Three-Dimensional Structure of Nucleoside Diphosphate Kinase

Three-dimensional structures are known from X-ray studies of the nucleoside diphosphate(NDP) kinase of many organisms from bacteria to human. All NDP kinases have subunits ofabout 150 residues with a very similar fold based on the sandwich orferredoxin fold.This fold is found in many nucleotide or polynucleotide-binding proteins with no sequencerelationship to NDP kinase. This common fold is augmented here with specificfeatures: asurface -helix hairpin, the Kpn loop, and the C-terminal extension. The -helix hairpin andKpn loop make up the nucleotide binding site, which is unique to NDP kinase and differentfrom that of other kinases or ATPases. The Kpn loop and the C-terminal extension are alsoinvolved in the quaternary structure. Whereas all known eukaryotic NDP kinases, includingmitochondral enzymes, are hexamers, some bacterial enzymes are tetramers. However,hexameric and tetrameric NDP kinases are built from the same dimer. The structural environmentof the active histidine is identical in all. The nucleotide binding site is also fully conserved,except for a feature implicating C-terminal residues in the hexamer, but not in the tetramer.Structural data on the native and phosphorylated enzyme, complexes with substrates, inhibitor,and a transition state analog, give a solid basis to a mechanism of phosphate transfer in whichthe largest contributors to catalysis are the 3-OH of the sugar and the bound Mg²⁺ in thenucleotide substrate. In contrast, we still lack structural data relating to DNA binding andother functions of NDP kinases.     

Phosphorylation of Anti-HIV Nucleoside Analogs by Nucleoside Diphosphate Kinase

Abstract

The reaction of NDP kinase with antiviral nucleoside triphosphates used in antiviral therapies was studied at the presteady state by fluorescence stopped-flow and compared with the steady-state parameters. The affinity of the analogs was determined by fluorescence titration of a mutated enzyme with an inserted Trp in the binding site. The lack of the 3′ hydroxyl in analogs is shown to decrease the k_cat more than the K_D.     

Nm23/Nucleoside Diphosphate Kinase in Human Cancers

Tumor metastasis is the leading cause of death in cancer patients. From a series of tumorcohort studies, low expression of Nm23/NDP kinase has been correlated with poor patientprognosis and survival, lymph node infiltration, and histopathological indicators of highmetastatic potential in a number of cancer types, including mammary and ovarian carcinomas andmelanoma. In other tumor types, no correlation has been established. Transfection ofNm23/NDP kinase cDNA into highly metastatic breast, melanoma, prostrate and squamous cellcarcinomas, and colon adenocarcinoma cells significantly reduced the metastatic competencyof the cells in vivo. In culture, cell motility, invasion, and colonization were inhibited, whereastumorigenicity and cellular proliferation were not affected, indicating that Nm23/NDP kinaseacts as a metastasis suppressor.     

A Nucleoside Diphosphate Kinase from Paramecium tetraurelia with Protein Kinase Activity

Nucleoside diphosphate kinase (NDP kinase) from Paramecium was purified to homogeneity. The native enzyme was 80 kDa (by gel filtration), with subunits of 18 and 20 kDa. Near the amino terminus, 15 of 20 residues were identical with those in human NDP kinase, and 17 of 20 with the awd gene product from Drosophila. NDP kinase bound α-labeled ATP and GTP, and a photoreactive GTP analog labeled both subunits. Purified NDP kinase underwent autophosphorylation on a histidine and a serine residue using either ATP or GTP as a substrate. The enzyme also catalyzed acid-stable phosphorylation of casein and phosvitin. This protein kinase activity is distinct from the histidine phosphorylation that is part of the NDP kinase catalytic cycle. Antiserum against the purified protein from Paramecium cross-reacted with 16- to 20-kDa proteins in most species tested, and with a larger protein (44 kDa) in Paramecium, Xenopus, and two human lines. The multiple forms (20 and 44 kDa) of the NDP kinase in Paramecium and its protein kinase activity, suggest that the protein is more than a housekeeping enzyme; it may have regulatory roles such as those of the NDP kinase-like awd protein of Drosophila and Nm23 protein of humans.     

