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The final step in the de novo synthesis of cytidine 5'-triphosphate (CTP) is catalyzed by CTP synthase (CTPS), which can form cytoophidia in all three domains of life. Recently, we have discovered that CTPS binds to ribonucleotides (NTPs) to form filaments, and have successfully resolved the structures of Drosophila melanogaster CTPS bound with NTPs. Previous biochemical studies have shown that CTPS can bind to deoxyribonucleotides (dNTPs) to produce 2'-deoxycytidine-5'-triphosphate (dCTP). However, the structural basis of CTPS binding to dNTPs is still unclear. In this study, we find that Drosophila CTPS can also form filaments with dNTPs. Using cryo-electron microscopy, we are able to resolve the structure of Drosophila melanogaster CTPS bound to dNTPs with a resolution of up to 2.7 Å. By combining these structural findings with biochemical analysis, we compare the binding and reaction characteristics of NTPs and dNTPs with CTPS. Our results indicate that the same enzyme can act bifunctionally as CTP/dCTP synthase in vitro, and provide a structural basis for these activities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

There are three key components at the core of the mpox virus (MPXV) DNA polymerase holoenzyme: DNA polymerase F8, processivity factors A22, and the Uracil-DNA glycosylase E4. The holoenzyme is recognized as a vital antiviral target because MPXV replicates in the cytoplasm of host cells. Nucleotide analogs such as cidofovir and cytarabine (Ara-C) have shown potential in curbing MPXV replication and they also display promise against other poxviruses. However, the mechanism behind their inhibitory effects remains unclear. Here, we present the cryo-EM structure of the DNA polymerase holoenzyme F8/A22/E4 bound with its competitive inhibitor Ara-C-derived cytarabine triphosphate (Ara-CTP) at an overall resolution of 3.0 Å and reveal its inhibition mechanism. Ara-CTP functions as a direct chain terminator in proximity to the deoxycytidine triphosphate (dCTP)-binding site. The extra hydrogen bond formed with Asn665 makes it more potent in binding than dCTP. Asn665 is conserved among eukaryotic B-family polymerases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 outbreak that is affecting the entire planet. As the pandemic is still spreading worldwide, with multiple mutations of the virus, it is of interest and of help to employ computational methods for identifying potential inhibitors of the enzymes responsible for viral replication. Attractive antiviral nucleotide analogue RNA-dependent RNA polymerase (RdRp) chain terminator inhibitors are investigated with this purpose. This study, based on molecular dynamics (MD) simulations, addresses the important aspects of the incorporation of an endogenously synthesized nucleoside triphosphate, ddhCTP, in comparison with the natural nucleobase cytidine triphosphate (CTP) in RdRp. The ddhCTP species is the product of the viperin antiviral protein as part of the innate immune response. The absence of the ribose 3'-OH in ddhCTP could have important implications in its inhibitory mechanism of RdRp. We built an in silico model of the RNA strand embedded in RdRp using experimental methods, starting from the cryo-electron microscopy structure and exploiting the information obtained by spectrometry on the RNA sequence. We determined that the model was stable during the MD simulation time. The obtained results provide deeper insights into the incorporation of nucleoside triphosphates, whose molecular mechanism by the RdRp active site still remains elusive.

Cytidine-5'-triphosphate (CTP) synthase (CTPS) is the class I glutamine-dependent amidotransferase (GAT) that catalyzes the last step in the de novo biosynthesis of CTP. Glutamine hydrolysis is catalyzed in the GAT domain and the liberated ammonia is transferred via an intramolecular tunnel to the synthase domain where the ATP-dependent amination of UTP occurs to form CTP. CTPS is unique among the glutamine-dependent amidotransferases, requiring an allosteric effector (GTP) to activate the GAT domain for efficient glutamine hydrolysis. Recently, the first cryo-electron microscopy structure of Drosophila CTPS was solved with bound ATP, UTP, and, notably, GTP, as well as the covalent adduct with 6-diazo-5-oxo-l-norleucine. This structural information, along with the numerous site-directed mutagenesis, kinetics, and structural studies conducted over the past 50 years, provide more detailed insights into the elaborate conformational changes that accompany GTP binding at the GAT domain and their contribution to catalysis. Interactions between GTP and the L2 loop, the L4 loop from an adjacent protomer, the L11 lid, and the L13 loop (or unique flexible "wing" region), induce conformational changes that promote the hydrolysis of glutamine at the GAT domain; however, direct experimental evidence on the specific mechanism by which these conformational changes facilitate catalysis at the GAT domain is still lacking. Significantly, the conformational changes induced by GTP binding also affect the assembly and maintenance of the NH 3 tunnel. Hence, in addition to promoting glutamine hydrolysis, the allosteric effector plays an important role in coordinating the reactions catalyzed by the GAT and synthase domains of CTPS.

The ParABS system is supposed to be responsible for plasmid partitioning and chromosome segregation in bacteria. ParABS ensures a high degree of fidelity in inheritance by dividing the genetic material equally between daughter cells during cell division. However, the molecular mechanisms underlying the assembly of the partition complex, representing the core of the ParABS system, are still far from being understood. Here we demonstrate that the partition complex is formed via liquid-liquid phase separation. Assembly of the partition complex is initiated by the formation of oligomeric ParB species, which in turn are regulated by CTP-binding. Phase diagrams and in vivo analysis show how the partition complex can further be spatially regulated by parS. By investigating the phylogenetic variation in phase separation and its regulation by CTP, we find a high degree of evolutionary conservation among distantly related prokaryotes. These results advance the understanding of partition complex formation and regulation in general, by confirming and extending recently proposed models., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Flavins play a central role in metabolism as molecules that catalyze a wide range of redox reactions in living organisms. Several variations in flavin biosynthesis exist among the domains of life, and their analysis has revealed many new structural and mechanistic insights till date. The cytidine triphosphate (CTP)-dependent riboflavin kinase in archaea is one such example. Unlike most kinases that use adenosine triphosphate, archaeal riboflavin kinases utilize CTP to phosphorylate riboflavin and produce flavin mononucleotide. In this study, we present the characterization of a new mesophilic archaeal CTP-utilizing riboflavin kinase homologue from Methanococcus maripaludis (MmpRibK), which is linked closely in sequence to the previously characterized thermophilic Methanocaldococcus jannaschii homologue. We reconstitute the activity of MmpRibK, determine its kinetic parameters and molecular factors that contribute to its unique properties, and finally establish the residues that improve its thermostability using computation and a series of experiments. Our work advances the molecular understanding of flavin biosynthesis in archaea by the characterization of the first mesophilic CTP-dependent riboflavin kinase. Finally, it validates the role of salt bridges and rigidifying amino acid residues in imparting thermostability to this unique structural fold that characterizes archaeal riboflavin kinase enzymes, with implications in enzyme engineering and biotechnological applications., (© 2021 Wiley-VCH GmbH.)

Despite the development of non-radioactive DNA/RNA labelling methods, radiolabelled nucleic acids are commonly used in studies focused on the determination of RNA fate. Nucleic acid fragments with radioactive nucleotide analoguesincorporated into the body or at the 5' or 3' terminus of the molecule can serve as probes in hybridization-based analyses of in vivo degradation and processing of transcripts. Radiolabelled oligoribonucleotides are utilized as substrates in biochemical assays of various RNA metabolic enzymes, such as exo- and endoribonucleases, nucleotidyltransferases or helicases. In some applications, the placement of the label is not a concern, while in other cases it is required that the radioactive mark is located at the 5'- or 3'-end of the molecule. An unsurpassed method for 5'-end RNA labelling employs T4 polynucleotide kinase (PNK) and [γ- 32 P]ATP. In the case of 3'-end labelling, several different possibilities exist. However, they require the use of costly radionucleotide analogues. Previously, we characterized an untypical nucleotidyltransferase named CutA, which preferentially incorporates cytidines at the 3'-end of RNA substrates. Here, we demonstrate that this unusual feature can be used for the development of a novel, efficient, reproducible and economical method of RNA 3'-end labelling by CutA-mediated cytidine tailing. The labelling efficiency is comparable to that achieved with the most common method applied to date, i.e . [5'- 32 P]pCp ligation to the RNA 3'-terminus catalysed by T4 RNA ligase I. We show the utility of RNA substrates labelled using our new method in exemplary biochemical assays assessing directionality of two well-known eukaryotic exoribonucleases, namely Dis3 and Xrn1.

