Publications

• Nucleoside selectivity of Aspergillus fumigatus nucleoside-diphosphate kinase.

  Nguyen S., Jovcevski B., Pukala T.L., Bruning J.B.

  Aspergillus fumigatus infections are rising at a disconcerting rate in tandem with antifungal resistance rates. Efforts to develop novel antifungals have been hindered by the limited knowledge of fundamental biological and structural mechanisms of A. fumigatus propagation. Biosynthesis of NTPs, th ... >> More

  Aspergillus fumigatus infections are rising at a disconcerting rate in tandem with antifungal resistance rates. Efforts to develop novel antifungals have been hindered by the limited knowledge of fundamental biological and structural mechanisms of A. fumigatus propagation. Biosynthesis of NTPs, the building blocks of DNA and RNA, is catalysed by NDK. An essential enzyme in A. fumigatus, NDK poses as an attractive target for novel antifungals. NDK exhibits broad substrate specificity across species, using both purines and pyrimidines, but the selectivity of such nucleosides in A. fumigatus NDK is unknown, impeding structure-guided inhibitor design. Structures of NDK in unbound- and NDP-bound states were solved, and NDK activity was assessed in the presence of various NTP substrates. We present the first instance of a unique substrate binding mode adopted by CDP and TDP specific to A. fumigatus NDK that illuminates the structural determinants of selectivity. Analysis of the oligomeric state reveals that A. fumigatus NDK adopts a hexameric assembly in both unbound- and NDP-bound states, contrary to previous reports suggesting it is tetrameric. Kinetic analysis revealed that ATP exhibited the greatest turnover rate (321 ± 33.0 s^-1 ), specificity constant (626 ± 110.0 mm^-1 ·s^-1 ) and binding free energy change (-37.0 ± 3.5 kcal·mol^-1 ). Comparatively, cytidine nucleosides displayed the slowest turnover rate (53.1 ± 3.7 s^-1 ) and lowest specificity constant (40.2 ± 4.4 mm^-1 ·s^-1 ). We conclude that NDK exhibits nucleoside selectivity whereby adenine nucleosides are used preferentially compared to cytidine nucleosides, and these insights can be exploited to guide drug design. ENZYMES: Nucleoside-diphosphate kinase (EC 2.7.4.6). DATABASE: Structural data are available in the PDB database under the accession numbers: Unbound-NDK (6XP4), ADP-NDK (6XP7), GDP-NDK (6XPS), IDP-NDK (6XPU), UDP-NDK (6XPT), CDP-NDK (6XPW), TDP-NDK (6XPV). << Less

  FEBS J 288:2398-2417(2021) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• The maize (Zea mays L.) nucleoside diphosphate kinase1 (ZmNDPK1) gene encodes a human NM23-H2 homologue that binds and stabilizes G-quadruplex DNA.

  Kopylov M., Bass H.W., Stroupe M.E.

  Noncanonical forms of DNA like the guanine quadruplex (G4) play important roles in regulating transcription and translation through interactions with their protein partners. Although potential G4 elements have been identified in or near genes from species diverse as bacteria, mammals, and plants, ... >> More

  Noncanonical forms of DNA like the guanine quadruplex (G4) play important roles in regulating transcription and translation through interactions with their protein partners. Although potential G4 elements have been identified in or near genes from species diverse as bacteria, mammals, and plants, little is known about how they might function as cis-regulatory elements or as binding sites for trans-acting protein partners. In fact, until now no G4 binding partners have been identified in the plant kingdom. Here, we report on the cloning and characterization of the first plant-kingdom gene known to encode a G4-binding protein, maize (Zea mays L.) nucleoside diphosphate kinase1 (ZmNDPK1). Structural characterization by X-ray crystallography reveals that it is a homohexamer, akin to other known NDPKs like the human homologue NM23-H2. Further probing into the G4-binding properties of both NDPK homologues suggests that ZmNDPK1 possesses properties distinct from that of NM23-H2, which is known to interact with a G-rich sequence element upstream of the c-myc gene and, in doing so, modulate its expression. Indeed, ZmNDPK1 binds the folded G4 with low nanomolar affinity but corresponding unfolded G-rich DNA more weakly, whereas NM23-H2 binds both folded and unfolded G4 with low nanomolar affinities; nonetheless, both homologues appear to stabilize folded DNAs whether they were prefolded or not. We also demonstrate that the G4-binding activity of ZmNDPK1 is independent of nucleotide binding and kinase activity, suggesting that the G4-binding region and the enzyme active sites are separate. Together, these findings establish a broad evolutionary conservation of some NDPKs as G4-DNA binding enzymes, but with potentially distinct biochemical properties that may reflect divergent evolution or species-specific deployment of these elements in gene regulatory processes. << Less

