The main sites of metal ion bonding in the latter species are the phosphate groups of the nucleotide, while in the coordination compounds - besides the phosphate groups - also the donor nitrogen atoms from the polyamines. In this paper we have also quantitatively calculated the effect of metal ions on the formation of the molecular complexes.

3.3-Å resolution cryo-EM structure of human ribonucleotide reductase with substrate and allosteric regulators bound.

Ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides, a reaction essential for DNA replication and repair. Human RNR requires two subunits for activity, the α subunit contains the active site, and the ß subunit houses the radical cofactor. Here, we present a 3.3-Å resolution structure by cryo-electron microscopy (EM) of a dATP-inhibited state of human RNR. This structure, which was determined in the presence of substrate CDP and allosteric regulators ATP and dATP, has three α2 units arranged in an α6 ring. At near-atomic resolution, these data provide insight into the molecular basis for CDP recognition by allosteric specificity effectors dATP/ATP. Additionally, we present lower-resolution EM structures of human α6 in the presence of both the anticancer drug clofarabine triphosphate and ß2. Together, these structures support a model for RNR inhibition in which ß2 is excluded from binding in a radical transfer competent position when α exists as a stable hexamer.

Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism.

It is thought that KRAS oncoproteins are constitutively active because their guanosine triphosphatase (GTPase) activity is disabled. Consequently, drugs targeting the inactive or guanosine 5'-diphosphate-bound conformation are not expected to be effective. We describe a mechanism that enables such drugs to inhibit KRAS(G12C) signaling and cancer cell growth. Inhibition requires intact GTPase activity and occurs because drug-bound KRAS(G12C) is insusceptible to nucleotide exchange factors and thus trapped in its inactive state. Indeed, mutants completely lacking GTPase activity and those promoting exchange reduced the potency of the drug. Suppressing nucleotide exchange activity downstream of various tyrosine kinases enhanced KRAS(G12C) inhibition, whereas its potentiation had the opposite effect. These findings reveal that KRAS(G12C) undergoes nucleotide cycling in cancer cells and provide a basis for developing effective therapies to treat KRAS(G12C)-driven cancers.

X-ray structure of a CDP-alcohol phosphatidyltransferase membrane enzyme and insights into its catalytic mechanism.

Phospholipids have major roles in the structure and function of all cell membranes. Most integral membrane proteins from the large CDP-alcohol phosphatidyltransferase family are involved in phospholipid biosynthesis across the three domains of life. They share a conserved sequence pattern and catalyse the displacement of CMP from a CDP-alcohol by a second alcohol. Here we report the crystal structure of a bifunctional enzyme comprising a cytoplasmic nucleotidyltransferase domain (IPCT) fused with a membrane CDP-alcohol phosphotransferase domain (DIPPS) at 2.65 Å resolution. The bifunctional protein dimerizes through the DIPPS domains, each comprising six transmembrane α-helices. The active site cavity is hydrophilic and widely open to the cytoplasm with a magnesium ion surrounded by four highly conserved aspartate residues from helices TM2 and TM3. We show that magnesium is essential for the enzymatic activity and is involved in catalysis. Substrates docking is validated by mutagenesis studies, and a structure-based catalytic mechanism is proposed.

A rare combination of ribonucleotide reductases in the social amoeba Dictyostelium discoideum.

Ribonucleotide reductases (RNRs) catalyze the only pathway for de novo synthesis of deoxyribonucleotides needed for DNA replication and repair. The vast majority of eukaryotes encodes only a class I RNR, but interestingly some eukaryotes, including the social amoeba Dictyostelium discoideum, encode both a class I and a class II RNR. The amino acid sequence of the D. discoideum class I RNR is similar to other eukaryotic RNRs, whereas that of its class II RNR is most similar to the monomeric class II RNRs found in Lactobacillus spp. and a few other bacteria. Here we report the first study of RNRs in a eukaryotic organism that encodes class I and class II RNRs. Both classes of RNR genes were expressed in D. discoideum cells, although the class I transcripts were more abundant and strongly enriched during mid-development compared with the class II transcript. The quaternary structure, allosteric regulation, and properties of the diiron-oxo/radical cofactor of D. discoideum class I RNR are similar to those of the mammalian RNRs. Inhibition of D. discoideum class I RNR by hydroxyurea resulted in a 90% reduction in spore formation and decreased the germination viability of the surviving spores by 75%. Class II RNR could not compensate for class I inhibition during development, and an excess of vitamin B12 coenzyme, which is essential for class II activity, did not improve spore formation. We suggest that class I is the principal RNR during D. discoideum development and growth and is important for spore formation, possibly by providing dNTPs for mitochondrial replication.