The naturally occurring nucleotide 3'-deoxy-3',4'-didehydro-cytidine-5'-triphosphate ( ddhCTP ) was recently found to exert potent and broad-spectrum antiviral activity. However, nucleoside 5'-triphosphates in general are not cell-permeable, which precludes the direct use of ddhCTP as a therapeutic. To harness the therapeutic potential of this endogenous antiviral nucleotide, we synthesized phosphoramidate prodrug HLB-0532247 ( 1 ) and found it to result in dramatically elevated levels of ddhCTP in cells. We compared 1 and 3'-deoxy-3',4'-didehydro-cytidine ( ddhC ) and found that 1 more effectively reduces titers of Zika and West Nile viruses in cell culture with minimal nonspecific toxicity to host cells. We conclude that 1 is a promising antiviral agent based on a novel strategy of facilitating elevated levels of the endogenous ddhCTP antiviral nucleotide.

NS5B is the RNA-dependent RNA polymerase that catalyzes the replication of the hepatitis C virus genome. It is a major target for antiviral drugs including nucleoside analogs, such as the prodrugs mericitabine and sofosbuvir, which get metabolized to 2'-fluoro-2'C-methylcytidine-5'-triphosphate and 2'-fluoro-2'C-methyluridine-5'-triphosphate, respectively. These analogs act as chain terminators after they are incorporated during RNA synthesis. Recently, it has been shown that NS5B can efficiently remove chain terminators by a nucleotide-mediated excision reaction that rescues RNA synthesis. In this study, we use transient-state kinetics to understand the efficiency of inhibition for five nucleoside analogs. We show that CTP analogs are readily incorporated into a growing primer by NS5B but are also efficiently excised. In contrast, although UMP analogs are more slowly incorporated, the excision of UMP is slow and inefficient, and modifications to the 2'-carbon of the UTP ribose ring further decreased rates of excision to an undetectable level. Taken together, these data suggest that the clinical effectiveness of sofosbuvir is largely a function of being intractable to nucleotide-mediated excision compared with similar nucleoside analogs., Competing Interests: Conflict of interest—K. A. J. is president of KinTek Corporation, which provided the RQF-3 rapid quench-flow instrument and KinTek Explorer software used in this study., (© 2020 Villalba et al.)

3'-Deoxynucleotides are an important class of drugs because they interfere with the metabolism of nucleotides, and their incorporation into DNA or RNA terminates cell division and viral replication. These compounds are generally produced by multi-step chemical synthesis, and an enzyme with the ability to catalyse the removal of the 3'-deoxy group from different nucleotides has yet to be described. Here, using a combination of HPLC, HRMS and NMR spectroscopy, we demonstrate that a thermostable fungal radical S-adenosylmethionine (SAM) enzyme, with similarity to the vertebrate antiviral enzyme viperin (RSAD2), can catalyse the transformation of CTP, UTP and 5-bromo-UTP to their 3'-deoxy-3',4'-didehydro (ddh) analogues. We show that, unlike the fungal enzyme, human viperin only catalyses the transformation of CTP to ddhCTP. Using electron paramagnetic resonance spectroscopy and molecular docking and dynamics simulations in combination with mutagenesis studies, we provide insight into the origin of the unprecedented substrate promiscuity of the enzyme and the mechanism of dehydration of a nucleotide. Our findings highlight the evolution of substrate specificity in a member of the radical-SAM enzymes. We predict that our work will help in using a new class of the radical-SAM enzymes for the biocatalytic synthesis of 3'-deoxy nucleotide/nucleoside analogues., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Viperin is a radical S -adenosylmethionine (SAM) enzyme that inhibits viral replication by converting cytidine triphosphate (CTP) into 3'-deoxy-3',4'-didehydro-CTP and by additional undefined mechanisms operating through its N- and C-terminal domains. Here, we describe crystal structures of viperin bound to a SAM analogue and CTP or uridine triphosphate (UTP) and report kinetic parameters for viperin-catalyzed reactions with CTP or UTP as substrates. Viperin orients the C4' hydrogen atom of CTP and UTP similarly for abstraction by a 5'-deoxyadenosyl radical, but the uracil moiety introduces unfavorable interactions that prevent tight binding of UTP. Consistently, k cat is similar for CTP and UTP whereas the K m for UTP is much greater. The structures also show that nucleotide binding results in ordering of the C-terminal tail and reveal that this region contains a P-loop that binds the γ-phosphate of the bound nucleotide. Collectively, the results explain the selectivity for CTP and reveal a structural role for the C-terminal tail in binding CTP and UTP.

Three sets of 7-deazaadenine and cytosine nucleosides and nucleoside triphosphates bearing either unsubstituted ferrocene, octamethylferrocene and ferrocenecarboxamide linked through an alkyne tether to position 7 or 5, respectively, were designed and synthesized. The modified dN FcX TPs were good substrates for KOD XL DNA polymerase in primer extension and were used for enzymatic synthesis of redox-labelled DNA probes. Square-wave voltammetry showed that the octamethylferrocene oxidation potential was shifted to lower values, whilst the ferrocenecarboxamide was shifted to higher potentials, as compared to ferrocene. Tailed PEX products containing different ratios of Fc-labelled A (dA Fc ) and FcPa-labelled C (dC FcPa ) were synthesized and hybridized with capture oligonucleotides immobilized on gold electrodes to study the electrochemistry of the redox-labelled DNA. Clearly distinguishable, fully orthogonal and ratiometric peaks were observed for the dA Fc and dC FcPa bases in DNA, demonstrating their potential for use in redox coding of nucleobases and for the direct electrochemical measurement of the relative ratio of nucleobases in an unknown sequence of DNA., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

ParABS systems facilitate chromosome segregation and plasmid partitioning in bacteria and archaea. ParB protein binds centromeric parS DNA sequences and spreads to flanking DNA. We show that ParB is an enzyme that hydrolyzes cytidine triphosphate (CTP) to cytidine diphosphate (CDP). parS DNA stimulates cooperative CTP binding by ParB and CTP hydrolysis. A nucleotide cocrystal structure elucidates the catalytic center of the dimerization-dependent ParB CTPase. Single-molecule imaging and biochemical assays recapitulate features of ParB spreading from parS in the presence but not absence of CTP. These findings suggest that centromeres assemble by self-loading of ParB DNA sliding clamps at parS ParB CTPase is not related to known nucleotide hydrolases and might be a promising target for developing new classes of antibiotics., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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 Mg 2+ 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., (open access.)