  Biochemistry 54:1743-1757(2015) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Saccharomyces cerevisiae nucleoside-diphosphate kinase: purification, characterization, and substrate specificity.

  Jong A.Y., Ma J.J.

  Nucleoside-diphosphate kinase is an enzyme which catalyzes the phosphorylation of nucleoside diphosphates into the corresponding triphosphates for nucleic acid biosynthesis. In this communication, we describe the purification and characterization of nucleoside-diphosphate kinase from yeast. The pu ... >> More

  Nucleoside-diphosphate kinase is an enzyme which catalyzes the phosphorylation of nucleoside diphosphates into the corresponding triphosphates for nucleic acid biosynthesis. In this communication, we describe the purification and characterization of nucleoside-diphosphate kinase from yeast. The purified protein appears to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel analysis, with a molecular weight of about 17,000-18,000. An estimate from the fast protein liquid chromatography Superose 12 gel filtration shows a native molecular weight of about 68,000 to 70,000. The results suggest that yeast nucleoside-diphosphate kinase is composed of four subunits. Substrate specificity studies show that the relative activity of nucleoside diphosphates (NDP) as phosphate acceptors is in the order of dTDP greater than CDP greater than UDP greater than dUDP greater than GDP greater than or equal to dGDP greater than dCDP greater than dADP greater than ADP; and the relative activity of triphosphate donors is in the order of UTP greater than dTTP greater than CTP greater than dCTP greater than dATP greater than ATP greater than or equal to dGTP greater than GTP. The Km and Vm of dTDP, dGDP, dCDP, dUDP, CDP, and UDP have been determined. The rate constant studies indicate that the purified NDP kinase prefers using, to a slight extent, dTDP (approximately 800 min-1) as the substrate rather than other tested deoxyribo- and ribonucleotides (350-450 min-1). The broad substrate specificity and kinetic data suggest that the enzyme is involved in both DNA and RNA metabolism. << Less

  Arch. Biochem. Biophys. 291:241-246(1991) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.

• The human adenylate kinase 9 is a nucleoside mono- and diphosphate kinase.

  Amiri M., Conserva F., Panayiotou C., Karlsson A., Solaroli N.

  Adenylate kinases regulate adenine nucleotide levels and are present in different intracellular compartments. These enzymes also participate in the activation of pharmacologically active nucleoside and nucleotide analogs. We have in the present study identified the ninth isoform of the adenylate k ... >> More

  Adenylate kinases regulate adenine nucleotide levels and are present in different intracellular compartments. These enzymes also participate in the activation of pharmacologically active nucleoside and nucleotide analogs. We have in the present study identified the ninth isoform of the adenylate kinase family of enzymes and accordingly named the protein adenylate kinase 9 (AK9). Initially a full-length cDNA of a hypothetical protein containing a predicted adenylate kinase domain was identified and subsequently cloned and expressed in Escherichia coli. The substrate specificity of the recombinant protein showed that the enzyme catalyzed the phosphorylation of AMP, dAMP, CMP and dCMP with ATP as phosphate donor, while only AMP and CMP were phosphorylated when GTP was the phosphate donor. The kinetic parameters of AK9 were determined for AMP, dAMP and CMP with ATP as phosphate donor. Interestingly, in addition to the diphosphate products, a nucleoside diphosphate kinase (NDPK) activity was also present with subsequent triphosphates formed. With ATP or GTP as phosphate donor it was possible to detect the production of ATP, CTP, GTP, UTP, dATP, dCTP, dGTP and TTP as enzymatic products from the corresponding diphosphate substrates. A number of previously characterized adenylate kinases were also tested and found to possess a broad phosphotransferase activity similar to AK9. These enzymes are accordingly suggested to be regarded as nucleoside mono- and diphosphate kinases with catalytic activities possibly determined by local substrate concentrations. << Less

  Int. J. Biochem. Cell Biol. 45:925-931(2013) [PubMed] [EuropePMC]

  This publication is cited by 23 other entries.

