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.     

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.     

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.     

Characterization and Detection of Endolysin Gene from Three Acinetobacter baumannii Bacteriophages Isolated from Sewage Water

Acinetobacter baumannii is an opportunistic pathogen that exists in hospital environments. The emergence of multidrug resistant A. baumannii (MDRAB) has been reported worldwide. It is necessary to find a novel and effective treatment for MDRAB infection. In this study, three bacteriophages, designated as ØABP-01, ØABP-02 and ØABP-04 were selected for analysis. Transmission electron microscopy showed that bacteriophage ØABP-01 belonged to the Podoviridae family and bacteriophage ØABP-02 and ØABP-04 are classified into the family Myoviridae. ØABP-01 had the widest host range. ØABP-01, ØABP-02 and ØABP-04 exhibited a latent period of 15, 20 and 20 min. The burst sizes of the three bacteriophages were 110, 120 and 150 PFU/cell. DNA restriction analysis using EcoRI, HindIII, PstI, SphI, BamHI and SmaI showed different DNA fragment patterns between the three bacteriophages. ØABP-01 and ØABP-04 was positive for the endolysin gene as determined by PCR. In conclusion, bacteriophage ØABP-01 showed broad host-specificity, good lytic activity and a short latency period, making it an appropriate candidate for studying the control and diagnosis associated with MDRAB infections.     

In Rat Liver Mitochondria All Nucleoside Diphosphate Kinase of the Outer Compartment Is Associated with the Outer Surface of the Outer Membrane

Data on localization of nucleoside diphosphate kinase (NDPK) in the outer mitochondrial compartment are contradictory. We have demonstrated that repeated quintuple wash of a mitochondrial pellet (protein concentration is about 2 mg/ml) solubilized only 60% of total NDPK activity. Since no release of adenylate kinase, the marker enzyme of the intermembrane space, was observed, it was concluded that the solubilized NDPK activity was associated with the outer surface of the outer mitochondrial membrane. Treatment of mitochondria with digitonin solutions in low (sucrose, mannitol) or high (KCl) ionic strength media revealed that solubilization of remaining NDPK activity basically coincided with the solubilization curve of monoamine oxidase, the marker enzyme of the outer mitochondrial membrane, but differed from solubilization behavior of adenylate kinase and malate dehydrogenase. We concluded that the remaining NDPK activity was also associated with the outer mitochondrial membrane and electrostatic interactions were not essential for NDPK binding to mitochondrial membranes. Results of polarographic determination of remaining adenylate kinase and NDPK activities of mitochondria incubated in ice for different time intervals and subjected to subsequent centrifugation suggest that all NDPK activity of the outer compartment of rat liver mitochondria is associated with the outer surface of the outer mitochondrial membrane. We suggest the existence of at least three NDPK fractions. They represent 70, 15, and 15% of total NDPK activity of the outer compartment and differ by tightness of membrane binding.     

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.     

分离自老年病房的鲍曼不动杆菌耐药机制初步研究*

目的:检测老年住院患者分离的鲍曼不动杆菌的主要耐药基因,并研究不同耐药基因型与耐药表型之间的对应关系。方法:用PCR方法检测分离自老年住院患者的不同标本来源的170例非重复鲍曼不动杆菌的耐药基因。检测的耐药基因包括D类碳青霉烯酶:bla_(OXA-51),bla_(OXA-23),bla_(OXA-24),bla_(OXA-58),B类金属碳青霉烯酶:bla_(VIM),bla_(IMP),bla_(SIM),bla_(GIM),bla_(DIM),bla_(NDM-1),以及A类超广谱β-内酰胺酶:blaKPC,共计11种。根据检测结果对菌株进行基因分型,并研究不同基因型与CRAB和CSAB这两种耐药表型之间的对应关系。结果:170株鲍曼不动杆菌的固有基因bla_(OXA-51)均为阳性,此外,主要检出基因为bla_(OXA-23),共124株。另外检测出blaKPC12株,bla OXA-58 6株,bla_(NDM-1)3株,bla_(SIM)2株,bla_(OXA-24)、bla_(VIM)和bla_(DIM)各1株,IMP和GIM未检出。根据检出耐药基因的不同组合,分为bla OXA-51+bla_(OXA-23)阳性为基础的A型(124株)及bla_(OXA-23)阴性为基础的B型(bla_(OXA-51),39株)、C型(bla_(OXA-51)+bla_(OXA-58),6株)、D型(bla_(OXA-51)+bla_(OXA-24),1株)共计四类基因型。从耐药表型来看,128株碳青霉烯耐药菌中有122株bla_(OXA-23)为阳性,在CRAB中占95.3%(122/128),42株碳青霉烯敏感株中,有40株bla_(OXA-23)为阴性,在CSAB中占95.2%(40/42)。结论:老年病房流行的耐碳青霉烯鲍曼不动杆菌的耐药基因型以bla_(OXA-23)阳性为主。其与鲍曼不动杆菌CRAB耐药表型、bla_(OXA-23)阴性与CSAB耐药表型之间有良好的对应关系。     