The leading cause of bacterial gastroenteritis, Campylobacter jejuni, is a Gram-negative pathogen that contains a unique O-methyl phosphoramidate (MeOPN) on its capsular polysaccharide. Previously, MeOPN has been linked to the evasion of host immune responses and serum resistance. Despite the involvement of MeOPN in pathogenicity, the complete biosynthesis of this modification is unknown; however, the first four enzymatic steps have been elucidated. The second enzyme in this pathway, Cj1416, is a CTP/phosphoglutamine cytididylyltransferase that catalyzes the displacement of pyrophosphate from MgCTP by l-glutamine phosphate to form CDP-l-glutamine. Initially, Cj1416 was predicted to use phosphoramidate to form cytidine diphosphoramidate, but no activity was detected with MgATP as a substrate. However, in the presence of MnCTP, Cj1416 can directly catalyze the formation of cytidine diphosphoramidate from phosphoramidate and MnCTP. Here we characterize the manganese-induced promiscuity of Cj1416. In the presence of Mn 2+ , Cj1416 catalyzes the formation of 12 different reaction products using l-glutamine phosphate, phosphoramidate, methyl phosphate, methyl phosphonate, phosphate, arsenate, ethanolamine phosphate, glycerol-1-phosphate, glycerol-2-phosphate, serinol phosphate, l-serine phosphate, or 3-phospho-d-glycerate as the nucleophile to displace pyrophosphate from CTP.

Discrimination of single nucleotides by a nanopore remains a challenge because of the minor difference among the four types of single nucleotides. Here, the blockade currents induced by the translocation of single nucleotides through a 1.8 nm diameter silicon nitride nanopore have been measured. It is found that the single nucleotides are driven through the nanopore by an electroosmotic flow instead of electrophoretic force when a bias voltage is applied. The blockade currents for the four types of single nucleotides are unique and differentiable, following the order of the nucleotide volume. Also, the dwell time for each single nucleotide can last for several hundred microseconds with the advantage of the electroosmotic flow, which is helpful for single nucleotide identification. The dwell-time distributions are found to obey the first-passage time distribution from the 1D Fokker-Planck equation, from which the velocity and diffusion constant of each nucleotide can be deduced. Interestingly, the larger nucleotide is found to translocate faster than the smaller one inside the nanopore because the larger nucleotide has a larger surface area, which may produce larger drag force induced by the electroosmotic flow, which is validated by molecular dynamics simulations.

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)(H x+y )(PA) (where H x+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 -PO 3 x- group deprotonation, which unambiguously confirms the dipole-dipole type of interaction. In the pH range of total deprotonation of NH x + 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)H x+y (PA) type coordination compounds and Cu(NucP)⋯(PA)(H x ) 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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Viral infections continue to represent major challenges to public health, and an enhanced mechanistic understanding of the processes that contribute to viral life cycles is necessary for the development of new therapeutic strategies 1 . Viperin, a member of the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes, is an interferon-inducible protein implicated in the inhibition of replication of a broad range of RNA and DNA viruses, including dengue virus, West Nile virus, hepatitis C virus, influenza A virus, rabies virus 2 and HIV 3,4 . Viperin has been suggested to elicit these broad antiviral activities through interactions with a large number of functionally unrelated host and viral proteins 3,4 . Here we demonstrate that viperin catalyses the conversion of cytidine triphosphate (CTP) to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), a previously undescribed biologically relevant molecule, via a SAM-dependent radical mechanism. We show that mammalian cells expressing viperin and macrophages stimulated with IFNα produce substantial quantities of ddhCTP. We also establish that ddhCTP acts as a chain terminator for the RNA-dependent RNA polymerases from multiple members of the Flavivirus genus, and show that ddhCTP directly inhibits replication of Zika virus in vivo. These findings suggest a partially unifying mechanism for the broad antiviral effects of viperin that is based on the intrinsic enzymatic properties of the protein and involves the generation of a naturally occurring replication-chain terminator encoded by mammalian genomes.

Noncanonical RNA nucleotidyltransferases (NTases), including poly(A), poly(U) polymerases (PAPs/PUPs), and C/U-adding enzymes, modify 3'-ends of different transcripts affecting their functionality and stability. They contain PAP/OAS1 substrate-binding domain (SBD) with inserted NTase domain. Aspergillus nidulans CutA (AnCutA), synthesizes C/U-rich 3'-terminal extensions in vivo. Here, using high-throughput sequencing of the 3'-RACE products for tails generated by CutA proteins in vitro in the presence of all four NTPs, we show that even upon physiological ATP excess synthesized tails indeed contain an unprecedented number of cytidines interrupted by uridines and stretches of adenosines, and that the majority end with two cytidines. Strikingly, processivity assays documented that in the presence of CTP as a sole nucleotide, the enzyme terminates after adding two cytidines only. Comparison of our CutA 3D model to selected noncanonical NTases of known structures revealed substantial differences in the nucleotide recognition motif (NRM) within PAP/OAS1 SBD. We demonstrate that CutA specificity toward CTP can be partially changed to PAP or PUP by rational mutagenesis within NRM and, analogously, Cid1 PUP can be converted into a C/U-adding enzyme. Collectively, we suggest that a short cluster of amino acids within NRM is a determinant of NTases' substrate preference, which may allow us to predict their specificity., (© 2017 Kobyłecki et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The divergence of archaea, bacteria and eukaryotes was a fundamental step in evolution. One marker of this event is a major difference in membrane lipid chemistry between these kingdoms. Whereas the membranes of bacteria and eukaryotes primarily consist of straight fatty acids ester-bonded to glycerol-3-phosphate, archaeal phospholipids consist of isoprenoid chains ether-bonded to glycerol-1-phosphate. Notably, the mechanisms underlying the biosynthesis of these lipids remain elusive. Here, we report the structure of the CDP-archaeol synthase (CarS) of Aeropyrum pernix (ApCarS) in the CTP- and Mg 2+ -bound state at a resolution of 2.4 Å. The enzyme comprises a transmembrane domain with five helices and cytoplasmic loops that together form a large charged cavity providing a binding site for CTP. Identification of the binding location of CTP and Mg 2+ enabled modeling of the specific lipophilic substrate-binding site, which was supported by site-directed mutagenesis, substrate-binding affinity analyses, and enzyme assays. We propose that archaeol binds within two hydrophobic membrane-embedded grooves formed by the flexible transmembrane helix 5 (TM5), together with TM1 and TM4. Collectively, structural comparisons and analyses, combined with functional studies, not only elucidated the mechanism governing the biosynthesis of phospholipids with ether-bonded isoprenoid chains by CTP transferase, but also provided insights into the evolution of this enzyme superfamily from archaea to bacteria and eukaryotes.

P2X receptors are trimeric ATP-gated cation channels involved in diverse physiological processes, ranging from muscle contraction to nociception. Despite the recent structure determination of the ATP-bound P2X receptors, the molecular mechanism of the nucleotide base specificity has remained elusive. Here, we present the crystal structure of zebrafish P2X4 in complex with a weak affinity agonist, CTP, together with structure-based electrophysiological and spectroscopic analyses. The CTP-bound structure revealed a hydrogen bond, between the cytosine base and the side chain of the basic residue in the agonist binding site, which mediates the weak but significant affinity for CTP. The cytosine base is further recognized by two main chain atoms, as in the ATP-bound structure, but their bond lengths seem to be extended in the CTP-bound structure, also possibly contributing to the weaker affinity for CTP over ATP. This work provides the structural insights for the nucleotide base specificity of P2X receptors.