• Nucleoside diphosphate kinase from bovine retina: purification, subcellular localization, molecular cloning, and three-dimensional structure.

  Abdulaev N.G., Karaschuk G.N., Ladner J.E., Kakuev D.L., Yakhyaev A.V., Tordova M., Gaidarov I.O., Popov V.I., Fujiwara J.H., Chinchilla D., Eisenstein E., Gilliland G.L., Ridge K.D.

  The biochemical and structural properties of bovine retinal nucleoside diphosphate kinase were investigated. The enzyme showed two polypeptides of approximately 17.5 and 18.5 kDa on SDS-PAGE, while isoelectric focusing revealed seven to eight proteins with a pI range of 7.4-8.2. Sedimentation equi ... >> More

  The biochemical and structural properties of bovine retinal nucleoside diphosphate kinase were investigated. The enzyme showed two polypeptides of approximately 17.5 and 18.5 kDa on SDS-PAGE, while isoelectric focusing revealed seven to eight proteins with a pI range of 7.4-8.2. Sedimentation equilibrium yielded a molecular mass of 96 +/-2 kDa for the enzyme. Carbohydrate analysis revealed that both polypeptides contained Gal, Man, GlcNAc, Fuc, and GalNac saccharides. Like other nucleoside diphosphate kinases, the retinal enzyme showed substantial differences in the Km values for various di- and triphosphate nucleotides. Immunogold labeling of bovine retina revealed that the enzyme is localized on both the membranes and in the cytoplasm. Screening of a retinal cDNA library yielded full-length clones encoding two distinct isoforms (NBR-A and NBR-B). Both isoforms were overexpressed in Escherichia coli and their biochemical properties compared with retinal NDP-kinase. The structures of NBR-A and NBR-B were determined by X-ray crystallography in the presence of guanine nucleotide(s). Both isoforms are hexameric, and the fold of the monomer is similar to other nucleoside diphosphate kinase structures. The NBR-A active site contained both a cGMP and a GDP molecule each bound at half occupancy while the NBR-B active site contained only cGMP. << Less

  Biochemistry 37:13958-13967(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The nucleoside diphosphate kinase from mimivirus: a peculiar affinity for deoxypyrimidine nucleotides.

  Jeudy S., Claverie J.-M., Abergel C.

  The first viral Nucleoside Diphosphate Kinase was recently identified in the giant double-stranded DNA virus Acanthamoeba polyphag a Mimivirus (ApM). Here we report its expression and detailed biochemical characterization. NDK(apm) exhibits unique features such as a shorter Kpn-loop, a structural ... >> More

  The first viral Nucleoside Diphosphate Kinase was recently identified in the giant double-stranded DNA virus Acanthamoeba polyphag a Mimivirus (ApM). Here we report its expression and detailed biochemical characterization. NDK(apm) exhibits unique features such as a shorter Kpn-loop, a structural motif previously reported to be part of the active site and involved in oligomer formation. Enzymatic activity measurements on the recombinant NDK(apm) revealed its preferential affinity for deoxypyrimidine nucleotides. This property might represent an adaptation of NDK(apm) to the production of the limiting TTP deoxynucleotide required for the replication of the large A+T rich (72%) viral genome. The NDK(apm) might also assume a role in dUTP detoxification to compensate for the surprising absence of Mimivirus dUTPase (deoxyuridine triphosphate pyrophosphatase) an important enzyme conserved in most viruses. Although the phylogenetic analysis of NDK sequences sampled through organisms from the three domains of life is only partially informative, it favors an ancestral origin for NDK(apm) over a recent acquisition from a eukaryotic organism by horizontal gene transfer. << Less

  J. Bioenerg. Biomembr. 38:247-254(2006) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.