Biochemical properties of the protein tyrosine kinase of the bacterium Acinetobacter johnsonii

The biochemical properties of the autophosphorylating protein tyrosine kinase of Acinetobacter johnsonii were analyzed in vitro. The study shows that the optimal pH value for the phosphorylation reaction is approximately 7. The enzyme activity is stimulated by magnesium and, to a lesser extent, by manganese ions, whereas calcium ions have no effect. The phosphorylation process is rapid reaching a maximum in < 2 min, and the enzyme is modified at multiple sites. Interestingly, the bacterial enzyme is insensitive to a series of molecules known to affect the activity of eukaryotic protein tyrosine kinases: genistein, quercetin, tosyllysine chloromethyl ketone, and vanadate. We concluded that, even though the overall phosphorylation reaction catalyzed by the A. johnsonii enzyme is identical to that occurring in eukaryotes, this bacterial kinase exhibits a number of specific properties and therefore probably belongs to a separate group in the general family of protein tyrosine kinases.     

The 90-kDa heat shock protein (hsp-90) possesses an ATP binding site and autophosphorylating activity

The 90-kDa heat shock protein (hsp-90) is an abundant cytosolic protein believed to play a role in maintenance of protein trafficking and closely associated with several steroid hormone receptors. Incubation of highly purified hsp-90 with [gamma-32P]ATP results in its autophosphorylation on serine residues. There are several lines of evidence which suggest that this activity is due to a kinase intrinsic to hsp-90 rather than some closely associated protein kinases: 1) the phosphorylation persists after the removal of casein kinase II by heparin chromatography and after immunoprecipitation of hsp-90 with anti-hsp-90 antibodies. 2) The approximate kM for ATP of the reaction is 0.16 mM, higher than that of many other protein kinases. 3) Phosphorylation is not affected by a number of activators and inhibitors of other known kinases which might associate with hsp-90. 4) The phosphorylation displays a unique cation dependence being most active in the presence of Ca2+ and practically inactive with Mg2+, although the autophosphorylation in the presence of Mg2+ is activated by histones and polyamines. 5) The activity is remarkably heat-stable; incubation of hsp-90 for 20 min at 95 degrees C results in only a 60% decrease in autophosphorylation. 6) Finally, and most importantly, purified hsp-90 can be labeled with azido-ATP and it is able to bind to ATP-agarose. The binding shows similar cation dependence to the autophosphorylation. These data are in agreement with the presence of a consensus sequence for ATP binding sites in the primary structure of the protein similar to that observed in the 70-kDa heat-shock proteins. Our data suggest the 90-kDa heat shock protein possesses an enzymatic activity analogous in many respects to the similar activity of the 70-kDa heat shock proteins. This may represent an important, previously unrecognized function of hsp-90.     

Characterization of restriction endonuclease AjoI from Acinetobacter johnsonii

Abstract A new type II restriction endonuclease, named Ajo I, was detected in Acinetobacter johnsonii . The enzyme Ajo I, an isoschizomer of Pst I, recognized the hexanucleotide sequence [5'-CTGCA/G-3'], with a cleavage site generating fragments of DNA with protruding cohesive 3' termini.     

ATP stimulates binding of retinol-cellular retinol binding protein complex to DNA-cellulose

In calf uterus cytosol a cellular retinol-binding protein (cRBP) was detected which was found to bind to DNA-cellulose. The binding to DNA-cellulose could be enhanced by ATP in a dose-dependent manner. ATP treatment did not change the physico-chemical properties of the retinol-cRBP complex. Our findings suggest a role for ATP in the binding of retinol-cRBP complex to DNA.     

Rat adrenocortical carcinoma 494 autophosphorylating protein kinase, autophosphorylating protein kinase 500. Purification, biochemical and immunological characterization, and substrate specificity