Glycerophosphoinositol (GPI) is a compatible solute present in a few hyperthermophiles. Interestingly, different GPI stereoisomers accumulate in Bacteria and Archaea, and the basis for this domain-dependent specificity was investigated herein. The archaeon Archaeoglobus fulgidus and the bacterium Aquifex aeolicus were used as model organisms. The synthesis of GPI involves glycerol phosphate cytidylyltransferase (GCT), which catalyzes the production of CDP-glycerol from CTP and glycerol phosphate, and di-myo-inositol phosphate-phosphate synthase (DIPPS), catalyzing the formation of phosphorylated GPI from CDP-glycerol and l-myo-inositol 1-phosphate. DIPPS of A. fulgidus recognized the two CDP-glycerol stereoisomers similarly. This feature and the ability of 31 P nuclear magnetic resonance (NMR) to distinguish the GPI diastereomers provided a means to study the stereospecificity of GCTs. The AF1418 gene and genes aq_185 and aq_1368 are annotated as putative GCT genes in the genomes of A. fulgidus and Aq. aeolicus, respectively. The functions of these genes were determined by assaying the activity of the respective recombinant proteins: AQ1368 and AQ185 are GCTs, while AF1418 has flavin adenine dinucleotide (FAD) synthetase activity. AQ185 is absolutely specific for sn-glycerol 3-phosphate, while AQ1368 recognizes the two enantiomers but has a 2:1 preference for sn-glycerol 3-phosphate. In contrast, the partially purified A. fulgidus GCT uses sn-glycerol 1-phosphate preferentially (4:1). Significantly, the predominant GPI stereoforms found in the bacterium and the archaeon reflect the distinct stereospecificities of the respective GCTs: i.e., A. fulgidus accumulates predominantly sn-glycero-1-phospho-3-l-myo-inositol, while Aq. aeolicus accumulates sn-glycero-3-phospho-3-l-myo-inositol., Importance: Compatible solutes of hyperthermophiles show high efficacy in thermal protection of proteins in comparison with solutes typical of mesophiles; therefore, they are potentially useful in several biotechnological applications. Glycerophosphoinositol (GPI) is synthesized from CDP-glycerol and l-myo-inositol 1-phosphate in a few hyperthermophiles. In this study, the molecular configuration of the GPI stereoisomers accumulated by members of the Bacteria and Archaea was established. The stereospecificity of glycerol phosphate cytidylyltransferase (GCT), the enzyme catalyzing the synthesis of CDP-glycerol, is crucial to the stereochemistry of GPI. However, the stereospecific properties of GCTs have not been investigated thus far. We devised a method to characterize GCT stereospecificity which does not require sn-glycerol 1-phosphate, a commercially unavailable substrate. This led us to understand the biochemical basis for the distinct GPI stereoisomer composition observed in archaea and bacteria., (Copyright © 2016 American Society for Microbiology.)

CTP synthase (CTPS) catalyzes the conversion of UTP to CTP and is a target for the development of antiviral, anticancer, antiprotozoal, and immunosuppressive agents. Exposure of cell lines to the antineoplastic cytidine analogue gemcitabine causes depletion of intracellular CTP levels, but the direct inhibition of CTPS by its metabolite gemcitabine-5'-triphosphate (dF-dCTP) has not been demonstrated. We show that dF-dCTP is a potent competitive inhibitor of Escherichia coli CTPS with respect to UTP [K i =(3.0±0.1) μm], and that its binding affinity exceeds that of CTP ≈75-fold. Site-directed mutagenesis studies indicated that Glu149 is an important binding determinant for both CTP and dF-dCTP. Comparison of the binding affinities of the 5'-triphosphates of 2'-fluoro-2'-deoxycytidine and 2'-fluoro-2'-deoxyarabinocytidine revealed that the 2'-F-arabino group contributes markedly to the strong binding of dF-dCTP. Geminal 2'-F substitution on UTP (dF-dUTP) did not result in an increase in binding affinity with CTPS. Remarkably, CTPS catalyzed the conversion of dF-dUTP into dF-dCTP, thus suggesting that dF-dCTP might be regenerated in vivo from its catabolite dF-dUTP., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Polyhedra represent an ancient system used by a number of insect viruses to protect virions during long periods of environmental exposure. We present high resolution crystal structures of polyhedra for seven previously uncharacterised types of cypoviruses, four using ab initio selenomethionine phasing (two of these required over 100 selenomethionine crystals each). Approximately 80% of residues are structurally equivalent between all polyhedrins (pairwise rmsd ⩽ 1.5 Å), whilst pairwise sequence identities, based on structural alignment, are as little as 12%. These structures illustrate the effect of 400 million years of evolution on a system where the crystal lattice is the functionally conserved feature in the face of massive sequence variability. The conservation of crystal contacts is maintained across most of the molecular surface, except for a dispensable virus recognition domain. By spreading the contacts over so much of the protein surface the lattice remains robust in the face of many individual changes. Overall these unusual structural constraints seem to have skewed the molecule's evolution so that surface residues are almost as conserved as the internal residues., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Human DNA polymerase β (polβ) has been suggested to play a role in cisplatin resistance, especially in polβ-overexpressing cancer cells. Polβ has been shown to accurately albeit slowly bypass the cisplatin-1,2-d(GpG) (Pt-GG) intramolecular cross-link in vitro. Currently, the structural basis for the inefficient Pt-GG bypass mechanism of polβ is unknown. To gain structural insights into the mechanism, we determined two ternary structures of polβ incorporating dCTP opposite the templating Pt-GG lesion in the presence of the active site Mg(2+) or Mn(2+). The Mg(2+)-bound structure shows that the bulky Pt-GG adduct is accommodated in the polβ active site without any steric hindrance. In addition, both guanines of the Pt-GG lesion form Watson-Crick base pairing with the primer terminus dC and the incoming dCTP, providing the structural basis for the accurate bypass of the Pt-GG adduct by polβ. The Mn(2+)-bound structure shows that polβ adopts a catalytically suboptimal semiclosed conformation during the insertion of dCTP opposite the templating Pt-GG, explaining the inefficient replication across the Pt-GG lesion by polβ. Overall, our studies provide the first structural insights into the mechanism of the potential polβ-mediated cisplatin resistance., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The pathogen Helicobacter pylori requires two nickel-containing enzymes, urease and [NiFe]-hydrogenase, for efficient colonization of the human gastric mucosa. These enzymes possess complex metallocenters that are assembled by teams of proteins in multistep pathways. One essential accessory protein is the GTPase HypB, which is required for Ni(II) delivery to [NiFe]-hydrogenase and participates in urease maturation. Ni(II) or Zn(II) binding to a site embedded in the GTPase domain of HypB modulates the enzymatic activity, suggesting a mechanism of regulation. In this study, biochemical and structural analyses of H. pylori HypB (HpHypB) revealed an intricate link between nucleotide and metal binding. HpHypB nickel coordination, stoichiometry, and affinity were modulated by GTP and GDP, an effect not observed for zinc, and biochemical evidence suggests that His-107 coordination to nickel toggles on and off in a nucleotide-dependent manner. These results are consistent with the crystal structure of HpHypB loaded with Ni(II), GDP, and Pi, which reveals a nickel site distinct from that of zinc-loaded Methanocaldococcus jannaschii HypB as well as subtle changes to the protein structure. Furthermore, Cys-142, a metal ligand from the Switch II GTPase motif, was identified as a key component of the signal transduction between metal binding and the enzymatic activity. Finally, potassium accelerated the enzymatic activity of HpHypB but had no effect on the other biochemical properties of the protein. Altogether, this molecular level information about HpHypB provides insight into its cellular function and illuminates a possible mechanism of metal ion discrimination.