核苷二磷酸激酶A的异构及其分子机制

对核苷二磷酸激酶A(NDPKA)的异构及其分子机制进行研究.还原和非还原SDSPAGE观察重组人核苷二磷酸激酶A(rhNDPKA)的异构;RPHPLC分析rhNDPKA异构体的反相色谱行为,并测定rhNDPKA异构体的酶活性;多角度激光散射法测定rhNDPKA异构体在溶液中的表观分子量;飞行质谱分析异构体的质量肽谱.结果发现,rhNDPKA在非还原SDSPAGE上表现为4条带,对应于NDPKA的氧化型、还原型、氧化型二聚体和还原型二聚体,其分子量分别为18.1kD、21.3kD、35.2kD和38.3kD.RPHPLC发现,还原型rhNDPKA和氧化型rhNDPKA疏水性有差异.新鲜制备的rhNDPKA在纯水溶液中,经空气氧化后,逐渐由还原型向氧化型过渡,而还原剂或生理盐水可使rhNDPKA稳定于还原型或氧化型.酶活测定结果表明,还原型rhNDPKA比活性为1965±166Umg,氧化型rhNDPKA比活性为974±53Umg.多角度激光散射检测发现,还原型rhNDPKA在溶液中仍可形成六聚体.质量肽谱结果证明,在氧化型rhNDPKA中,C4和C145位巯基形成二硫键,而C109位巯基游离存在.根据本文所确定的NDPKA单体中的二硫键位置,推导出rhNDPKA单体异构体和二聚体异构体的变构原理,这为进一步研究NDPKA的多能性调节机制打下了良好基础.     

Role of AWD/Nucleoside Diphosphate Kinase in Drosophila Development

The abnormal wing discs gene of Drosophila encodes a soluble protein with nucleosidediphosphate kinase activity. This enzymic activity is necessary for the biological function ofthe abnormal wing discs gene product. Complete loss of function, i.e., null, mutations causelethality after the larval stage. Most larval organs in such null mutant larvae appear to benormal, but the imaginal discs are small and incapable of normal differentiation.Killer-of-prune is a neomorphic mutation in the abnormal wing discs gene. It causes dominant lethalityin larvae that lack prune gene activity. The Killer-of-prune mutant protein may have alteredsubstrate specificity. Null mutant larvae have a low level of nucleoside diphosphate kinaseactivity. This suggests that there may be additional Drosophila genes that encode proteinswith nucleoside dipthosphate kinase activity. Candidate genes have been found in theDrosophila genome.     

NM23/Nucleoside Diphosphate Kinase and Signal Transduction

NM23s (or NDP kinases) regulate a fascinating variety of cellular activities, includingproliferation, development, and differentiation. All these processes are modulated by external stimuli,leading to the idea that this family of proteins modulates transmembrane signaling pathways.This review summarizes the evidence indicating that NM23/NDP kinases participate intransmembrane signaling in eukaryotic cells and discusses the molecular mechanisms proposed toaccount for these actions.     

Dissecting the Unique Nucleotide Specificity of Mimivirus Nucleoside Diphosphate Kinase