A novel autophosphorylating protein kinase, autophosphorylating protein kinase 500, independent of cyclic AMP, cyclic GMP, calcium, and calmodulin was purified from rat adrenocortical carcinoma 494 by ammonium sulfate fractionation followed by the chromatographic steps of DEAE-cellulose, gel filtration, cyclic AMP-epoxy Sepharose, and phosphocellulose. Sometimes two additional chromatographic purification steps of chromatofocusing and gel filtration were necessary for complete purification. The enzyme was homogeneous as evidenced by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sucrose density sedimentation studies indicated that Mr of the enzyme was 490,000, while ultracentrifugal analysis demonstrated a value of 481,400 (+/-7%). The protein was composed of two identical subunits each with Mr = 250,000. The enzyme molecule was slightly asymmetric with frictional and sedimentation coefficients of 1.28 and 18.20, respectively, and a Stokes radius of 66 A. Isoelectric focusing electrophoresis revealed a single peak with pI 4.6, indicating acidity of the protein. The enzyme self phosphorylated one or more of its serine residues. The reaction utilized the terminal phosphate of ATP; GTP was inactive. Divalent cations (5 mM Mn2+ or 10 mM Mg2+) were essential for optimum activity. Autophosphorylating protein kinase 500 did not phosphorylate the commonly used exogenous substrates such as histones, casein, phosvitin, or protamine. Analysis of autophosphorylating protein kinase 500 with rabbit anti-autophosphorylating protein kinase 500 IgG by immunoelectrophoresis and crossed immune electrophoresis demonstrated single arcs of precipitation, confirming the biochemical demonstration of enzyme purification and homogeneity. Indirect immunofluorescence studies revealed an intracytoplasmic localization of the enzyme in cultured and freshly isolated adrenocortical carcinoma 494 cells. Both cell types revealed an intensity of perinuclear enzyme fluorescence, but an absence of the enzyme in the nuclei or nucleoli. The anti-autophosphorylating protein kinase 500 IgG blocked the self-catalyzed phosphorylation of autophosphorylating protein kinase 500, providing immunological support of the biochemical results that autophosphorylation is an intrinsic characteristic of the enzyme. When autophosphorylating protein kinase 500 was incubated with membrane-bound ribosomes, it phosphorylated a Mr = 31,000 protein. This phosphorylation was blocked by the anti-autophosphorylating protein kinase 500 IgG.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genome shuffling improves production of the low-temperature alkalophilic lipase by Acinetobacter johnsonii

The production of a low-temperature alkalophilic lipase from Acinetobacter johnsonii was improved using genome shuffling. The starting populations, obtained by UV irradiation and diethyl sulfate mutagenesis, were subjected to recursive protoplast fusion. The optimal conditions for protoplast formation and regeneration were 0.15 mg lysozyme/ml for 45 min at 37°C. The protoplasts were inactivated under UV for 20 min or heated at 60°C for 60 min and a fusant probability of ~98% was observed. The positive colonies were created by fusing the inactivated protoplasts. After two rounds of genome shuffling, one strain, F22, with a lipase activity of 7 U/ml was obtained.     

用ID-SVM预测蛋白质的ATP结合位点

从蛋白质序列出发,对经Dr．G．P．S．Raghava整理和使用过的168条非冗余的ATP与蛋白质结合氨基酸序列进行分段,对ATP与蛋白质结合位点进行了统计分析。在此基础上,利用20种氨基酸的亲疏水性将20种氨基酸约化为6类。以氨基酸组分和6类亲疏水紧邻为参数,用多样性增量（ID）方法将氨基酸组分和6类亲疏水紧邻降维并将降维后的特征参数输入支持向量机中运算,本文运算结果显示用氨基酸组分ID值和6类亲疏水紧邻ID值共同作为特征参数结果最优,在七交叉检验下的预测总精度达到了99．67％,相关系数达到0．9934,好于前人的预测结果。     

Autoregulation of kinase dephosphorylation by ATP binding to AGC protein kinases

AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non-ATP-competitive kinase inhibitors that discriminate within and between protein kinase families.Key words: inhibitors hijacking kinase activation, activation loop phosphorylation, dephosphorylation, phosphatase resistance, PKA, PKB, PKC     

Integrated approach for production of recombinant acetylacetone dioxygenase from Acinetobacter johnsonii

The C–C bond-cleaving acetylacetone dioxygenase Dke1 (EC 1.13.11.50) is a Fe^2?+?-dependent enzyme from Acinetobacter johnsonii that activates oxygen to convert a range of β-dicarbonyl substrates into α-oxo-aldehyde and acid products. Previous methods of downstream processing yielded Dke1 with substoichiometric Fe^2?+? content. This paper reports the integration of enzyme production in E. coli and affinity chromatography to prepare recombinant Dke1 that is completely loaded with its metal cofactor. The specific activity of Dke1 in E. coli cell extracts could be increased up to 20-fold, compared to optimized enzyme production with the natural host. Introduction of an affinity-tag allowed the isolation of fully active Dke1 in a single purification step with high yield (70%). Mass spectrometric analysis revealed at the level of >80% sequence coverage that the isolated enzyme corresponded exactly to the predicted gene product. Tagged Dke1 is shown to have retained the functional properties of native Dke1.     

