Use of adenylate kinase as a solubility tag for high level expression of T4 DNA ligase in Escherichia coli.

The discovery of T4 DNA ligase in 1960s was pivotal in the spread of molecular biotechnology. The enzyme has become ubiquitous for recombinant DNA routinely practiced in biomedical research around the globe. Great efforts have been made to express and purify T4 DNA ligase to meet the world demand, yet over-expression of soluble T4 DNA ligase in E. coli has been difficult. Here we explore the use of adenylate kinase to enhance T4 DNA ligase expression and its downstream purification. E.coli adenylate kinase, which can be expressed in active form at high level, was fused to the N-terminus of T4 DNA ligase. The resulting His-tagged AK-T4 DNA ligase fusion protein was greatly over-expressed in E. coli, and readily purified to near homogeneity via two purification steps consisting of Blue Sepharose and Ni-NTA chromatography. The purified AK-T4 DNA ligase not only is fully active for DNA ligation, but also can use ADP in addition to ATP as energy source since adenylate kinase converts ADP to ATP and AMP. Thus adenylate kinase may be used as a solubility tag to facilitate recombinant protein expression as well as their downstream purification.

Structural modification of proteins by direct electric current from low voltage.

The interaction of direct electric current (dc) and proteins is a little explored topic. We had reported that exposure of Crotalus atrox venom to dc caused irreversible inactivation of phospholipase A(2) and metalloprotease and that the eukaryote adenylate kinases (AK) precipitate in nondenaturing gel electrophoresis. AK1 displays an elevated percent difference of CHarged versus POlar amino acid content (CH-PO 14). Commercial AK1 and other 17 enzymes with various CH-PO values were exposed in solution to dc (0-0.7 mA) from low voltage (0-10 V), then enzymatic activity was assayed. The enzymes with CH-PO higher than 10.0 were irreversibly inactivated by current exposure; those with CH-PO between +3 and -5 were not. Inactivation was dependent on the ionic strength of the medium and not on the net charge of the protein. Circular dichroic spectroscopy showed a structural modification in some of the inactivated enzymes. CH-PO could be a crucial, although rough, parameter for predicting protein inactivation by low-voltage exposure. The observed effect seems due to the current density. Enzymatic activity maybe a more accurate sensor of conformational changes than circular dichroism spectroscopy. A better understanding of efficacy of many electrical devices utilized in medical practice may follow.

Cobalt-, zinc- and iron-bound forms of adenylate kinase (AK) from the sulfate-reducing bacterium Desulfovibrio gigas: purification, crystallization and preliminary X-ray diffraction analysis.

Adenylate kinase (AK; ATP:AMP phosphotransferase; EC 2.7.4.3) is involved in the reversible transfer of the terminal phosphate group from ATP to AMP. AKs contribute to the maintenance of a constant level of cellular adenine nucleotides, which is necessary for the energetic metabolism of the cell. Three metal ions, cobalt, zinc and iron(II), have been reported to be present in AKs from some Gram-negative bacteria. Native zinc-containing AK from Desulfovibrio gigas was purified to homogeneity and crystallized. The crystals diffracted to beyond 1.8 A resolution. Furthermore, cobalt- and iron-containing crystal forms of recombinant AK were also obtained and diffracted to 2.0 and 3.0 A resolution, respectively. Zn(2+)-AK and Fe(2+)-AK crystallized in space group I222 with similar unit-cell parameters, whereas Co(2+)-AK crystallized in space group C2; a monomer was present in the asymmetric unit for both the Zn(2+)-AK and Fe(2+)-AK forms and a dimer was present for the Co(2+)-AK form. The structures of the three metal-bound forms of AK will provide new insights into the role and selectivity of the metal in these enzymes.

ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli.

Except for apyrases, ATPases generally target only the gamma-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1/10,000 of the level of the ATPase. AK catalyzes conversion of two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP/ADP-dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP-dependent activities of ADP-dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide-free and binds to both Q and S ion exchange matrixes.

Acyclic phosphonate nucleotides and human adenylate kinases: impact of a borano group on alpha-P position.

Adenylate kinases are involved in the activation of antiviral drugs such as the acyclic phosphonates analogs PMEA and (R)PMPA. We examine the in vitro phosphorylation of PMEA and PMPA bearing a borano- or a H- group on the phosphorus atom. The alpha-borano or alpha-H on PMEA and PMPA were detrimental to the activity of recombinant human AMP kinases 1 and 2. Docking PMEA to the active site of AMP kinase 1 indicated that the borano group may prevent two conserved critical Arg interactions with the alpha-phosphate, resulting in substrate bad positioning.