The analysis of the Acanthamoeba polyphaga mimivirus genome revealed the first virus-encoded nucleoside diphosphate kinase (NDK), an enzyme that is central to the synthesis of RNA and DNA, ubiquitous in cellular organisms, and well conserved among the three domains of life. In contrast with the broad specificity of cellular NDKs for all types of ribo- and deoxyribonucleotides, the mimivirus enzyme exhibits a strongly preferential affinity for deoxypyrimidines. In order to elucidate the molecular basis of this unique substrate specificity, we determined the three-dimensional (3D) structure of the Acanthamoeba polyphaga mimivirus NDK alone and in complex with various nucleotides. As predicted from a sequence comparison with cellular NDKs, the 3D structure of the mimivirus enzyme exhibits a shorter Kpn loop, previously recognized as a main feature of the NDK active site. The structure of the viral enzyme in complex with various nucleotides also pinpointed two residue changes, both located near the active site and specific to the viral NDK, which could explain its stronger affinity for deoxynucleotides and pyrimidine nucleotides. The role of these residues was explored by building a set of viral NDK variants, assaying their enzymatic activities, and determining their 3D structures in complex with various nucleotides. A total of 26 crystallographic structures were determined at resolutions ranging from 2.8 Å to 1.5 Å. Our results suggest that the mimivirus enzyme progressively evolved from an ancestral NDK under the constraints of optimizing its efficiency for the replication of an AT-rich (73%) viral genome in a thymidine-limited host environment.Mimivirus, a DNA virus infecting Acanthamoeba, is the largest and most complex virus isolated to date (8, 37). It is the first representative and prototype member of the Mimiviridae, the latest addition to the large nucleocytoplasmic DNA viruses, including the poxviruses, the phycodnaviruses, (infecting algae), the iridoviruses (infecting invertebrates and fishes), and asfarvirus (the agent of a swine fever in Africa) (18). The mimivirus''s record genome size (1.2 Mb) and gene content (911 encoded proteins), as well as the presence of genes previously thought to be specific to cellular organisms (such as aminoacyl-tRNA synthetases [3]), revived the debate about the evolutionary origin of DNA viruses and their putative role in the emergence of the eukaryote nucleus (reviewed in reference 7) or in the advent of DNA genomes (13).In this peculiar context, we found the discovery of the first virus-encoded nucleoside diphosphate kinase (NDK) within the mimivirus genome of great interest and warranting a detailed study of the structural and biochemical properties of this unique viral enzyme. Ubiquitous in cellular organisms, NDKs are responsible for the last step of 2′-deoxynucleoside triphosphate (dNTP) pathways and as such play an essential role in the replication of DNA by providing the basic precursors for its synthesis. Acting indiscriminately on ribonucleotides and deoxyribonucleotides, the cellular NDKs are also responsible for supplying energy to various essential synthetic pathways, producing NTPs for RNA synthesis, CTP for lipid synthesis, UTP for polysaccharide synthesis, and GTP for protein synthesis elongation, signal transduction, and microtubules polymerization. Besides their direct role in the above metabolic pathways, cellular NDKs have been involved in the regulation of cell growth and differentiation in vertebrates (22).Cellular NDKs are small proteins of about 150 amino acids, the sequences of which are highly conserved among the three domains of life (>40% identity). They are most often hexameric enzymes, with a few occurrences of tetrameric and dimeric NDK structures in bacteria (19, 25, 26, 31, 38). They all catalyze the transfer of a phosphate group from an NTP onto a nucleotide diphosphate (NDP) through an Mg²⁺-dependent reaction. In vivo, the phosphate donor is usually the nonlimiting ATP nucleotide.In agreement with their implication in various metabolic pathways, cellular NDKs exhibit little substrate specificity and are equally able to act on purine and pyrimidine nucleotides, in their 2′ OH and deoxyribonucleotide forms. In clear contrast, our characterization of the mimivirus NDK revealed its enhanced affinity for deoxypyrimidine nucleotides (20). This marked difference between the viral and cellular NDKs offered a good opportunity to explore the sequence and structure features governing substrate specificity. For instance, cellular NDKs exhibit a conserved loop, the Kpn loop, involved both in substrate binding and in oligomerization of the enzyme (19). Interestingly, a sequence comparison predicted this loop to be shorter in the Acanthamoeba polyphaga mimivirus NDK (NDK_apm) sequence. However, many other single-residue changes could also be involved in modifying the enzyme properties. To explore these issues, we performed a detailed structure-function analysis of the NDK_apm protein in a variety of mutated forms and substrate-enzyme complexes. Despite its markedly different sequence, the three-dimensional structure of the mimivirus NDK was found to be very similar to that of cellular enzymes. Its peculiar substrate specificity is not attributable to a single sequence feature but rather appears to result from the conjunction of several factors, suggesting the progressive optimization of an ancestral enzyme for the replication of an AT-rich (73%) genome in a thymidine-limited host environment.     

