Magnesium Adenosine Triphosphate Research Articles

ATP-induced reconfiguration of the micro-viscoelasticity of cardiac and skeletal myosin solutions.

We study the high-frequency, micro-mechanical response of suspensions composed of cardiac and skeletal muscle myosin by optical trapping interferometry. We observe that in low ionic strength solutions, upon the addition of magnesium adenosine triphosphate (MgATP2-), myosin suspensions radically change their micro-mechanics properties, generating a viscoelastic fluid characterized by a complex modulus similar to a suspension of worm-like micelles. This transduction of energy, from chemical to mechanical, may be related to the relaxed states of myosin, which regulate muscle contractility and can be involved in the etiology of many myopathies. Within an analogous generic mechanical response, cardiac and skeletal myosin suspensions provide different stress relaxation times, elastic modulus values, and characteristic lengths. These discrepancies probably rely on the dissimilar physiological functions of cardiac and skeletal muscle, on the different MgATPase hydrolysis rates of cardiac and skeletal myosins, and on the observed distinct cooperative behavior of their myosin heads in the super-relaxed state. In vitro studies like these allow us to understand the foundations of muscle cell mechanics on the micro-scale, and may contribute to the engineering of biological materials whose micro-mechanics can be activated by energy regulators.

ATP:Mg2+ shapes material properties of protein-RNA condensates and their partitioning of clients

Many cellular condensates are heterotypic mixtures of proteins and RNA formed in complex environments. Magnesium ions (Mg2+) and ATP can impact RNA folding, and local intracellular levels of these factors can vary significantly. However, the effect of ATP:Mg2+ on the material properties of protein-RNA condensates is largely unknown. Here, we use an in vitro condensate model of nucleoli, made from nucleophosmin 1 (NPM1) proteins and ribosomal RNA (rRNA), to study the effect of ATP:Mg2+. While NPM1 dynamics remain unchanged at increasing Mg2+ concentrations, the internal RNA dynamics dramatically slowed until a critical point, where gel-like states appeared, suggesting the RNA component alone forms a viscoelastic network that undergoes maturation driven by weak multivalent interactions. ATP reverses this arrest and liquefies the gel-like structures. ATP:Mg2+ also influenced the NPM1-rRNA composition of condensates and enhanced the partitioning of two clients: an arginine-rich peptide and a small nuclear RNA. By contrast, larger ribosome partitioning shows dependence on ATP:Mg2+ and can become reversibly trapped around NPM1-rRNA condensates. Lastly, we show that dissipative enzymatic reactions that deplete ATP can be used to control the shape, composition, and function of condensates. Our results illustrate how intracellular environments may regulate the state and client partitioning of RNA-containing condensates.

Capillary electrophoretic assay of human acetyl-coenzyme A carboxylase 2.

Human acetyl-coenzyme A carboxylase 2 catalyzes the carboxylation of acetyl coenzyme A to form malonyl coenzyme A, along with the conversion of magnesium-adenosine triphosphate complex to magnesium-adenosine diphosphate complex. A simple off-column capillary electrophoresis assay for human acetyl-coenzyme A carboxylase 2 was developed based on the separation of magnesium-adenosine triphosphate complex, magnesium-adenosine diphosphate complex, acetyl coenzyme A and malonyl coenzyme A with detection by ultraviolet absorption at 256 nm. When Mg2+ was absent from the separation buffer, the zones due to magnesium-adenosine triphosphate complex and magnesium-adenosine diphosphate complex both split and migrated as two separate peaks. With Mg2+ added to the separation buffer, magnesium-adenosine triphosphate complex and magnesium-adenosine diphosphate complex produced single peaks, and the reproducibility of peak shape and area improved for human acetyl-coenzyme A carboxylase 2 assay components. The final separation buffer used was 30.0mM HEPES, 3.0mM MgCl2 , 2.5mM KHCO3 , and 2.5mM potassium citrate at pH 7.50. The same buffer was used for the enzyme-catalyzed reaction (off-column). Inhibition of human acetyl-coenzyme A carboxylase 2 by CP-640186, a known inhibitor, was detected using the capillary electrophoresis assay.