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.     

地黄核苷二磷酸激酶基因的克隆及生物信息学分析

核苷二磷酸激酶(NDPK)是一种高度保守的多功能蛋白,具有催化底物磷酸化的作用,能够参与植物的生长发育、非生物胁迫、感病应激、光合作用和能量代谢等过程。为了解地黄核苷二磷酸激酶基因(RgNDPKⅠ)的结构、功能和性质,该研究利用地黄转录组学数据,通过电子克隆的方法获得了RgNDPKⅠ基因的全长cDNA序列,长度为765 bp。生物信息学分析结果表明,RgNDPKⅠ基因的开放阅读框长度为447 bp,编码148个氨基酸,具有典型的核苷二磷酸激酶活性结构域和其他磷酸化活性位点。RgNDPKⅠ基因编码的蛋白质定位于细胞质,是无跨膜区域的亲水性蛋白,该蛋白质与芝麻、紫花风铃的核苷二磷酸激酶相似性较高,分别为97%和96%。在多种生物中已经克隆得到了核苷二磷酸激酶基因,且不同植物核苷二磷酸激酶氨基酸序列中存在多个相似的保守结构域,推测RgNDPKⅠ基因所编码的蛋白质为核苷二磷酸激酶超家族成员。该研究结果为进一步探明RgNDPKⅠ的性质、结构、功能及表达机制提供了重要的理论依据。     

Structural and serological characterisation of the O-antigenic polysaccharide of the lipopolysaccharide from Acinetobacter baumannii strain 24

Extraction of dry bacteria of Acinetobacter baumannii strain 24 by phenol-water yielded a lipopolysaccharide (LPS) that was studied by serological methods and fatty acid analysis. After immunisation of BALB/c mice with this strain, monoclonal antibody S48-3-13 (IgG(3) isotype) was obtained, which reacted with the LPS in western blot and characterized it as S-form LPS. Degradation of the LPS in aqueous 1% acetic acid followed by GPC gave the O-antigenic polysaccharide, whose structure was determined by compositional analyses and NMR spectroscopy of the polysaccharide and O-deacylated polysaccharide as [carbohydrate structure: see text] where QuiN4N is 2,4-diamino-2,4,6-trideoxyglucose and GalNAcA 2-acetamido-2-deoxygalacturonic acid. The amino group at C-4 of the QuipN4N residues is acetylated in about 2/3 of LPS molecules and (S)-3-hydroxybutyrylated in the rest.     

Comparative genomic analysis of Acinetobacter baumannii clinical isolates reveals extensive genomic variation and diverse antibiotic resistance determinants

Background

Acinetobacter baumannii is an important nosocomial pathogen that poses a serious health threat to immune-compromised patients. Due to its rapid ability to develop multidrug resistance (MDR), A. baumannii has increasingly become a focus of attention worldwide. To better understand the genetic variation and antibiotic resistance mechanisms of this bacterium at the genomic level, we reported high-quality draft genome sequences of 8 clinical isolates with various sequence types and drug susceptibility profiles.