Structural and Functional Characterization of Acinetobacter baumannii Nucleoside Diphosphate Kinase

近年来,鲍曼不动杆菌(Acinetobacter baumannii)在医院里越来越受到人们的关注,尤其是在重症监护病房(ICUs)．它以强大的多重耐药性(multiresistance)而闻名．核苷二磷酸激酶(nucleoside diphosphate kinase,NDK)是一种进化上非常保守的酶,它能催化核苷之间磷酸基团的转移．我们解析了鲍曼不动杆菌NDK野生型和C端氨基酸残基Arg141-Thr142-Arg143(RTR)截短突变体的结构．通过和黄色黏菌(Myxococcus xanthus)NDK的三维结构进行比较,推断鲍曼不动杆菌NDK的催化机制和黄色黏菌类似．通过激酶活性实验和圆二色谱实验,发现鲍曼不动杆菌NDK E28A突变体二级结构发生了改变,从而导致蛋白催化活性降低,说明Glu28是鲍曼不动杆菌NDK结构中非常关键的氨基酸残基．鲍曼不动杆菌NDK C端RTR截短突变体显示出催化活性极大的降低,这可能与C端RTR残基介导的二体间相互作用有关．虽然RTR截短突变体中的Lys33伸向了和野生型中不同的方向,和Val15产生相互作用弥补了一部分因为RTR截短丢失的相互作用,维持了RTR截短突变体和野生型类似的结构．但是,Lys33产生的相互作用依然太弱,不足以维持蛋白在催化的动态过程中整体结构的高效转换．我们解析的鲍曼不动杆菌NDK晶体高分辨率结构将有助于科学家设计针对鲍曼不动杆菌的药物．     

重组人核苷二磷酸激酶A的理化性质

对重组人核苷二磷酸激酶A（rhNDPK-A）进行纯化,并对重组产物的理化性质及在溶液中的聚合状态进行鉴定。NDPK-A工程菌发酵后的菌体高压匀浆,然后微孔过滤、超滤浓缩,所得样品经DEAE阴离子交换、Cibacron Blue亲和层析、分子筛层析三步纯化后,以SDS-PAGE和RP-HPLC分析纯化产物的纯度,RP-HPLC测定酶活性。合格制品以基质辅助激光解析飞行时间质谱测定相对分子质量（MW）;Edman降解法测定N末端序列;多角度激光散射法测定重组产物在溶液中的表观分子量。结果表明,rhNDPK-A纯化产物的SDS-PAGE纯度为97.3%,RP-HPLC纯度为99.2%;比活性为（900±100）u/mg;单体相对分子质量为17017,与NDPKA分子量理论值相差132。测序结果表明,rhNDPK-A N末端缺失Met残基,其理论分子量为17017,与飞行质谱测定结果完全一致。表观分子量测定结果表明,rhNDPK-A在溶液中形成六聚体,表观分子量为102kD。上述结果说明, NDPK-A重组产物具与天然产物相同的自发形成六聚体性质,这为NDPK-A新药开发和机理研究打下了良好基础。     

A New Subfamily of Polyphosphate Kinase 2 (Class III PPK2) Catalyzes both Nucleoside Monophosphate Phosphorylation and Nucleoside Diphosphate Phosphorylation

Inorganic polyphosphate (polyP) is a linear polymer of tens to hundreds of phosphate (P_i) residues linked by “high-energy” phosphoanhydride bonds as in ATP. PolyP kinases, responsible for the synthesis and utilization of polyP, are divided into two families (PPK1 and PPK2) due to differences in amino acid sequence and kinetic properties. PPK2 catalyzes preferentially polyP-driven nucleotide phosphorylation (utilization of polyP), which is important for the survival of microbial cells under conditions of stress or pathogenesis. Phylogenetic analysis suggested that the PPK2 family could be divided into three subfamilies (classes I, II, and III). Class I and II PPK2s catalyze nucleoside diphosphate and nucleoside monophosphate phosphorylation, respectively. Here, we demonstrated that class III PPK2 catalyzes both nucleoside monophosphate and nucleoside diphosphate phosphorylation, thereby enabling us to synthesize ATP from AMP by a single enzyme. Moreover, class III PPK2 showed broad substrate specificity over purine and pyrimidine bases. This is the first demonstration that class III PPK2 possesses both class I and II activities.     