Purification and functional characterization of tomato mosaic virus 130K protein expressed in silkworm pupae using a baculovirus vector

Tomato mosaic virus (ToMV; genus, Tobamovirus) is a member of the alpha-like virus superfamily of positive-strand RNA viruses, which includes many plant and animal viruses of agronomical and clinical importance. The genomes of alpha-like viruses encode replication-associated proteins that contain methyltransferase, helicase and/or polymerase domains. The three-dimensional structure of the helicase domain fragment of ToMV has been determined, but the structures of the other domains of alpha-like virus replication proteins are not available. In this study, we expressed full-length ToMV replication-associated protein 130 K, which contains the methyltransferase and helicase domains, using the baculovirus-silkworm expression system and purified the recombinant protein to near homogeneity. Purified 130 K, which was stable in phosphate buffer containing magnesium ions and ATP, formed a dimer in solution and hydrolyzed nucleoside 5′-triphosphates.

ATP-Driven Contraction of Phage T3 Capsids with DNA Incompletely Packaged In Vivo.

Adenosine triphosphate (ATP) cleavage powers packaging of a double-stranded DNA (dsDNA) molecule in a pre-assembled capsid of phages that include T3. Several observations constitute a challenge to the conventional view that the shell of the capsid is energetically inert during packaging. Here, we test this challenge by analyzing the in vitro effects of ATP on the shells of capsids generated by DNA packaging in vivo. These capsids retain incompletely packaged DNA (ipDNA) and are called ipDNA-capsids; the ipDNA-capsids are assumed to be products of premature genome maturation-cleavage. They were isolated via preparative Nycodenz buoyant density centrifugation. For some ipDNA-capsids, Nycodenz impermeability increases hydration and generates density so low that shell hyper-expansion must exist to accommodate associated water. Electron microscopy (EM) confirmed hyper-expansion and low permeability and revealed that 3.0 mM magnesium ATP (physiological concentration) causes contraction of hyper-expanded, low-permeability ipDNA-capsids to less than mature size; 5.0 mM magnesium ATP (border of supra-physiological concentration) or more disrupts them. Additionally, excess sodium ADP reverses 3.0 mM magnesium ATP-induced contraction and re-generates hyper-expansion. The Nycodenz impermeability implies assembly perfection that suggests selection for function in DNA packaging. These findings support the above challenge and can be explained via the assumption that T3 DNA packaging includes a back-up cycle of ATP-driven capsid contraction and hyper-expansion.

GlmU (N-acetylglucosamine-1-phosphate uridyltransferase) bound to three magnesium ions and ATP at the active site.

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU), a bifunctional enzyme exclusive to prokaryotes, belongs to the family of sugar nucleotidyltransferases (SNTs). The enzyme binds GlcNAc-1-P and UTP, and catalyzes a uridyltransfer reaction to synthesize UDP-GlcNAc, an important precursor for cell-wall biosynthesis. As many SNTs are known to utilize a broad range of substrates, substrate specificity in GlmU was probed using biochemical and structural studies. The enzymatic assays reported here demonstrate that GlmU is specific for its natural substrates UTP and GlcNAc-1-P. The crystal structure of GlmU bound to ATP and GlcNAc-1-P provides molecular details for the inability of the enzyme to utilize ATP for the nucleotidyltransfer reaction. ATP binding results in an inactive pre-catalytic enzyme-substrate complex, where it adopts an unusual conformation such that the reaction cannot be catalyzed; here, ATP is shown to be bound together with three Mg2+ ions. Overall, this structure represents the binding of an inhibitory molecule at the active site and can potentially be used to develop new inhibitors of the enzyme. Further, similar to DNA/RNA polymerases, GlmU was recently recognized to utilize two metal ions, MgA2+ and MgB2+, to catalyze the uridyltransfer reaction. Interestingly, displacement of MgB2+ from its usual catalytically competent position, as noted in the crystal structure of RNA polymerase in an inactive state, was considered to be a key factor inhibiting the reaction. Surprisingly, in the current structure of GlmU MgB2+ is similarly displaced; this raises the possibility that an analogous inhibitory mechanism may be operative in GlmU.

A 3D localized surface plasmon resonance biosensor for the study of trivalent arsenic binding to the ArsA ATPase