Tangled up in knots: structures of inactivated forms of E. coli class Ia ribonucleotide reductase.

Ribonucleotide reductases (RNRs) provide the precursors for DNA biosynthesis and repair and are successful targets for anticancer drugs such as clofarabine and gemcitabine. Recently, we reported that dATP inhibits E. coli class Ia RNR by driving formation of RNR subunits into α4ß4 rings. Here, we present the first X-ray structure of a gemcitabine-inhibited E. coli RNR and show that the previously described α4ß4 rings can interlock to form an unprecedented (α4ß4)2 megacomplex. This complex is also seen in a higher-resolution dATP-inhibited RNR structure presented here, which employs a distinct crystal lattice from that observed in the gemcitabine-inhibited case. With few reported examples of protein catenanes, we use data from small-angle X-ray scattering and electron microscopy to both understand the solution conditions that contribute to concatenation in RNRs as well as present a mechanism for the formation of these unusual structures.

Role of arginine 293 and glutamine 288 in communication between catalytic and allosteric sites in yeast ribonucleotide reductase.

Ribonucleotide reductases (RRs) catalyze the rate-limiting step of de novo deoxynucleotide (dNTP) synthesis. Eukaryotic RRs consist of two proteins, RR1 (α) that contains the catalytic site and RR2 (ß) that houses a diferric-tyrosyl radical essential for ribonucleoside diphosphate reduction. Biochemical analysis has been combined with isothermal titration calorimetry (ITC), X-ray crystallography and yeast genetics to elucidate the roles of two loop 2 mutations R293A and Q288A in Saccharomyces cerevisiae RR1 (ScRR1). These mutations, R293A and Q288A, cause lethality and severe S phase defects, respectively, in cells that use ScRR1 as the sole source of RR1 activity. Compared to the wild-type enzyme activity, R293A and Q288A mutants show 4% and 15%, respectively, for ADP reduction, whereas they are 20% and 23%, respectively, for CDP reduction. ITC data showed that R293A ScRR1 is unable to bind ADP and binds CDP with 2-fold lower affinity compared to wild-type ScRR1. With the Q288A ScRR1 mutant, there is a 6-fold loss of affinity for ADP binding and a 2-fold loss of affinity for CDP compared to the wild type. X-ray structures of R293A ScRR1 complexed with dGTP and AMPPNP-CDP [AMPPNP, adenosine 5-(ß,γ-imido)triphosphate tetralithium salt] reveal that ADP is not bound at the catalytic site, and CDP binds farther from the catalytic site compared to wild type. Our in vivo functional analyses demonstrated that R293A cannot support mitotic growth, whereas Q288A can, albeit with a severe S phase defect. Taken together, our structure, activity, ITC and in vivo data reveal that the arginine 293 and glutamine 288 residues of ScRR1 are crucial in facilitating ADP and CDP substrate selection.

Biochemical characterization of the CDP-D-arabinitol biosynthetic pathway in Streptococcus pneumoniae 17F.

Streptococcus pneumoniae is a major human pathogen associated with many diseases worldwide. Capsular polysaccharides (CPSs) are the major virulence factor. The biosynthetic pathway of D-arabinitol, which is present in the CPSs of several S. pneumoniae serotypes, has never been identified. In this study, the genes abpA (previously known as abp1) and abpB (previously known as abp2), which have previously been reported to be responsible for nucleoside diphosphate (NDP)-D-arabinitol (the nucleotide-activated form of D-arabinitol) synthesis, were cloned. The enzyme products were overexpressed, purified, and analyzed for their respective activities. Novel products produced by AbpA- and AbpB-catalyzing reactions were detected by capillary electrophoresis, and the structures of the products were elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. As a result, abpA was identified to be a D-xylulose-5-phosphate cytidylyltransferase-encoding gene, responsible for the transfer of CTP to D-xylulose-5-phosphate (D-Xlu-5-P) to form CDP-D-xylulose, and abpB was characterized to be a CDP-D-xylulose reductase-encoding gene, responsible for the conversion of CDP-D-xylulose to CDP-D-arabinitol as the final product. The kinetic parameters of AbpA for the substrates D-Xlu-5-P and CTP and those of AbpB for the substrate CDP-D-xylulose and the cofactors NADH or NADPH were measured, and the effects of temperature, pH, and cations on the two enzymes were analyzed. This study confirmed the involvement of the genes abpA and abpB and their products in the biosynthetic pathway of CDP-D-arabinitol.