Mycobacterial Nucleoside Diphosphate Kinase Blocks Phagosome Maturation in Murine Raw 264.7 Macrophages

Background

Microorganisms capable of surviving within macrophages are rare, but represent very successful pathogens. One of them is Mycobacterium tuberculosis (Mtb) whose resistance to early mechanisms of macrophage killing and failure of its phagosomes to fuse with lysosomes causes tuberculosis (TB) disease in humans. Thus, defining the mechanisms of phagosome maturation arrest and identifying mycobacterial factors responsible for it are key to rational design of novel drugs for the treatment of TB. Previous studies have shown that Mtb and the related vaccine strain, M. bovis bacille Calmette-Guérin (BCG), disrupt the normal function of host Rab5 and Rab7, two small GTPases that are instrumental in the control of phagosome fusion with early endosomes and late endosomes/lysosomes respectively.

Methodology/Principal Findings

Here we show that recombinant Mtb nucleoside diphosphate kinase (Ndk) exhibits GTPase activating protein (GAP) activity towards Rab5 and Rab7. Then, using a model of latex bead phagosomes, we demonstrated that Ndk inhibits phagosome maturation and fusion with lysosomes in murine RAW 264.7 macrophages. Maturation arrest of phagosomes containing Ndk-beads was associated with the inactivation of both Rab5 and Rab7 as evidenced by the lack of recruitment of their respective effectors EEA1 (early endosome antigen 1) and RILP (Rab7-interacting lysosomal protein). Consistent with these findings, macrophage infection with an Ndk knocked-down BCG strain resulted in increased fusion of its phagosome with lysosomes along with decreased survival of the mutant.

Conclusion

Our findings provide evidence in support of the hypothesis that mycobacterial Ndk is a putative virulence factor that inhibits phagosome maturation and promotes survival of mycobacteria within the macrophage.     

The Binding Mode of Human Nucleoside Diphosphate Kinase B to Single-Strand DNA

In this paper, we studied the interaction of the human isoform B of nucleoside diphosphatekinase (NDP kinase B) with the nuclease hypersensitive element (NHE) present in the promoterelement of the c-myc oncogene. The DNA-binding properties of NDP kinase B and otherNDP kinases are compared and the nucleotide requirement for binding are discussed. Usingquantitative methods, we identified the DNA-binding sites on the protein and we proposed astructural model for a complex of one hexameric NDP kinase B with an oligonucleotide.     

Nucleoside Diphosphate Kinase Is a Possible Component of the Ethylene Signal Transduction Pathway

In etiolated seedlings of Pisum sativum and leaves of Arabidopsis thaliana, in vivo ethylene treatment resulted in an increase in in vitro phosphorylation of 17 kD (P. sativum) or 16 and 17 kD (A. thaliana) polypeptides. These polypeptides were identified as nucleoside diphosphate kinase (NDPK) based on both biochemical properties and interaction with antibodies against NDPK from P. sativum. Using the receptor-directed antagonist of ethylene action 2,5-norbornadiene and the ethylene-insensitive mutants of A. thaliana etr1-1 and eti5, ethylene specificity and receptor dependence of NDPK phosphorylation have been demonstrated. In pea epicotyls, ethylene treatment also led to increase in nucleoside transferase activity unlike in A. thaliana leaves. The increases in nucleoside transferase activity and NDPK phosphorylation were very rapid and transient. The results suggest a role for NDPK as a possible component of the ethylene signal transduction chain.     