RNAs are an important class of cellular regulatory elements, and they are well characterized by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy in their folded or bound states. However, the apo or unfolded states are more difficult to characterize by either method. Particularly, effective NMR spectroscopy studies of RNAs in the past were hampered by chemical shift overlap of resonances and associated rapid signal loss due to line broadening for RNAs larger than the median size found in the PDB (~25 nt); most functional riboswitches are bigger than this median size. Incorporation of selective site-specific (13)C/(15)N-labeled nucleotides into RNAs promises to overcome this NMR size limitation. Unlike previous isotopic enrichment methods such as phosphoramidite, de novo, uniform-labeling, and selective-biomass approaches, this newer chemical-enzymatic selective method presents a number of advantages for producing labeled nucleotides over these other methods. For example, total chemical synthesis of nucleotides, followed by solid-phase synthesis of RNA using phosphoramidite chemistry, while versatile in incorporating isotope labels into RNA at any desired position, faces problems of low yields (<10%) that drop precipitously for oligonucleotides larger than 50 nt. The alternative method of de novo pyrimidine biosynthesis of NTPs is also a robust technique, with modest yields of up to 45%, but it comes at the cost of using 16 enzymes, expensive substrates, and difficulty in making some needed labeling patterns such as selective labeling of the ribose C1' and C5' and the pyrimidine nucleobase C2, C4, C5, or C6. Biomass-produced, uniformly or selectively labeled NTPs offer a third method, but suffer from low overall yield per labeled input metabolite and isotopic scrambling with only modest suppression of (13)C-(13)C couplings. In contrast to these four methods, our current chemo-enzymatic approach overcomes most of these shortcomings and allows for the synthesis of gram quantities of nucleotides with >80% yields while using a limited number of enzymes, six at most. The unavailability of selectively labeled ribose and base precursors had prevented the effective use of this versatile method until now. Recently, we combined an improved organic synthetic approach that selectively places (13)C/(15)N labels in the pyrimidine nucleobase (either (15)N1, (15)N3, (13)C2, (13)C4, (13)C5, or (13)C6 or any combination) with a very efficient enzymatic method to couple ribose with uracil to produce previously unattainable labeling patterns (Alvarado et al., 2014). Herein we provide detailed steps of both our chemo-enzymatic synthesis of custom nucleotides and their incorporation into RNAs with sizes ranging from 29 to 155 nt and showcase the dramatic improvement in spectral quality of reduced crowding and narrow linewidths. Applications of this selective labeling technology should prove valuable in overcoming two major obstacles, chemical shift overlap of resonances and associated rapid signal loss due to line broadening, that have impeded studying the structure and dynamics of large RNAs such as full-length riboswitches larger than the ~25 nt median size of RNA NMR structures found in the PDB.

The Hfq protein forms a doughnut-shaped homohexamer that possesses RNA-binding activity. There are two distinct RNA-binding surfaces located on the proximal and the distal sides of the hexamer. The proximal side is involved in the binding of mRNA and small noncoding RNAs (sRNAs), while the distal side has an affinity for A-rich RNA sequences. In this work, the ability of various ribonucleotides to form complexes with Hfq from Pseudomonas aeruginosa has been tested using X-ray crystallography. ATP and ADPNP have been located in the distal R-site, which is a site for poly(A) RNA binding. UTP has been found in the so-called lateral RNA-binding site at the proximal surface. CTP has been found in both the distal R-site and the proximal U-binding site. GTP did not form a complex with Hfq under the conditions tested. The results have demonstrated the power of the crystallographic method for locating ribonucleotides and predicting single-stranded RNA-binding sites on the protein surface.

The enzyme CTP:phosphocholine cytidylyltransferase (CCT) is essential in the lipid biosynthesis of Plasmodia (Haemosporida), presenting a promising antimalarial target. Here, we identified two independent gene duplication events of CCT within Apicomplexa and characterized a truncated construct of Plasmodium falciparum CCT that forms a dimer resembling the molecular architecture of CCT enzymes from other sources. Based on biophysical and enzyme kinetics methods, our data show that the CDP-choline product of the CCT enzymatic reaction binds to the enzyme considerably stronger than either substrate (CTP or choline phosphate). Interestingly, in the presence of Mg²⁺ , considered to be a cofactor of the enzyme, the binding of the CTP substrate is attenuated by a factor of 5. The weaker binding of CTP:Mg²⁺ , similarly to the related enzyme family of aminoacyl tRNA synthetases, suggests that, with lack of Mg²⁺ , positively charged side chain(s) of CCT may contribute to CTP accommodation. Thermodynamic investigations by isothermal titration calorimetry and fluorescent spectroscopy studies indicate that accommodation of the choline phosphate moiety in the CCT active site is different when it appears on its own as one of the substrates or when it is linked to the CDP-choline product. A tryptophan residue within the active site is identified as a useful internal fluorescence sensor of enzyme-ligand binding. Results indicate that the catalytic mechanism of Plasmodium falciparum CCT may involve conformational changes affecting the choline subsite of the enzyme., (© 2013 FEBS.)

Reactive oxygen species are produced as side products of oxygen utilization and can lead to the oxidation of nucleic acids and their precursor nucleotides. Among the various oxidized bases, 8-oxo-7,8-dihydroguanine seems to be the most critical during the transfer of genetic information because it can pair with both cytosine and adenine. During the de novo synthesis of guanine nucleotides, GMP is formed first, and it is converted to GDP by guanylate kinase. This enzyme hardly acts on an oxidized form of GMP (8-oxo-GMP) formed by the oxidation of GMP or by the cleavage of 8-oxo-GDP and 8-oxo-GTP by MutT protein. Although the formation of 8-oxo-GDP from 8-oxo-GMP is thus prevented, 8-oxo-GDP itself may be produced by the oxidation of GDP by reactive oxygen species. The 8-oxo-GDP thus formed can be converted to 8-oxo-GTP because nucleoside-diphosphate kinase and adenylate kinase, both of which catalyze the conversion of GDP to GTP, do not discriminate 8-oxo-GDP from normal GDP. The 8-oxo-GTP produced in this way and by the oxidation of GTP can be used for RNA synthesis. This misincorporation is prevented by MutT protein, which has the potential to cleave 8-oxo-GTP as well as 8-oxo-GDP to 8-oxo-GMP. When (14)C-labeled 8-oxo-GTP was applied to CaCl2-permeabilized cells of a mutT(-) mutant strain, it could be incorporated into RNA at 4% of the rate for GTP. Escherichia coli cells appear to possess mechanisms to prevent misincorporation of 8-oxo-7,8-dihydroguanine into RNA.

Hepatitis C virus (HCV) RNA-dependent RNA polymerase replicates the viral genomic RNA and is a primary drug target for antiviral therapy. Previously, we described the purification of an active and stable polymerase-primer-template elongation complex. Here, we show that, unexpectedly, the polymerase elongation complex can use NTPs to excise the terminal nucleotide in nascent RNA. Mismatched ATP, UTP, or CTP could mediate excision of 3'-terminal CMP to generate the dinucleoside tetraphosphate products Ap(4)C, Up(4)C, and Cp(4)C, respectively. Pre-steady-state kinetic studies showed that the efficiency of NTP-mediated excision was highest with ATP. A chain-terminating inhibitor, 3'deoxy-CMP, could also be excised through this mechanism, suggesting important implications for nucleoside drug potency and resistance. The nucleotide excision reaction catalyzed by recombinant hepatitis C virus polymerase was 100-fold more efficient than the corresponding reaction observed with HIV reverse transcriptase.

CTP:phosphocholine cytidylyltransferase (CCT), an amphitropic enzyme that regulates phosphatidylcholine synthesis, is composed of a catalytic head domain and a regulatory tail. The tail region has dual functions as a regulator of membrane binding/enzyme activation and as an inhibitor of catalysis in the unbound form of the enzyme, suggesting conformational plasticity. These functions are well conserved in CCTs across diverse phyla, although the sequences of the tail regions are not. CCT regulatory tails of diverse origins are composed of a long membrane lipid-inducible amphipathic helix (m-AH) followed by a highly disordered segment, reminiscent of the Parkinson disease-linked protein, α-synuclein, which we show shares a novel sequence motif with vertebrate CCTs. To unravel features required for silencing, we created chimeric enzymes by fusing the catalytic domain of rat CCTα to the regulatory tail of CCTs from Drosophila, Caenorhabditis elegans, or Saccharomyces cerevisiae or to α-synuclein. Only the tail domains of the two invertebrate CCTs were competent for both suppression of catalytic activity and for activation by lipid vesicles. Thus, both silencing and activating functions of the m-AH can tolerate significant changes in length and sequence. We identified a highly amphipathic 22-residue segment in the m-AH with features conserved among animal CCTs but not yeast CCT or α-synuclein. Deletion of this segment from rat CCT increased the lipid-independent V(max) by 10-fold, equivalent to the effect of deleting the entire tail, and severely weakened membrane binding affinity. However, membrane binding was required for additional increases in catalytic efficiency. Thus, full activation of CCT may require not only loss of a silencing conformation in the m-AH but a gain of an activating conformation, promoted by membrane binding.