A new type of metal-binding site in cobalt- and zinc-containing adenylate kinases isolated from sulfate-reducers Desulfovibrio gigas and Desulfovibrio desulfuricans ATCC 27774.

Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the "LID" domain. The sequence 129Cys-X5-His-X15-Cys-X2-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.

Adenylate kinase-independent thiamine triphosphate accumulation under severe energy stress in Escherichia coli.

BACKGROUND: Thiamine triphosphate (ThTP) exists in most organisms and might play a role in cellular stress responses. In E. coli, ThTP is accumulated in response to amino acid starvation but the mechanism of its synthesis is still a matter of controversy. It has been suggested that ThTP is synthesized by an ATP-dependent specific thiamine diphosphate kinase. However, it is also known that vertebrate adenylate kinase 1 catalyzes ThTP synthesis at a very low rate and it has been postulated that this enzyme is responsible for ThTP synthesis in vivo. RESULTS: Here we show that bacterial, as vertebrate adenylate kinases are able to catalyze ThTP synthesis, but at a rate more than 106-fold lower than ATP synthesis. This activity is too low to explain the high rate of ThTP accumulation observed in E. coli during amino acid starvation. Moreover, bacteria from the heat-sensitive CV2 strain accumulate high amounts of ThTP (>50% of total thiamine) at 37 degrees C despite complete inactivation of adenylate kinase and a subsequent drop in cellular ATP. CONCLUSION: These results clearly demonstrate that adenylate kinase is not responsible for ThTP synthesis in vivo. Furthermore, they show that E. coli accumulate large amounts of ThTP under severe energy stress when ATP levels are very low, an observation not in favor of an ATP-dependent mechanisms for ThTP synthesis.

Partial characterization of the proteome of the mouse striatum.

Many diseases of the mammalian CNS, including Parkinson's (PD) and Lesch Nyhan disease (LND), are associated with programmatic neurodegeneration or dysfunction of dopaminergic neurons in the mesencephalon, the nigrostriatal pathway, and its projections in the striatum [1-4]. Proteomic studies on brain tissue of both animal models and human PD patients have provided evidence for dysfunction and damage of many pathways, including oxidative stress-related damage, ubiquitin-proteasome dysfunction, mitochondrial energy metabolism deficiencies, and synaptic function [5-11]. To date no such proteomic studies have been reported in the related and rare basal ganglia disorder LND, a developmental rather than a neurodegenerative neurological disorder caused by deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) that regulates a major step in the purine salvage pathway [12]. Many studies have demonstrated that the both human LND patients and a mouse knockout model of HPRT deficiency have significantly reduced levels and uptake of dopamine in the striatum [4, 13-16] that is likely to be the principal cause of the CNS disorder. The precise molecular and cellular mechanisms that underlie this neurotransmitter defect are unknown.

Expression of adenylate kinase 1 in bovine retinal cytosol.

Adenylate kinases (AKs) are ubiquitous phosphotransferases that contribute to homeostasis of adenine nucleotide composition in cells. Six AK isoforms were found in vertebrates. We report that soluble AK isoform 1 is expressed in the cytosol of bovine retina consistently devoid of rod outer segments. Immunoblotting analysis with a polyclonal antibody raised against soluble adenylate kinase and subsequent sequencing of eluted peptide by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry allowed enzyme isolation by joining purification methods to two-dimensional electrophoresis. In this study, we found that cytosolic adenylate kinase isoform 1 is expressed in bovine retina. Cytoplasmic AK1 would physiologically contribute to retinal energy metabolism.

A novel nuclear-localized protein with special adenylate kinase properties from Caenorhabditis elegans.

The adrenal gland protein AD-004 like protein (ADLP) from Caenorhabditis elegans was cloned and expressed in Escherichia coli. Enzyme assays showed that ADLP has special adenylate kinase (AK) properties, with ATP and dATP as the preferred phosphate donors. In contrast to all other AK isoforms, AMP and dAMP were the preferred substrates of ADLP; CMP, TMP and shikimate acid were also good substrates. Subcellular localization studies showed a predominant nuclear localization for this protein, which is different from AK1-AK5, but similar to that of human AK6. These results suggest that ADLP is more likely a member of the AK6 family. Furthermore, RNAi experiments targeting ADLP were conducted and showed that RNAi treatment resulted in the suppression of worm growth.