Results

We sequenced 7 MDR and 1 drug-sensitive clinical A. baumannii isolates and performed comparative genomic analysis of these draft genomes with 16 A. baumannii complete genomes from GenBank. We found a high degree of variation in A. baumannii, including single nucleotide polymorphisms (SNPs) and large DNA fragment variations in the AbaR-like resistance island (RI) regions, the prophage and the type VI secretion system (T6SS). In addition, we found several new AbaR-like RI regions with highly variable structures in our MDR strains. Interestingly, we found a novel genomic island (designated as GI_BJ4) in the drug-sensitive strain BJ4 carrying metal resistance genes instead of antibiotic resistance genes inserted into the position where AbaR-like RIs commonly reside in other A. baumannii strains. Furthermore, we showed that diverse antibiotic resistance determinants are present outside the RIs in A. baumannii, including antibiotic resistance-gene bearing integrons, the bla_OXA-23-containing transposon Tn2009, and chromosomal intrinsic antibiotic resistance genes.

Conclusions

Our comparative genomic analysis revealed that extensive genomic variation exists in the A. baumannii genome. Transposons, genomic islands and point mutations are the main contributors to the plasticity of the A. baumannii genome and play critical roles in facilitating the development of antibiotic resistance in the clinical isolates.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1163) contains supplementary material, which is available to authorized users.     

Cloning, expression, and enzymatic activity of Acinetobacter baumannii and Klebsiella pneumoniae acetyl-coenzyme A carboxylases

Pathogenic Gram-negative bacteria are a major public health concern because they are causative agents of life-threatening hospital-acquired infections. Due to the increasing rates of resistance to available antibiotics, there is an urgent need to develop new drugs. Acetyl-coenzyme A carboxylase (ACCase) is a promising target for the development of novel antibiotics. We describe here the expression, purification, and enzymatic activity of recombinant ACCases from two clinically relevant Gram-negative pathogens, Acinetobacter baumannii and Klebsiella pneumoniae. Recombinant ACCase subunits (AccAD, AccB, and AccC) were expressed and purified, and the holoenzymes were reconstituted. ACCase enzyme activity was monitored by direct detection of malonyl-coenzyme A (malonyl-CoA) formation by liquid chromatography tandem mass spectrometry (LC–MS/MS). Steady-state kinetics experiments showed similar k_cat and K_M values for both enzymes. In addition, similar IC₅₀ values were observed for inhibition of both enzymes by a previously reported ACCase inhibitor. To provide a higher throughput assay suitable for inhibitor screening, we developed and validated a luminescence-based ACCase assay that monitors ATP depletion. Finally, we established an enzyme activity assay for the isolated AccAD (carboxyltransferase) subunit, which is useful for determining whether novel ACCase inhibitors inhibit the biotin carboxylase or carboxyltransferase site of ACCase. The methods described here could be applied toward the identification and characterization of novel inhibitors.     

Nucleoside diphosphate kinase from pea chloroplasts: purification,cDNA cloning and import into chloroplasts

Nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) was enriched 1900-fold from purified pea (Pisum sativum L. cv. Golf.) chloroplasts. The active enzyme preparation contained two polypeptides of apparent molecular weight 18.5 kDa and 17.4kDa. Both proteins were enzymatically active and were recognized by an antiserum raised against NDPK from spinach chloroplasts, suggesting the existence of two isoforms in pea chloroplasts. The N-terminal protein sequence data were obtained for both polypeptides and compared with the nucleotide sequence of a cDNA clone isolated from a pea cDNA library. The analysis revealed that the two NDPK forms are encoded for by one mRNA, indicating that the lower-molecular-weight form could represent a proteolytic breakdown product of the 18.5-kDa NDPK. The pea chloroplastic NDPK is made as a larger precursor protein which is imported into chloroplasts. The NDPK precursor is then processed by the stromal processing peptidase to yield the 18.5-kDa form.Abbreviations NDPK nucleoside diphosphate kinase - preNDPK precursor NDPK - ps-NDPK cDNA coding for Pisum sativum NDPK II We thank Dr. Schmidt, University Göttingen, Germany, for doing the protein sequencing. This work was supported in part by grants from the Deutsche Forschungsgemeinschaft.     