Polyuridylation is emerging as a ubiquitous post-translational modification with important roles in multiple aspects of RNA metabolism. These poly (U) tails are added by poly (U) polymerases with homology to poly (A) polymerases; nevertheless, the selection for UTP over ATP remains enigmatic. We report the structures of poly (U) polymerase Cid1 from Schizoscaccharomyces pombe alone and in complex with UTP, CTP, GTP and 3'-dATP. These structures reveal that each of the 4 nt can be accommodated at the active site; however, differences exist that suggest how the polymerase selects UTP over the other nucleotides. Furthermore, we find that Cid1 shares a number of common UTP recognition features with the kinetoplastid terminal uridyltransferases. Kinetic analysis of Cid1's activity for its preferred substrates, UTP and ATP, reveal a clear preference for UTP over ATP. Ultimately, we show that a single histidine in the active site plays a pivotal role for poly (U) activity. Notably, this residue is typically replaced by an asparagine residue in Cid1-family poly (A) polymerases. By mutating this histidine to an asparagine residue in Cid1, we diminished Cid1's activity for UTP addition and improved ATP incorporation, supporting that this residue is important for UTP selectivity.

Cytidine triphosphate (CTP) stabilized-Au nanoparticles (CTP-AuNPs) allow a quantitative assay of Ca(2+) down to a concentration of 10(-4) M with high selectivity towards Ca(2+) ions over various metal ions including Mg(2+) and the detection range of this probe also can be easily tuned by changing the concentration of CTP., (This journal is © The Royal Society of Chemistry 2011)

Bacterial pathogens deliver effector proteins with diverse biochemical activities into host cells, thereby modulating various host functions. Legionella pneumophila hijacks host vesicle trafficking to avoid phagosome-lysosome fusion, a mechanism that is dependent on the Legionella Dot/Icm type IV secretion system. SidM/DrrA, a Legionella type IV effector, is important for the interactions of Legionella-containing vacuoles with host endoplasmic reticulum-derived vesicles. SidM is the only known protein that catalyzes both the exchange of GDP for GTP and GDI displacement from small GTPase Rab1. We determined the crystal structures of SidM alone (residues 317-647) and SidM (residues 193-550) in complex with nucleotide-free WT Rab1. The SidM structure contains an N-terminal helical domain with a potential new function, a Rab1-activation domain, and a C-terminal phosphatidylinositol 4-phosphate-binding P4M domain. The Rab1-activation domain has extensive strong interactions mainly with Rab1 switch I and II regions that undergo substantial conformational changes on SidM binding. Mutations of switch-contacting residues in SidM attenuate both the nucleotide exchange and GDI displacement activities. Structural comparisons of Rab1 in the SidM complex with Rab1-GDP and Ypt1-GDP in the GDI complex identify key conformational changes that disrupt the nucleotide and GDI binding of Rab1. Further biochemical and structural analyses reveal a unique mechanism of coupled GDP release and GDI displacement likely triggered by the SidM-induced drastic displacement of switch I of Rab1.

Ribonucleotide reductase (RNR) from Lactobacillus leichmannii, a 76 kDa monomer using adenosylcobalamin (AdoCbl) as a cofactor, catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is rapidly (<30 s) inactivated by 1 equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F(2)CTP). [1'-(3)H]- and [5-(3)H]F(2)CTP were synthesized and used independently to inactivate RNR. Sephadex G-50 chromatography of the inactivation mixture revealed that 0.47 equiv of a sugar was covalently bound to RNR and that 0.71 equiv of cytosine was released. Alternatively, analysis of the inactivated RNR by SDS-PAGE without boiling resulted in 33% of RNR migrating as a 110 kDa protein. Inactivation of RNR with a mixture of [1'-(3)H]F(2)CTP and [1'-(2)H]F(2)CTP followed by reduction with NaBH(4), alkylation with iodoacetamide, trypsin digestion, and HPLC separation of the resulting peptides allowed isolation and identification by MALDI-TOF mass spectrometry (MS) of a (3)H/(2)H-labeled peptide containing C(731) and C(736) from the C-terminus of RNR accounting for 10% of the labeled protein. The MS analysis also revealed that the two cysteines were cross-linked to a furanone species derived from the sugar of F(2)CTP. Incubation of [1'-(3)H]F(2)CTP with C119S-RNR resulted in 0.3 equiv of sugar being covalently bound to the protein, and incubation with NaBH(4) subsequent to inactivation resulted in trapping of 2'-fluoro-2'-deoxycytidine. These studies and the ones in the preceding paper (DOI: 10.1021/bi9021318 ) allow proposal of a mechanism of inactivation of RNR by F(2)CTP involving multiple reaction pathways. The proposed mechanisms share many common features with F(2)CDP inactivation of the class I RNRs.

Ribonucleotide reductase (RNR, 76 kDa) from Lactobacillus leichmannii is a class II RNR that requires adenosylcobalamin (AdoCbl) as a cofactor. It catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is 100% inactivated by 1 equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F(2)CTP) in <2 min. Sephadex G-50 chromatography of the inactivation reaction mixture for 2 min revealed that 0.47 equiv of a sugar moiety is covalently bound to RNR and 0.25 equiv of a cobalt(III) corrin is tightly associated, likely through a covalent interaction with C(419) (Co-S) in the active site of RNR [Lohman, G. J. S., and Stubbe, J. (2010) Biochemistry 49, DOI: 10.1021/bi902132u ]. After 1 h, a similar experiment revealed 0.45 equiv of the Co-S adduct associated with the protein. Thus, at least two pathways are associated with RNR inactivation: one associated with alkylation by the sugar of F(2)CTP and the second with AdoCbl destruction. To determine the fate of [1'-(3)H]F(2)CTP in the latter pathway, the reaction mixture at 2 min was reduced with NaBH(4) (NaB(2)H(4)) and the protein separated from the small molecules using a centrifugation device. The small molecules were dephosphorylated and analyzed by HPLC to reveal 0.25 equiv of a stereoisomer of cytidine, characterized by mass spectrometry and NMR spectroscopy, indicating the trapped nucleotide had lost both of its fluorides and gained an oxygen. High-field ENDOR studies with [1'-(2)H]F(2)CTP from the reaction quenched at 30 s revealed a radical that is nucleotide-based. The relationship between this radical and the trapped cytidine analogue provides insight into the nonalkylative pathway for RNR inactivation relative to the alkylative pathway.