An expanded adenylate kinase gene family in the protozoan parasite Trypanosoma cruzi.

Adenylate kinases supply energy routes in cellular energetic homeostasis. In this work, we identified and characterized the adenylate kinase activity in extracts from the flagellated parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Adenylate kinase activity was detected in different subcellular fractions and the cytosolic isoform was biochemically characterized. Cytosolic adenylate kinase specific activity increases continuously during the epimastigote growth and is down-regulated when other soluble phosphotransferase, arginine kinase, is overexpressed. Six different genes of adenylate kinase isoforms were identified and the mRNA expression was confirmed by RT-PCR and Northern Blot. Three open reading frames coding for different enzyme isoforms named TzADK1, TzADK2 and TzADK5 were cloned and functionally expressed in E. coli. This work reports an unusually large number of genes of adenylate kinases and suggests a coordinated regulation of phosphotransferase-mediated ATP regenerating pathways in the unicellular parasite Trypanosoma cruzi.

Adenylate kinase of Escherichia coli, a component of the phage T4 dNTP synthetase complex.

Adenylate kinase, which catalyzes the reversible ATP-dependent phosphorylation of AMP to ADP and dAMP to dADP, can also catalyze the conversion of nucleoside diphosphates to the corresponding triphosphates. Lu and Inouye (Lu, Q., and Inouye, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5720-5725) showed that an Escherichia coli ndk mutant, lacking nucleoside diphosphate kinase, can use adenylate kinase as an alternative source of nucleoside triphosphates. Bacteriophage T4 can reproduce in an Escherichia coli ndk mutant, implying that adenylate kinase can meet a demand for deoxyribonucleoside triphosphates that increases by up to 10-fold as a result of T4 infection. In terms of kinetic linkage and specific protein-protein associations, NDP kinase is an integral component of T4 dNTP synthetase, a multienzyme complex containing phage-coded enzymes, which facilitates the synthesis of dNTPs and their flow into DNA. Here we asked whether, by similar criteria, adenylate kinase of the host cell is also a specific component of the complex. Experiments involving protein affinity chromatography, immunoprecipitation, optical biosensor measurements, and glutathione S-transferase pulldowns demonstrated direct interactions between adenylate kinase and several phage-coded enzymes, as well as E. coli nucleoside diphosphate kinase. These results identify adenylate kinase as a specific component of the complex. The rate of DNA synthesis after infection of an ndk mutant was found to be about 40% of the rate seen in wild-type infection, implying that complementation of the missing NDP kinase function by adenylate kinase is fairly efficient, but that adenylate kinase becomes rate-limiting for DNA synthesis when it is the sole source of dNTPs.

Structural stability of adenylate kinase from the sulfate-reducing bacteria Desulfovibrio gigas.

A novel adenylate kinase (AK) has recently been purified from Desulfovibrio gigas and characterized as a Co(2+)/Zn(2+)-containing enzyme: this is an unusual characteristic for AKs from Gram-negative bacteria, in which these enzymes are normally devoid of metals. Here, we studied the conformational stability of holo- and apo-AK as a function of temperature by differential scanning calorimetry (DSC), circular dichroism (CD), and intrinsic fluorescence spectroscopy. The thermal unfolding of AK is a cooperative two-state process, and is sufficiently reversible in the 9-11 pH range, that can be correctly interpreted in terms of a simple two-state thermodynamic model. The spectral parameters as monitored by ellipticity changes in the CD spectra of the enzyme as well as the decrease in tryptophan intensity emission upon heating were seen to be good complements to the highly sensitive but integral DSC-method.

Molecular and functional characterization of adenylate kinase 2 gene from Leishmania donovani.

ATP-regenerating enzymes may have an important role in maintaining ATP levels in mitochondria-like kinetoplast organelle and glycosomes in parasitic protozoa. Adenylate kinase (AK) (ATP:AMP phosphotransferase) catalyses the reversible transfer of the gamma-phosphate group from ATP to AMP, releasing two molecules of ADP. This study describes cloning and functional characterization of the gene encoding AK2 from a genomic library of Leishmania donovani and also its expression in leishmania promastigote cultures. AK2 was localized on an approximately 1.9-Mb chromosomal band as a single copy gene. L. donovani AK2 gene is expressed as a single 1.9-kb mRNA transcript that is developmentally regulated and accumulated during the early log phase. The overexpression of L. donovani AKgene in Escherichia coli yielded a 26-kDa polypeptide that could be refolded to a functional protein with AK activity. The recombinant protein was purified to apparent homogeneity. Kinetic analysis of purified L. donovani AK showed hyperbolic behaviour for both ATP and AMP, with Km values of 104 and 74 microM, respectively. The maximum enzyme activity (Vmax) was 0.18 micromol.min(-1).mg(-1) protein. P1,P5-(bis adenosine)-5'-pentaphosphate (Ap5A), the specific inhibitor of AK, competitively inhibited activity of the recombinant enzymes with estimated Ki values of 190 nM and 160 nM for ATP and AMP, respectively. Ap5A also inhibited the growth of L. donovani promastigotes in vitro which could be only partially reversed by the addition of ADP. Thus, presence of a highly regulated AK2, which may have role in maintenance of ADP/ATP levels in L. donovani, has been demonstrated.

Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli.

The 5'-AMP-activated protein kinase (AMPK) plays a critical role in the regulation of cellular energy homeostasis. AMPK is a heterotrimer composed of a catalytic subunit (alpha) and two regulatory subunits (beta and gamma). To date, purified AMPK has only been obtained in small, microgram quantities from tissues. Here, we describe an expression and purification system for production of functional AMPK in Escherichia coli. A plasmid carrying all three subunits of AMPK (alpha1, beta1, and gamma1) for T7 RNA polymerase-driven transcription of a single tricistronic messenger was constructed, allowing spontaneous formation of the heterotrimeric complex in the bacterial cytosol. AMPK was purified from the bacterial lysates by single-step nickel-ion chromatography, utilizing a poly-histidine tag fused to the N-terminus of the alpha-subunit. The recombinant AMPK complex was monodisperse, as shown by gel filtration chromatography with elution of a single peak at a Stokes radius of 52A. Bacterially expressed AMPK was entirely inactive, yet it could be activated by upstream kinase in the presence of AMP. Sufficient quantities of purified functional AMPK should prove to be an invaluable tool to solve many of the pertinent questions about its molecular structure and function, in particular facilitating protein crystallization for X-ray structure analysis.

Nucleoside diphosphate kinase-like activity in adenylate kinase of Mycobacterium tuberculosis.

Ak (adenylate kinase) is a ubiquitous enzyme that catalyses a reversible high-energy phosphoryl-transfer reaction between ATP and AMP to form ADP. In the present study, the Ak gene (adk) of Mycobacterium tuberculosis was cloned, expressed in Escherichia coli and purified as a glutathione S-transferase fusion protein. Purified Ak converted AMP into ADP in the presence of [gamma-32P]ATP or [gamma-32P]GTP. Replacement of arginine-88 of adk with glycine resulted in the loss of enzymic activity. The purified protein also showed Ndk (nucleoside diphosphate kinase)-like activity as it transferred terminal phosphate from [gamma-32P]ATP to all nucleoside diphosphates, converting them into corresponding triphosphates. However, Ndk-like activity of Ak was not observed with [gamma-32P]GTP. Immunoblot analysis of various cellular fractions of M. tuberculosis H37Rv revealed that Ak is a cytoplasmic protein. The dual activity of Ak as both nucleoside mono- and di-phosphate kinases suggested that this enzyme may have a role in RNA and DNA biosynthesis in addition to its role in intracellular nucleotide metabolism.

Novel trimeric adenylate kinase from an extremely thermoacidophilic archaeon, Sulfolobus solfataricus: molecular cloning, nucleotide sequencing, expression in Escherichia coli, and characterization of the recombinant enzyme.

A gene coding for adenylate kinase was cloned from an extremely thermoacidophilic archaeon Sulfolobus solfataricus. The open reading frame of the sequenced gene consisted of 585 nucleotides coding for a polypeptide of 195 amino acid residues with a calculated molecular weight of 21,325. Although the S. solfataricus adenylate kinase, which belonged to the small variants of the adenylate kinase family, had low sequence identities with bacterial and eukaryotic enzymes, a functionally important glycine-rich region and also two invariant arginine residues were conserved in the sequence of the S. solfataricus enzyme. The recombinant enzyme, overexpressed in Escherichia coli and purified to homogeneity, had high affinity for AMP and high thermal stability, comparable to the extremely thermostable enzyme from a similar archaeon, S. acidocaldarius. Furthermore, gel filtration and sedimentation analyses showed that the S. solfataricus adenylate kinase was a homotrimer in solution, which is a novel subunit structure for nucleoside monophosphate kinases.

[Identification and cloning of adenylate kinase gene, a novel gene of Schistosoma japonicum].