In Vitro ATP Regeneration from Polyphosphate and AMP by Polyphosphate:AMP Phosphotransferase and Adenylate Kinase from Acinetobacter johnsonii 210A

In vitro enzyme-based ATP regeneration systems are important for improving yields of ATP-dependent enzymatic reactions for preparative organic synthesis and biocatalysis. Several enzymatic ATP regeneration systems have been described but have some disadvantages. We report here on the use of polyphosphate:AMP phosphotransferase (PPT) from Acinetobacter johnsonii strain 210A in an ATP regeneration system based on the use of polyphosphate (polyP) and AMP as substrates. We have examined the substrate specificity of PPT and demonstrated ATP regeneration from AMP and polyP using firefly luciferase and hexokinase as model ATP-requiring enzymes. PPT catalyzes the reaction polyP_n + AMP → ADP + polyP_n−1. The ADP can be converted to ATP by adenylate kinase (AdK). Substrate specificity with nucleoside and 2′-deoxynucleoside monophosphates was examined using partially purified PPT by measuring the formation of nucleoside diphosphates with high-pressure liquid chromatography. AMP and 2′-dAMP were efficiently phosphorylated to ADP and 2′-dADP, respectively. GMP, UMP, CMP, and IMP were not converted to the corresponding diphosphates at significant rates. Sufficient AdK and PPT activity in A. johnsonii 210A cell extract allowed demonstration of polyP-dependent ATP regeneration using a firefly luciferase-based ATP assay. Bioluminescence from the luciferase reaction, which normally decays very rapidly, was sustained in the presence of A. johnsonii 210A cell extract, MgCl₂, polyP_n=35, and AMP. Similar reaction mixtures containing strain 210A cell extract or partially purified PPT, polyP, AMP, glucose, and hexokinase formed glucose 6-phosphate. The results indicate that PPT from A. johnsonii is specific for AMP and 2′-dAMP and catalyzes a key reaction in the cell-free regeneration of ATP from AMP and polyP. The PPT/AdK system provides an alternative to existing enzymatic ATP regeneration systems in which phosphoenolpyruvate and acetylphosphate serve as phosphoryl donors and has the advantage that AMP and polyP are stabile, inexpensive substrates.     

In vitro ATP regeneration from polyphosphate and AMP by polyphosphate:AMP phosphotransferase and adenylate kinase from Acinetobacter johnsonii 210A

In vitro enzyme-based ATP regeneration systems are important for improving yields of ATP-dependent enzymatic reactions for preparative organic synthesis and biocatalysis. Several enzymatic ATP regeneration systems have been described but have some disadvantages. We report here on the use of polyphosphate:AMP phosphotransferase (PPT) from Acinetobacter johnsonii strain 210A in an ATP regeneration system based on the use of polyphosphate (polyP) and AMP as substrates. We have examined the substrate specificity of PPT and demonstrated ATP regeneration from AMP and polyP using firefly luciferase and hexokinase as model ATP-requiring enzymes. PPT catalyzes the reaction polyP(n) + AMP --> ADP + polyP(n-1). The ADP can be converted to ATP by adenylate kinase (AdK). Substrate specificity with nucleoside and 2'-deoxynucleoside monophosphates was examined using partially purified PPT by measuring the formation of nucleoside diphosphates with high-pressure liquid chromatography. AMP and 2'-dAMP were efficiently phosphorylated to ADP and 2'-dADP, respectively. GMP, UMP, CMP, and IMP were not converted to the corresponding diphosphates at significant rates. Sufficient AdK and PPT activity in A. johnsonii 210A cell extract allowed demonstration of polyP-dependent ATP regeneration using a firefly luciferase-based ATP assay. Bioluminescence from the luciferase reaction, which normally decays very rapidly, was sustained in the presence of A. johnsonii 210A cell extract, MgCl(2), polyP(n=35), and AMP. Similar reaction mixtures containing strain 210A cell extract or partially purified PPT, polyP, AMP, glucose, and hexokinase formed glucose 6-phosphate. The results indicate that PPT from A. johnsonii is specific for AMP and 2'-dAMP and catalyzes a key reaction in the cell-free regeneration of ATP from AMP and polyP. The PPT/AdK system provides an alternative to existing enzymatic ATP regeneration systems in which phosphoenolpyruvate and acetylphosphate serve as phosphoryl donors and has the advantage that AMP and polyP are stabile, inexpensive substrates.     