Neospora caninum:Tachyzoites Express a Potent Type-I Nucleoside Triphosphate Hydrolase,but Lack Nucleoside Diphosphate Hydrolase Activity

Asai, T., Howe, D. K., Nakajima, K., Nozaki, T., Takeuchi, T., and Sibley, L. D.Neospora caninum: Tachyzoites Express Type-I Nucleoside Triphosphate Hydrolase1. But Lack Nucleoside Diphosphate Hydrolase Activity.Experimental Parasitology90,277–285. We have identified type I nucleoside triphosphate hydrolase (NTPase; EC 3.6.1.3) activity, previously thought to be restricted to the virulent strains ofToxoplasma gondii, in the cell extracts ofNeospora caninumtachyzoites. Sequence analysis of a complete cDNA from Nc-1 strain indicated thatN. caninumNTPases shared approximately 69% identity to the NTPases ofT. gondiiand are most similar to the NTPase-I isozyme. Southern blot analysis of genomic DNA and sequence analysis of two independentNTPclones from the Nc-1 strain revealed the presence of multiple genes, at least two of which are transcribed. Substrate specificity andK_mvalues for MgATP²⁻and MgADP⁻hydrolysis for recombinant or partially purified native NcNTPase were the same as those for the type I isozyme (NTPase-I). Significantly, no type II enzyme (NTPase-II) activity for NDP hydrolysis was detected in cell extracts ofN. caninum, although it is universally present in allT. gondiistrains that have been tested. This intriguing difference between these two closely related apicomplexan parasites may provide insight into the function of the NTPases during intracellular parasitism.     

Structural analysis of Arabidopsis thaliana nucleoside diphosphate kinase-2 for phytochrome-mediated light signaling

In plants, nucleoside diphosphate kinases (NDPKs) play a key role in the signaling of both stress and light. However, little is known about the structural elements involved in their function. Of the three NDPKs (NDPK1-NDPK3) expressed in Arabidopsis thaliana, NDPK2 is involved in phytochrome-mediated signal transduction. In this study, we found that the binding of dNDP or NTP to NDPK2 strengthens the interaction significantly between activated phytochrome and NDPK2. To better understand the structural basis of the phytochrome-NDPK2 interaction, we determined the X-ray structures of NDPK1, NDPK2, and dGTP-bound NDPK2 from A.thaliana at 1.8A, 2.6A, and 2.4A, respectively. The structures showed that nucleotide binding caused a slight conformational change in NDPK2 that was confined to helices alphaA and alpha2. This suggests that the presence of nucleotide in the active site and/or the evoked conformational change contributes to the recognition of NDPK2 by activated phytochrome. In vitro binding assays showed that only NDPK2 interacted specifically with the phytochrome and the C-terminal regulatory domain of phytochrome is involved in the interaction. A domain swap experiment between NDPK1 and NDPK2 showed that the variable C-terminal region of NDPK2 is important for the activation by phytochrome. The structure of Arabidopsis NDPK1 and NDPK2 showed that the isoforms share common electrostatic surfaces at the nucleotide-binding site, but the variable C-terminal regions have distinct electrostatic charge distributions. These findings suggest that the binding of nucleotide to NDPK2 plays a regulatory role in phytochrome signaling and that the C-terminal extension of NDPK2 provides a potential binding surface for the specific interaction with phytochromes.     

Development of a pyrosequencing assay for rapid assessment of quinolone resistance in Acinetobacter baumannii isolates

Rapid and reliable assessment of Acinetobacter baumannii resistance to quinolones was successfully achieved through pyrosequencing of the gyrA and parC quinolone-resistance determining regions. A strong correlation was found between quinolone resistance and mutations in gyrA codon 83 and/or in the parC gene (codons 80 or 84). Absence of QRDR mutations was associated with susceptibility to quinolones.     

Nucleoside diphosphate kinase and GTP-binding proteins. Possible mechanisms of coupling

The results of numerous investigations during the last 20 years show that nucleoside diphosphate kinase (NDP kinase) is a multifunctional protein. In this paper, the current data are analyzed indicating that one of the possible mechanisms by which NDP kinase manifests its multifunctional role is its participation in the activation (or regulation) of heterotrimeric GTP-binding proteins (G proteins). We demonstrate that one of the NDP kinase isoforms dynamically interacts with the retinal rod G protein transducin (Gt) and phosphorylates its β-subunit at histidine residue (His 266). It is also shown that it leads to the consecutive transfer of the phosphate group to GDP in the active center of G protein α-subunit and G protein activation. The advantages of this mechanism are considered as compared to the classic G protein activation mechanism, GDP/GTP exchange.