The enzyme CMP-Kdo synthetase (KdsB) catalyzes the addition of 2-keto-3-deoxymanno-octulonic acid (Kdo) to CTP to form CMP-Kdo, a key reaction in the biosynthesis of lipopolysaccharide. The reaction catalyzed by KdsB and the related CMP-acylneuraminate synthase is unique among the sugar-activating enzymes in that the respective sugars are directly coupled to a cytosine monophosphate. Using inhibition studies, in combination with isothermal calorimetry, we show the substrate analogue 2beta-deoxy-Kdo to be a potent competitive inhibitor. The ligand-free Escherichia coli KdsB and ternary complex KdsB-CTP-2beta-deoxy-Kdo crystal structures reveal that Kdo binding leads to active site closure and repositioning of the CTP phosphates and associated Mg(2+) ion (Mg-B). Both ligands occupy conformations compatible with an S(n)2-type attack on the alpha-phosphate by the Kdo 2-hydroxyl group. Based on strong similarity with DNA/RNA polymerases, both in terms of overall chemistry catalyzed as well as active site configuration, we postulate a second Mg(2+) ion (Mg-A) is bound by the catalytically competent KdsB-CTP-Kdo ternary complex. Modeling of this complex reveals the Mg-A coordinated to the conserved Asp(100) and Asp(235) in addition to the CTP alpha-phosphate and both the Kdo carboxylic and 2-hydroxyl groups. EPR measurements on the Mn(2+)-substituted ternary complex support this model. We propose the KdsB/CNS sugar-activating enzymes catalyze the formation of activated sugars, such as the abundant CMP-5-N-acetylneuraminic acid, by recruitment of two Mg(2+) to the active site. Although each metal ion assists in correct positioning of the substrates and activation of the alpha-phosphate, Mg-A is responsible for activation of the sugar-hydroxyl group.

CTP:phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in the synthesis of phosphatidylcholine, the most abundant phospholipid in eukaryotic cell membranes. The CCT-catalyzed transfer of a cytidylyl group from CTP to phosphocholine to form CDP-choline is regulated by a membrane lipid-dependent mechanism imparted by its C-terminal membrane binding domain. We present the first analysis of a crystal structure of a eukaryotic CCT. A deletion construct of rat CCTalpha spanning residues 1-236 (CCT236) lacks the regulatory domain and as a result displays constitutive activity. The 2.2-A structure reveals a CCT236 homodimer in complex with the reaction product, CDP-choline. Each chain is composed of a complete catalytic domain with an intimately associated N-terminal extension, which together with the catalytic domain contributes to the dimer interface. Although the CCT236 structure reveals elements involved in binding cytidine that are conserved with other members of the cytidylyltransferase superfamily, it also features nonconserved active site residues, His-168 and Tyr-173, that make key interactions with the beta-phosphate of CDP-choline. Mutagenesis and kinetic analyses confirmed their role in phosphocholine binding and catalysis. These results demonstrate structural and mechanistic differences in a broadly conserved protein fold across the cytidylyltransferase family. Comparison of the CCT236 structure with those of other nucleotidyltransferases provides evidence for substrate-induced active site loop movements and a disorder-to-order transition of a loop element in the catalytic mechanism.

The CCA-adding enzyme synthesizes the CCA sequence at the 3' end of tRNA without a nucleic acid template. The crystal structures of class II Thermotoga maritima CCA-adding enzyme and its complexes with CTP or ATP were determined. The structure-based replacement of both the catalytic heads and nucleobase-interacting neck domains of the phylogenetically closely related Aquifex aeolicus A-adding enzyme by the corresponding domains of the T. maritima CCA-adding enzyme allowed the A-adding enzyme to add CCA in vivo and in vitro. However, the replacement of only the catalytic head domain did not allow the A-adding enzyme to add CCA, and the enzyme exhibited (A, C)-adding activity. We identified the region in the neck domain that prevents (A, C)-adding activity and defines the number of nucleotide incorporations and the specificity for correct CCA addition. We also identified the region in the head domain that defines the terminal A addition after CC addition. The results collectively suggest that, in the class II CCA-adding enzyme, the head and neck domains collaboratively and dynamically define the number of nucleotide additions and the specificity of nucleotide selection.

Background: Targeting the biosynthetic pathway of Coenzyme A (CoA) for drug development will compromise multiple cellular functions of the tubercular pathogen simultaneously. Structural divergence in the organization of the penultimate and final enzymes of CoA biosynthesis in the host and pathogen and the differences in their regulation mark out the final enzyme, dephosphocoenzyme A kinase (CoaE) as a potential drug target., Methodology/principal Findings: We report here a complete biochemical and biophysical characterization of the M. tuberculosis CoaE, an enzyme essential for the pathogen's survival, elucidating for the first time the interactions of a dephosphocoenzyme A kinase with its substrates, dephosphocoenzyme A and ATP; its product, CoA and an intrinsic yet novel inhibitor, CTP, which helps modulate the enzyme's kinetic capabilities providing interesting insights into the regulation of CoaE activity. We show that the mycobacterial enzyme is almost 21 times more catalytically proficient than its counterparts in other prokaryotes. ITC measurements illustrate that the enzyme follows an ordered mechanism of substrate addition with DCoA as the leading substrate and ATP following in tow. Kinetic and ITC experiments demonstrate that though CTP binds strongly to the enzyme, it is unable to participate in DCoA phosphorylation. We report that CTP actually inhibits the enzyme by decreasing its Vmax. Not surprisingly, a structural homology search for the modeled mycobacterial CoaE picks up cytidylmonophosphate kinases, deoxycytidine kinases, and cytidylate kinases as close homologs. Docking of DCoA and CTP to CoaE shows that both ligands bind at the same site, their interactions being stabilized by 26 and 28 hydrogen bonds respectively. We have also assigned a role for the universal Unknown Protein Family 0157 (UPF0157) domain in the mycobacterial CoaE in the proper folding of the full length enzyme., Conclusions/significance: In view of the evidence presented, it is imperative to assign a greater role to the last enzyme of Coenzyme A biosynthesis in metabolite flow regulation through this critical biosynthetic pathway.

Uracil phosphoribosyltransferase catalyzes the conversion of 5-phosphoribosyl-alpha-1-diphosphate (PRPP) and uracil to uridine monophosphate (UMP) and diphosphate (PP(i)). The tetrameric enzyme from Sulfolobus solfataricus has a unique type of allosteric regulation by cytidine triphosphate (CTP) and guanosine triphosphate (GTP). Here we report two structures of the activated state in complex with GTP. One structure (refined at 2.8-A resolution) contains PRPP in all active sites, while the other structure (refined at 2.9-A resolution) has PRPP in two sites and the hydrolysis products, ribose-5-phosphate and PP(i), in the other sites. Combined with three existing structures of uracil phosphoribosyltransferase in complex with UMP and the allosteric inhibitor cytidine triphosphate (CTP), these structures provide valuable insight into the mechanism of allosteric transition from inhibited to active enzyme. The regulatory triphosphates bind at a site in the center of the tetramer in a different manner and change the quaternary arrangement. Both effectors contact Pro94 at the beginning of a long beta-strand in the dimer interface, which extends into a flexible loop over the active site. In the GTP-bound state, two flexible loop residues, Tyr123 and Lys125, bind the PP(i) moiety of PRPP in the neighboring subunit and contribute to catalysis, while in the inhibited state, they contribute to the configuration of the active site for UMP rather than PRPP binding. The C-terminal Gly216 participates in a hydrogen-bond network in the dimer interface that stabilizes the inhibited, but not the activated, state. Tagging the C-terminus with additional amino acids generates an endogenously activated enzyme that binds GTP without effects on activity.

A method was developed for the quantification of araCTP, CTP and dCTP in a human follicular lymphoma cell line. This method involves solid phase extraction (SPE) using a weak anion-exchanger (WAX) cartridge, a porous graphitic carbon high-performance liquid chromatography (HPLC) column separation, and tandem mass spectrometry (MS/MS) detection. By using a triple quadrupole mass spectrometer operating in negative ion multiple reaction monitoring (MRM) mode, the method was able to achieve a lower limit of quantification (LLOQ) of 0.1 microg mL(-1) for araCTP and of 0.01 microg mL(-1) for both CTP and dCTP. The method was validated and used to determine the amount of araCTP, CTP and dCTP formed after incubation of araC and an araCMP prodrug in the human follicular lymphoma cell line RL.