Unique self-anhydride formation in the degradation of cytidine-5'-monophosphosialic acid (CMP-Neu5Ac) and cytidine-5'-diphosphosialic acid (CDP-Neu5Ac) and its application in CMP-sialic acid analogue synthesis.

Sialyloligosaccharides are synthesised by various glycosyltransferases and sugar nucleotides. All of these nucleotides are diphosphate compounds except for cytidine-5'-monophosphosialic acid (CMP-Neu5Ac). To obtain an insight into why cytidine-5'-diphosphosialic acid (CDP-Neu5Ac) has not been used for the sialyltransferase reaction and why it is not found in biological organisms, the compound was synthesised. This synthesis provided the interesting finding that the carboxylic acid moiety of the sialic acid attacks the attached phosphate group. This interaction yields an activated anhydride between carboxylic acid and the phosphate group and leads to hydrolysis of the pyrophosphate linkage. The mechanism was demonstrated by stable isotope-labelling experiments. This finding suggested that CMP-Neu5Ac might also form the corresponding anhydride structure between carboxylic acid and phosphate, and this seems to be the reason why CMP-Neu5Ac is acid labile in relation to other sugar nucleotides. To confirm the role of the carboxylic acid, CMP-Neu5Ac derivatives in which the carboxylic acid moiety in the sialic acid was substituted with amide or ester groups were synthesised. These analogues clearly exhibited resistance to acid hydrolysis. This result indicated that the carboxylic acid of Neu5Ac is associated with its stability in solution. This finding also enabled the development of a novel chemical synthetic method for CMP-Neu5Ac and CMP-sialic acid derivatives.

Ligand chirality effects on the dynamics of human 3-phosphoglycerate kinase: comparison between D- and L-nucleotides.

l-nucleoside analogues are now largely used as antiviral drugs for the treatment of viral infections like HBV, HCV and HIV. However, in order to be fully active, they need to be phosphorylated by cellular or viral kinases. Human 3-phosphogycerate kinase (hPGK) was shown to catalyze the last step of activation of l-enantiomers and thus constitutes an attractive target for theoretical predictions of its phosphorylation efficiency. Molecular dynamics simulations were carried out with four different nucleotides (d-/l-ADP and d-/l-CDP) in complex with hPGK and 1,3-bisphospho-d-glycerate (bPG). The binding affinities of CDPs (both enantiomers) for hPGK were found very weak while d- and l-ADP were better substrates. Interestingly, the binding affinity of the bPG substrate was found to be lower in presence of d-ADP than l-ADP which indicates a potential antagonistic effect on one substrate to the other. A detailed analysis of the simulations unravels important dynamic conditions for efficient phosphorylation. Indeed, as previously described for the natural substrate, the hinge bending motion of the domains upon substrates binding should be more correlated and directional. Interestingly, the unforeseen finding was the larger dynamics freedoms observed for the substrates that was favored by the protein atoms flexibility around the nucleobase binding site.

Homology modeling of Mycobacterium tuberculosis 2C-methyl-D-erythritol-4-phosphate cytidylyltransferase, the third enzyme in the MEP pathway for isoprenoid biosynthesis.