Intersubunit Ionic Interactions Stabilize the Nucleoside Diphosphate Kinase of Mycobacterium tuberculosis

Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg⁸⁰-Asp⁹³ as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved.     

Subcellular Localization and Characterization of Nucleoside Diphosphate Kinase in Rat Retina: Effect of Diabetes

Nucleoside diphosphate kinase (NDP kinase) catalyzes the transfer of terminal phosphate from nucleotide triphosphates (e.g. ATP) to nucleotide diphosphates (e.g. GDP) to yield nucleotide triphosphates (e.g. GTP). Since guanine nucleotides play critical role(s) in GTP-binding protein (G-protein)-mediated signal transduction mechanisms in retina, we quantitated NDP kinase activity in subcellular fraction-derived from normal rat retina. A greater than 85% of the total specific activity was present in the soluble fraction, which was stimulated (up to 7 fold) by 2 mM magnesium. NDP kinase exhibited saturation kinetics towards di- and tri-phosphate substrates, and was inhibited by known inhibitors of NDP kinase, uridine diphosphate (UDP) or cromoglycate (CRG). We have previously reported significant abnormalities in the activation of G-proteins in streptozotocin (STZ)-diabetic rat retina (Kowluru et al. Diabetologia 35:624–631, 1992). Since NDP kinase hasbeen implicated in direct interaction with and/or activation of various G-proteins, we quantitated both basal and magnesium-stimulated NDP kinase activity in soluble and particulate fractions of retina derived from STZ-diabetic rats to examine whether abnormalities in G-protein function in diabetes are attributable to alterations in retinal NDP kinase. There was no effect of diabetes either on the basal or the magnesium-activated retinal NDP kinase activity. This study represents the first characterization of NDP kinase activity in rat retina, and suggests that in diabetes, this enzyme may not be rate-limiting and/or causal for the observed alterations in retinal G-protein functions.     

Functional Coupling between Nucleoside Diphosphate Kinase of the Outer Mitochondrial Compartment and Oxidative Phosphorylation

In rat liver mitochondria all nucleoside diphosphate kinase of the outer compartment is associated with the outer surface of the outer membrane (Lipskaya, T. Yu., and Plakida, K. N. (2003) Biochemistry (Moscow), 68, 1136-1144). In the present study, three systems operating as ADP donors for oxidative phosphorylation have been investigated. The outer membrane bound nucleoside diphosphate kinase was the first system tested. Two others employed yeast hexokinase and yeast nucleoside diphosphate kinase. The two enzymes exhibited the same activity but could not bind to mitochondrial membranes. In all three systems, muscle creatine phosphokinase was the external agent competing with the oxidative phosphorylation system for ADP. Determination of mitochondrial respiration rate in the presence of increasing quantities of creatine phosphokinase revealed that at large excess of creatine phosphokinase activity over other kinase activities (of the three systems tested) and oxidative phosphorylation the creatine phosphokinase reaction reached a quasi-equilibrium state. Under these conditions equilibrium concentrations of all creatine phosphokinase substrates were determined and K(eq)app of this reaction was calculated for the system with yeast hexokinase. In samples containing active mitochondrial nucleoside diphosphate kinase the concentrations of ATP, creatine, and phosphocreatine were determined and the quasi-equilibrium concentration of ADP was calculated using the K(eq)app value. At balance of quasi-equilibrium concentrations of ADP and ATP/ADP ratio the mitochondrial respiration rate in the system containing nucleoside diphosphate kinase was 21% of the respiration rate assayed in the absence of creatine phosphokinase; in the system containing yeast hexokinase this parameter was only 7% of the respiration rate assayed in the absence of creatine phosphokinase. Substitution of mitochondrial nucleoside diphosphate kinase with yeast nucleoside diphosphate kinase abolished this difference. It is concluded that oxidative phosphorylation is accompanied by appearance of functional coupling between mitochondrial nucleoside diphosphate kinase and the oxidative phosphorylation system. Possible mechanisms of this coupling are discussed.