Aspartate transcarbamoylase has emerged as a textbook example of an allosteric enzyme whose binding of active-site substrates can be explained on the basis of the classical Monod-Wyman-Changeux (MWC) model of allostery. There is still debate, however, regarding the mode of action of ATP and cytidine triphosphate (CTP)--allosteric effectors that bind at regulatory sites 60 A away from the nearest active site. A large body of data for nucleotide binding is consistent with the MWC model, including a previous NMR study showing a shift in the allosteric equilibrium between R and T states that is predicted by this scheme. The possibility of binding-promoted changes to the structures of the active sites, while not within the framework of the MWC model, cannot be excluded, however. Here, the effects of binding of nucleotides are monitored in a series of (1)H-(13)C methyl transverse relaxation optimized spectroscopy spectra recorded on the 300-kDa aspartate transcarbamoylase holoenzyme in both the absence and the presence of saturating amounts of ATP or CTP. No changes in shifts of methyl probes of the catalytic chains (c-chains) that include the active sites are observed, consistent with a lack of structural changes. In addition, methyl (1)H-(13)C residual dipolar couplings are measured that are exquisitely sensitive to methyl axis orientations, and correlations between couplings measured on samples with and without nucleotide show no changes in structure of the c-chains. These results indicate that the mechanism of action of ATP and CTP can be explained fully by the MWC model and that any scheme invoking structural changes of the c-chains is not correct.

Recent polymorphism analyses of Mycobacterium tuberculosis strains have identified missense mutations unique to the W-Beijing lineage in genes belonging to the Nudix hydrolase superfamily. This study investigates the structure and function of one of these Nudix hydrolases, MutT2, and examines the effect that the W-Beijing mutation (G58R) has on enzyme characteristics. MutT2 has a preference for cytidine triphosphates, and although the G58R mutation does not alter nucleotide specificity, it reduces the protein's affinity for divalent cations. The K(D) of free Mg(2+) is 79-fold higher for the G58R mutant (3.30 +/- 0.19 mM) compared with that for the wild-type (41.7 +/- 1.4 microM). Circular dichroism and nuclear magnetic resonance spectroscopy measurements show that while the mutation does not perturb the overall structure of the protein, protein stability is significantly compromised by the presence of the arginine with DeltaG (H(2)O), the free-energy of unfolding, being reduced by 2.48 kcal mol(-1) in the G58R mutant. Homology modeling of MutT2 shows that Gly-58 is in close proximity (10.8 A) to the Mg(2+) binding site formed by the highly conserved Nudix box residues and hydrogen bonds with Ala-54 in the preceding alpha-helix. This may explain the increased divalent cation requirement and decreased stability observed when an arginine is substituted for glycine at this position. A role for MutT2 in the regulation of cytidine-triphosphates available for nucleotide-dependent reactions is postulated, and the impact that the G58R mutation may have on these reactions is discussed.

We describe herein a method for isolating fully-modified 4'-thioRNA aptamers against human alpha-thrombin using the SELEX protocol. In order to isolate the desired aptamers, in vitro transcription was examined in the presence of four kinds of 4'-thioribonucleoside triphosphates (4'-thioNTPs) in an effort to afford the fully-modified 4'-thioRNA. The transcription efficiency of the 4'-thioNTPs was first compared with that of the nucleoside 5'-(alpha-thio)triphosphates (alphaSNTPs) and found to be less effective than that of the alphaSNTPs, especially when GTP and/or ATP were substituted for 4'-thioGTP and/or 4'-thioATP. Further attempts to improve its efficiency, including the use of a mutant RNA polymerase instead of the wild type, various additives, and 4'-thioNTP concentrations were unsuccessful. Accordingly, the transcription was performed in the presence of 4'-thioNTPs together with the natural GTP and ATP at the appropriate concentrations. Although this attempt furnished a highly-modified 4'-thioRNA, but not a fully-modified 4'-thioRNA, we eventually succeeded in isolating the fully-modified 4'-thioRNA aptamers by SELEX using optimized transcription conditions, followed by post-modification of the resulting aptamers.

CTP:phosphocholine cytidylyltransferase (CCT), a rate-limiting enzyme in phosphatidylcholine synthesis, is regulated by reversible membrane interactions mediated by an amphipathic helical domain (M) that binds selectively to anionic lipids. CCT is a dimer; thus the functional unit has two M domains. To probe the functional contribution of each domain M we prepared a CCT heterodimer composed of one full-length subunit paired with a CCT subunit truncated before domain M that was also catalytically dead. We compared this heterodimer to the full-length homodimer with respect to activation by anionic vesicles, vesicle binding affinities, and promotion of vesicle aggregation. Surprisingly for all three functions the dimer with just one domain M behaved similarly to the dimer with two M domains. Full activation of the wild-type subunit was not impaired by loss of one domain M in its partner. Membrane binding affinities were the same for dimers with one versus two M domains, suggesting that the two M domains of the dimer do not engage a single bilayer simultaneously. Vesicle cross-bridging was also unhindered by loss of one domain M, suggesting that another motif couples with domain M for cross-bridging anionic membranes. Mutagenesis revealed that the positively charged nuclear localization signal sequence constitutes that second motif for membrane cross-bridging. We propose that the two M domains of the CCT dimer engage a single bilayer via an alternating binding mechanism. The tethering function involves the cooperation of domain M and the nuclear localization signal sequence, each engaging separate membranes. Membrane binding of a single M domain is sufficient to fully activate the enzymatic activity of the CCT dimer while sustaining the low affinity, reversible membrane interaction required for regulation of CCT activity.

Aspartate carbamoyltransferase (ATCase) is a paradigm for allosteric regulation of enzyme activity. B-class ATCases display very similar homotropic allosteric behaviour, but differ extensively in their heterotropic patterns. The ATCase from the thermoacidophilic archaeon Sulfolobus acidocaldarius, for example, is strongly activated by its metabolic pathway's end product CTP, in contrast with Escherichia coli ATCase which is inhibited by CTP. To investigate the structural basis of this property, we have solved the crystal structure of the S. acidocaldarius enzyme in complex with CTP. Structure comparison reveals that effector binding does not induce similar large-scale conformational changes as observed for the E. coli ATCase. However, shifts in sedimentation coefficients upon binding of the bi-substrate analogue PALA show the existence of structurally distinct allosteric states. This suggests that the so-called "Nucleotide-Perturbation model" for explaining heterotropic allosteric behaviour, which is based on global conformational strain, is not a general mechanism of B-class ATCases.

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&#95;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&#95;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&#95;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&#95;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.

The enzyme aspartate transcarbamoylase (ATCase, EC 2.1.3.2 of Escherichia coli), which catalyzes the committed step of pyrimidine biosynthesis, is allosterically regulated by all four ribonucleoside triphosphates (NTPs) in a nonlinear manner. Here, we dissect this regulation using the recently developed approach of random sampling-high-dimensional model representation (RS-HDMR). ATCase activity was measured in vitro at 300 random NTP concentration combinations, each involving (consistent with in vivo conditions) all four NTPs being present. These data were then used to derive a RS-HDMR model of ATCase activity over the full four-dimensional NTP space. The model accounted for 90% of the variance in the experimental data. Its main elements were positive ATCase regulation by ATP and negative by CTP, with the negative effects of CTP dominating the positive ones of ATP when both regulators were abundant (i.e., a negative cooperative effect of ATP x CTP). Strong sensitivity to both ATP and CTP concentrations occurred in their physiological concentration ranges. UTP had only a slight effect, and GTP had almost none. These findings support a predominant role of CTP and ATP in ATCase regulation. The general approach provides a new paradigm for dissecting multifactorial regulation of biological molecules and processes.