Tuberculosis is one of the leading infectious diseases in humans. Discovering new treatments for this disease is urgently required, especially in view of the emergence of multiple drug resistant organisms and to reduce the total duration of current treatments. The synthesis of isoprenoids in Mycobacterium tuberculosis has been reported as an interesting pathway to target, and particular attention has been focused on the methylerythritol phosphate (MEP) pathway comprising the early steps of isoprenoid biosynthesis. In this context we have studied the enzyme 2C-methyl-D-erythritol-4-phosphate cytidylyltransferase (CMS), the third enzyme in the MEP pathway, since the lack of a resolved structure of this protein in M. tuberculosis has seriously limited its use as a drug target. We performed homology modeling of M. tuberculosis CMS in order to provide a reliable model for use in structure-based drug design. After evaluating the quality of the model, we performed a thorough study of the catalytic site and the dimerization interface of the model, which suggested the most important sites (conserved and non-conserved) that could be useful for drug discovery and mutagenesis studies. We found that the metal coordination of CDP-methylerythritol in M. tuberculosis CMS differs substantially with respect to the Escherichia coli variant, consistent with the fact that the former is able to utilize several metal ions for catalysis. Moreover, we propose that electrostatic interactions could explain the higher affinity of the MEP substrate compared with the cytosine 5'-triphosphate substrate in the M. tuberculosis enzyme as reported previously.

Mechanism of inactivation of human ribonucleotide reductase with p53R2 by gemcitabine 5'-diphosphate.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside 5'-diphosphates to the corresponding deoxynucleotides supplying the dNTPs required for DNA replication and DNA repair. Class I RNRs require two subunits, alpha and beta, for activity. Humans possess two beta subunits: one involved in S phase DNA replication (beta) and a second in mitochondrial DNA replication (beta' or p53R2) and potentially DNA repair. Gemcitabine (F(2)C) is used clinically as an anticancer agent, and its phosphorylated metabolites target many enzymes involved in nucleotide metabolism, including RNR. The present investigation with alpha (specific activity of 400 nmol min(-1) mg(-1)) and beta' (0.6 Y./beta'2 and a specific activity of 420 nmol min(-1) mg(-1)) establishes that F(2)CDP is a substoichiometric inactivator of RNR. Incubation of this alpha/beta' with [1'-(3)H]-F(2)CDP or [5-(3)H]-F(2)CDP and reisolation of the protein by Sephadex G-50 chromatography resulted in recovery 0.5 equiv of covalently bound sugar and 0.03 equiv of tightly associated cytosine to alpha2. SDS-PAGE analysis (loaded without boiling) of the inactivated RNR showed that 60% of alpha migrates as a 90 kDa protein and 40% as a 120 kDa protein. Incubation of [1'-(3)H]-F(2)CDP with active site mutants C444S/A, C218S/A, and E431Q/D-alpha and the C-terminal tail C787S/A and C790S/A mutants reveals that no sugar label is bound to the active site mutants of alpha and that, in the case of C218S-alpha, alpha migrates as a 90 kDa protein. Analysis of the inactivated wt-alpha/beta' RNR by size exclusion chromatography indicates a quaternary structure of alpha6beta'6. A mechanism of inactivation common with halpha/beta is presented.

The wall teichoic acid polymerase TagF is non-processive in vitro and amenable to study using steady state kinetic analysis.

Wall teichoic acids are a chemically diverse group of anionic polymers that constitute up to 50% of the Gram-positive cell wall. These polymers play a pivotal role in virulence and have been implicated in a diverse range of physiological functions. The TagF-like family of enzymes has been shown to be responsible for wall teichoic acid priming and polymerization events. Although many such enzymes are well validated therapeutic targets, a mechanistic understanding of this enzyme family has remained elusive. TagF is the prototypical teichoic acid polymerase and uses CDP-glycerol to catalyze synthesis of the linear (1,3)-linked poly(glycerol phosphate) teichoic acid in Bacillus subtilis 168. Here we used a synthetic soluble analog of the natural substrate of the enzyme, Lipid , to conduct the first detailed mechanistic investigation of teichoic acid polymerization. Through the use of a new high pressure liquid chromatography-based assay to monitor single glycerol phosphate incorporations into the Lipid analog, we conducted a detailed analysis of reaction product formation patterns and unequivocally showed TagF to be non-processive in vitro. Furthermore by monitoring the kinetics of polymerization, we showed that Lipid analog species varying in size have the same K(m) value of 2.6 microm and validated use of Bi Bi velocity expressions to model the TagF enzyme system. Initial rate analysis showed that TagF catalyzes a sequential Bi Bi mechanism where both substrates are added to the enzyme prior to product release consistent with a single displacement chemical mechanism.

Structure of the nucleotide radical formed during reaction of CDP/TTP with the E441Q-alpha2beta2 of E. coli ribonucleotide reductase.

The Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleotides and requires a diferric-tyrosyl radical cofactor for catalysis. RNR is composed of a 1:1 complex of two homodimeric subunits: alpha and beta. Incubation of the E441Q-alpha mutant RNR with substrate CDP and allosteric effector TTP results in loss of the tyrosyl radical and formation of two new radicals on the 200 ms to min time scale. The first radical was previously established by stopped flow UV/vis spectroscopy and pulsed high field EPR spectroscopy to be a disulfide radical anion. The second radical was proposed to be a 4'-radical of a 3'-keto-2'-deoxycytidine 5'-diphosphate. To identify the structure of the nucleotide radical [1'-(2)H], [2'-(2)H], [4'-(2)H], [5'-(2)H], [U-(13)C, (15)N], [U-(15)N], and [5,6 -(2)H] CDP and [beta-(2)H] cysteine-alpha were synthesized and incubated with E441Q-alpha2beta2 and TTP. The nucleotide radical was examined by 9 GHz and 140 GHz pulsed EPR spectroscopy and 35 GHz ENDOR spectroscopy. Substitution of (2)H at C4' and C1' altered the observed hyperfine interactions of the nucleotide radical and established that the observed structure was not that predicted. DFT calculations (B3LYP/IGLO-III/B3LYP/TZVP) were carried out in an effort to recapitulate the spectroscopic observations and lead to a new structure consistent with all of the experimental data. The results indicate, unexpectedly, that the radical is a semidione nucleotide radical of cytidine 5'-diphosphate. The relationship of this radical to the disulfide radical anion is discussed.

New efficient synthesis of nucleoside diphosphate glycopyranoses.

A new access for the synthesis of nucleoside diphosphate glycopyranoses has been developed based on the cycloSal-concept. Using this approach, excellent chemical yields were obtained within short reaction times. In comparison to other methods the cycloSal-concept allows a fast and efficient preparation of anomerically defined nucleoside diphosphate glycopyranoses.

Functional and structural characterization of DR_0079 from Deinococcus radiodurans, a novel Nudix hydrolase with a preference for cytosine (deoxy)ribonucleoside 5'-Di- and triphosphates.

The genome of the extremely radiation resistant bacterium Deinococcus radiodurans encodes 21 Nudix hydrolases, of which only two have been characterized in detail. Here we report the activity and crystal structure for DR_0079, the first Nudix hydrolase observed to have a marked preference for cytosine ribonucleoside 5'-diphosphate (CDP) and cytosine ribonucleoside 5'-triphosphate (CTP). After CDP and CTP, the next most preferred substrates for DR_0079, with a relative activity of <50%, were the corresponding deoxyribose nucleotides, dCDP and dCTP. Hydrolase activity at the site of the phosphodiester bond was corroborated using (31)P NMR spectroscopy to follow the phosphorus resonances for three substrates, CDP, IDP, and CTP, and their hydrolysis products, CMP + P(i), IMP + P(i), and CMP + PP(i), respectively. Nucleophilic substitution at the beta-phosphorus of CDP and CTP was established, using (31)P NMR spectroscopy, by the appearance of an upfield-shifted P(i) resonance and line-broadened PP(i) resonance, respectively, when the hydrolysis was performed in 40% H(2)(18)O-enriched water. The optimal activity for CDP was at pH 9.0-9.5 with the reaction requiring divalent metal cation (Mg(2+) > Mn(2+) > Co(2+)). The biochemical data are discussed with reference to the crystal structure for DR_0079 that was determined in the metal-free form at 1.9 A resolution. The protein contains nine beta-strands, three alpha-helices, and two 3(10)-helices organized into three subdomains: an N-terminal beta-sheet, a central Nudix core, and a C-terminal helix-turn-helix motif. As observed for all known structures of Nudix hydrolases, the alpha-helix of the "Nudix box" is one of two helices that sandwich a "four-strand" mixed beta-sheet. To identify residues potentially involved in metal and substrate binding, NMR chemical shift mapping experiments were performed on (15)N-labeled DR_0079 with the paramagnetic divalent cation Co(2+) and the nonhydrolyzable substrate thymidine 5'-O-(alpha,beta-methylenediphosphate) and the results mapped onto the crystal structure.