OBJECTIVE: To subclone a novel gene of Schistosoma japonicum (Sj), adenylate kinase (AK) cDNA, which was identified through expressed sequence tag (EST) strategy and homology search, so as to prepare for further functional study of this gene. METHOD: The inserted cDNA fragment was sequenced and searched with BLASTn program. Two PCR primers were designed according to the sequence of this Sj AK cDNA and the cloning sites in pET32a (+) plasmid, with the product purified before linkage with pMD 18-T vector. The recombinant T-vector was digested with EcoRI /XhoI to obtain Sj AK cDNA, which was then introduced into the expression plasmid pET32a (+). RESULTS: The novel gene possessed 86% homology with Sm AK cDNA, and the PCR product is of expected length. Double digestion with EcoR I and Xho I proved that the recombinant T-vector and the expression plasmid had the insert with length identical to that of the target fragment. CONCLUSION: The novel cDNA codes for adenylate kinase of Schistosoma japonicum, and the recombinant expression plasmid pET32a (+)-Sj AK have been successfully constructed.

Adenylate kinase as a virulence factor of Pseudomonas aeruginosa.

Adenylate kinase (AK; ATP:AMP phosphotransferase, EC 2.7.4.3) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in eukaryotic and prokaryotic cells. AK catalyzes the reversible reaction Mg. ATP + AMP <--> Mg. ADP + ADP. In this study we show that AK secreted by the pathogenic strains of Pseudomonas aeruginosa appears to play an important role in macrophage cell death. We purified and characterized AK from the growth medium of a cystic fibrosis isolate strain of P. aeruginosa 8821 and hyperproduced it as a fusion protein with glutathione S-transferase. We demonstrated enhanced macrophage cell death in the presence of both the secreted and recombinant purified AK and its substrates AMP plus ATP or ADP. These data suggested that AK converts its substrates to a mixture of AMP, ADP, and ATP, which are potentially more cytotoxic than ATP alone. In addition, we observed increased macrophage killing in the presence of AK and ATP alone. Since the presence of ATPase activity on the macrophages was confirmed in the present work, external macrophage-effluxed ATP is converted to ADP, which in turn can be transformed by AK into a cytotoxic mixture of three adenine nucleotides. Evidence is presented in this study that secreted AK was detected in macrophages during infection with P. aeruginosa. Thus, the possible role of secreted AK as a virulence factor is in producing and keeping an intact pool of toxic mixtures of AMP, ADP, and ATP, which allows P. aeruginosa to exert its full virulence.

Enzymes of adenylate metabolism and their role in hibernation of the white-tailed prairie dog, Cynomys leucurus.

AMP deaminase (AMPD) and adenylate kinase (AK) were purified from skeletal muscle of the white-tailed prairie dog, Cynomus leucurus, and enzyme properties were assayed at temperatures characteristic of euthermia (37 degrees C) and hibernation (5 degrees C) to analyze their role in adenylate metabolism during hibernation. Total adenylates decreased in muscle of torpid individuals from 6.97 +/- 0. 31 to 4.66 +/- 0.58 micromol/g of wet weight due to a significant drop in ATP but ADP, AMP, IMP, and energy charge were unchanged. The affinity of prairie dog AMPD for AMP was not affected by temperature and did not differ from that of rabbit muscle AMPD, used for comparison. However, both prairie dog and rabbit AMPD showed much stronger inhibition by ions and GTP at 5 degrees C, versus 37 degrees C, and inhibition by inorganic phosphate, NH(4)Cl, and (NH(4))(2)SO(4) was much stronger at 5 degrees C for the prairie dog enzyme. Furthermore, ATP and ADP, which activated AMPD at 37 degrees C, were strong inhibitors of prairie dog AMPD at 5 degrees C, with I(50) values of 1 and 14 microM, respectively. ATP also inhibited rabbit AMPD at 5 degrees C (I(50) = 103 microM). Strong inhibition of AMPD at 5 degrees C by several effectors suggests that enzyme function is specifically suppressed in muscle of hibernating animals. By contrast, AK showed properties that would maintain or even enhance its function at low temperature. K(m) values for substrates (ATP, ADP, AMP) decreased with decreasing temperature, the change in K(m) ATP paralleling the decrease in muscle ATP concentration. AK inhibition by ions was also reduced at 5 degrees C. The data suggest that adenylate degradation via AMPD is blocked during hibernation but that AK maintains its function in stabilizing energy charge.