Nucleotide binding to the chaperonin GroEL: non-cooperative binding of ATP analogs and ADP, and cooperative effect of ATP

Chaperonin-assisted protein folding proceeds through cycles of ATP binding and hydrolysis by GroEL, which undergoes a large structural change by the ATP binding or hydrolysis. One of the main concerns of GroEL is the mechanism of the productive and cooperative structural change of GroEL induced by the nucleotide. We studied the cooperative nature of GroEL by nucleotide titration using isothermal titration calorimetry and fluorescence spectroscopy. Our results indicated that the binding of ADP and ATP analogs to a single ring mutant (SR1), as well as that to GroEL, was non-cooperative. Only ATP induces an apparently cooperative conformational change in both proteins. Furthermore, the fluorescence changes of pyrene-labeled GroEL indicated that GroEL has two kinds of nucleotide binding sites. The fluorescence titration result fits well with a model in which two kinds of binding sites are both non-cooperative and independent of each other. These results suggest that the binding and hydrolysis of ATP may be necessary for the cooperative transition of GroEL.     

Cooperative binding of ATP and RNA substrates to the DEAD/H protein DbpA

Unlike most DEAD/H proteins, the purified Escherichia coli protein DbpA demonstrates high specificity for its 23S rRNA substrate in vitro. Here we describe several assays designed to characterize the interaction of DbpA with its RNA and ATP substrates. Electrophoretic mobility shift assays reveal a sub-nanomolar binding affinity for a 153 nucleotide RNA substrate (R153) derived from the 23S rRNA. High affinity RNA binding requires both hairpin 92 and helix 90, as substrates lacking these structures bind DbpA with lower affinity. AMPPNP inhibition assays and ATP/ADP binding assays provide binding constants for ATP and ADP to DbpA with and without RNA substrates. These data have been used to describe a minimal thermodynamic scheme for the binding of the RNA and ATP substrates to DbpA, which reveals cooperative binding between larger RNAs and ATP with cooperative energies of approximately 1.3 kcal mol(-1). This cooperativity is lost upon removal of helix 89 from R153, suggesting this helix is either the preferred target for DbpA's helicase activity or is a necessary structural element for organization of the target site within R153.     

Characterization of the 70-kDa peroxisomal membrane protein, an ATP binding cassette transporter

The 70-kDa peroxisomal membrane protein (PMP70) is one of the major components of rat liver peroxisomal membranes and belongs to a superfamily of proteins known as ATP binding cassette transporters. PMP70 is markedly induced by administration of hypolipidemic agents in parallel with peroxisome proliferation and induction of peroxisomal fatty acid beta-oxidation enzymes. To characterize the role of PMP70 in biogenesis and function of peroxisomes, we transfected the cDNA of rat PMP70 into Chinese hamster ovary cells and established cell lines stably expressing PMP70. The content of PMP70 in the transfectants increased about 5-fold when compared with the control cells. A subcellular fractionation study showed that overexpressed PMP70 was enriched in peroxisomes. This peroxisomal localization was confirmed by immunofluorescence and immunoelectron microscopy. The number of immuno-gold particles corresponding to PMP70 on peroxisomes increased markedly in the transfectants, but the size and the number of peroxisomes were essentially the same in both the transfectants and the control cells. beta-Oxidation of palmitic acid increased about 2-3-fold in the transfectants, whereas the oxidation of lignoceric acid decreased about 30-40%. When intact peroxisomes prepared from both the cell lines were incubated with palmitoyl-CoA, oxidation was stimulated with ATP, but the degree of the stimulation was higher in the transfectants than in the control cells. Furthermore, we established three Chinese hamster ovary cell lines stably expressing mutant PMP70. In these cells, beta-oxidation of palmitic acid decreased markedly. These results suggest that PMP70 is involved in metabolic transport of long chain acyl-CoA across peroxisomal membranes and that increase of PMP70 is not associated with proliferation of peroxisomes.     

Characterization of two phosphate transport systems in Acinetobacter johnsonii 210A.

The transport of P(i) was characterized in Acinetobacter johnsonii 210A, which is able to accumulate an excessive amount of phosphate as polyphosphate (polyP) under aerobic conditions. P(i) is taken up against a concentration gradient by energy-dependent, carrier-mediated processes. A. johnsonii 210A, grown under P(i) limitation, contains two uptake systems with Kt values of 0.7 +/- 0.2 microM and 9 +/- 1 microM. P(i) uptake via the high-affinity component is drastically reduced by N,N'-dicyclohexylcarbodiimide, an inhibitor of H(+)-ATPase, and by osmotic shock. Together with the presence of P(i)-binding activity in concentrated periplasmic protein fractions, these results suggest that the high-affinity transport system belongs to the group of ATP-driven, binding-protein-dependent transport systems. Induction of this transport system upon transfer of cells grown in the presence of excess P(i) to P(i)-free medium results in a 6- to 10-fold stimulation of the P(i) uptake rate. The constitutive low-affinity uptake system for P(i) is inhibited by uncouplers and can mediate counterflow of P(i), indicating its reversible, secondary nature. The presence of an inducible high-affinity uptake system for P(i) and the ability to decrease the free internal P(i) pool by forming polyP enable A. johnsonii 210A to reduce the P(i) concentration in the aerobic environment to micromolar levels. Under anaerobic conditions, polyP is degraded again and P(i) is released via the low-affinity secondary transport system.     

Re-evaluation of the binding of ATP to metallothionein

In a recent paper Jiang et al. (Jiang, L. J., Maret, W. & Vallee, B. L. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9146-9149) reported that metallothionein interacts with adenosine triphosphate (ATP) to form a 1:1 complex with a dissociation constant of K(d) = 176 +/- 33 microM at pH 7.4. In an effort to characterize further this interaction using nuclear magnetic resonance spectroscopy, titration calorimetry, gel-filtration chromatography, affinity chromatography, and ultrafiltration, we were unable to find any evidence for the binding of ATP to metallothionein.     

Interaction of recA protein with a photoaffinity analogue of ATP, 8-azido-ATP: determination of nucleotide cofactor binding parameters and of the relationship between ATP binding and ATP hydrolysis

The binding and cross-linking of the ATP photoaffinity analogue 8-azidoadenosine 5'-triphosphate (azido-ATP) with recA protein have been investigated, and through cross-linking inhibition studies, the binding of other nucleotide cofactors to recA protein has also been studied. The azido-ATP molecule was shown to be a good ATP analogue with regard to recA protein binding and enzymatic function by three criteria: first, the cross-linking follows a simple hyperbolic binding curve with a Kd of 4 microM and a cross-linking efficiency ranging from 10% to 70% depending on conditions; second, ATP, dATP, and adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) specifically inhibit the cross-linking of azido-ATP to recA protein; third, azido-ATP is a substrate for recA protein ATPase activity. Quantitative analysis of the cross-linking inhibition studies using a variety of nucleotide cofactors as competitors has shown that the binding affinity of adenine-containing nucleotides for recA protein decreases in the following order: ATP-gamma-S greater than dATP greater than ATP greater than adenylyl beta,gamma-imidodiphosphate (AMP-PNP) much greater than adenylyl beta,gamma-methylenediphosphate (AMP-PCP) approximately adenine. Similar competition studies also showed that nearly all of the other nucleotide triphosphates also bind to recA protein, with the affinity decreasing in the following order: UTP greater than GTP approximately equal to dCTP greater than dGTP greater than CTP. In addition, studies performed in the presence of single-stranded DNA demonstrated that the affinity of ATP, dATP, ATP-gamma-S, and AMP-PNP for recA protein is significantly increased. These results are discussed in terms of the reciprocal effects that nucleotide cofactors have on the modulation of recA protein--single-stranded DNA binding affinity and vice versa. In addition, it is demonstrated that nucleotide and DNA binding are necessary though not sufficient conditions for ATPase activity. The significance of this result in terms of the possible requirement of critically sized clusters of 15 or more recA protein molecules contiguously bound to DNA for ATPase activity is discussed.     

ATP/ADP binding to a novel nucleotide binding domain of the reticulocyte-binding protein Py235 of Plasmodium yoelii

The mechanism by which a malaria merozoite recognizes a suitable host cell is mediated by a cascade of receptor-ligand interactions. In addition to the availability of the appropriate receptors, intracellular ATP plays an important role in determining whether erythrocytes are suitable for merozoite invasion. Recent work has shown that ATP secreted from erythrocytes signals a number of cellular processes. To determine whether ATP signaling might be involved in merozoite invasion, we investigated whether known plasmodium invasion proteins contain nucleotide binding motifs. Domain mapping identified a putative nucleotide binding region within all members of the reticulocyte-binding protein homologue (RBL) family analyzed. A representative domain, termed here nucleotide binding domain 94 (NBD94), was expressed and demonstrated to specifically bind to ATP. Nucleotide affinities of NBD94 were determined by fluorescence correlation spectroscopy, where an increase in the binding of ATP is observed compared with ADP analogues. ATP binding was reduced by the known F1F0-ATP synthase inhibitor 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Fluorescence quenching and circular dichroism spectroscopy of NBD94 after binding of different nucleotides provide evidence for structural changes in this protein. Our data suggest that different structural changes induced by ATP/ADP binding to RBL could play an important role during the invasion process.