A self-assembled 3D hydrogel–nanoparticle composite integrated surface plasmon resonance (SPR) sensor is reported here. The novel assembled substrate was developed by means of a surface mediated radical co-polymerization process to obtain a highly sensitive hydrogel-based thin film that possesses specific binding sites for target analytes. Initially, amino group modified gold nanoparticles (AuNPs) were covalently linked to acrylic acid monomer. Following this, N-isopropylacrylamide (NIPAAm) and AuNPs linked acrylic acid (AAc) monomers were randomly co-polymerized by the “grafting from” method in the presence of initiator and crosslinker onto the sensing surface. Surface charecterization techniques were utilized to evaluate the thickness and composition of the hydrogel-nanoparticle film. The sensing platform was employed to study the binding kinetics and conformational changes of the ArsA ATPase as a consequence of binding trivalent arsenicals under a variety of conditions. ArsA, the catalytic subunit of the ArsAB arsenite (As(III)) translocating ATPase, is one of the five proteins encoded by the arsenical resistance (ars) operon of plasmid R773 in cells of Escherichia coli, that confers resistance to trivalent and pentavalent salts of the metalloid arsenic. SPR measurements indicate that the 3D hydrogel-nanoparticle coated sensors exhibited a higher sensitivity than that of the 2D AuNPs decorated sensors. Binding of As(III) to ArsA is greatly facilitated by the presence of magnesium ion and ATP.

Structure and allostery of the PKA RIIβ tetrameric holoenzyme.

In its physiological state, cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is a tetramer that contains a regulatory (R) subunit dimer and two catalytic (C) subunits. We describe here the 2.3 angstrom structure of full-length tetrameric RIIβ(2):C(2) holoenzyme. This structure showing a dimer of dimers provides a mechanistic understanding of allosteric activation by cAMP. The heterodimers are anchored together by an interface created by the β4-β5 loop in the RIIβ subunit, which docks onto the carboxyl-terminal tail of the adjacent C subunit, thereby forcing the C subunit into a fully closed conformation in the absence of nucleotide. Diffusion of magnesium adenosine triphosphate (ATP) into these crystals trapped not ATP, but the reaction products, adenosine diphosphate and the phosphorylated RIIβ subunit. This complex has implications for the dissociation-reassociation cycling of PKA. The quaternary structure of the RIIβ tetramer differs appreciably from our model of the RIα tetramer, confirming the small-angle x-ray scattering prediction that the structures of each PKA tetramer are different.

Mechanism of Mo-Dependent Nitrogenase

Nitrogen-fixing bacteria catalyze the reduction of dinitrogen (N(2)) to two ammonia molecules (NH(3)), the major contribution of fixed nitrogen to the biogeochemical nitrogen cycle. The most widely studied nitrogenase is the molybdenum (Mo)-dependent enzyme. The reduction of N(2) by this enzyme involves the transient interaction of two component proteins, designated the iron (Fe) protein and the MoFe protein, and minimally requires 16 magnesium ATP (MgATP), eight protons, and eight electrons. The current state of knowledge on how these proteins and small molecules together effect the reduction of N(2) to ammonia is reviewed. Included is a summary of the roles of the Fe protein and MgATP hydrolysis, information on the roles of the two metal clusters contained in the MoFe protein in catalysis, insights gained from recent success in trapping substrates and inhibitors at the active-site metal cluster FeMo cofactor, and finally, considerations of the mechanism of N(2) reduction catalyzed by nitrogenase.

Characterization of an Alternative Splice Variant of LKB1

LKB1 is an upstream activating kinase for the AMP-activated protein kinase (AMPK) and at least 12 other AMPK-related kinases. LKB1 therefore acts as a master kinase regulating the activity of a wide range of downstream kinases, which themselves have diverse physiological roles. Here we identify a second form of LKB1 generated by alternative splicing of the LKB1 gene. The two LKB1 proteins have different C-terminal sequences generating a 50-kDa form (termed LKB1L) and a 48-kDa form (LKB1S). LKB1L is widely expressed in mouse tissues, whereas LKB1S has a restricted tissue distribution with predominant expression in the testis. LKB1S, like LKB1L, forms a complex with MO25 and STRAD, and phosphorylates and activates AMPK both in vitro and in intact cells. A phosphorylation site (serine 431 in mouse) and a farnesylation site (cysteine 433 in mouse) within LKB1L are not conserved in LKB1S raising the possibility that these sites might be involved in differential regulation and/or localization of the two forms of LKB1. However, we show that phosphorylation of serine 431 has no effect on LKB1L activity and that both LKB1L and LKB1S have similar patterns of subcellular localization. These results indicate that the physiological significance of the different forms of LKB1 is not related directly to differences in the C-terminal sequences but may be due to their differential patterns of tissue distribution.

The MTHFD1 p.Arg653Gln variant alters enzyme function and increases risk for congenital heart defects

Methylenetetrahydrofolate dehydrogenase)methenyltetrahydrofolate cyclohydrolase)formyltetrahydrofolate synthetase (MTHFD1) is a trifunctional enzyme that interconverts tetrahydrofolate (THF) derivatives for nucleotide synthesis. A common variant in MTHFD1, p.Arg653Gln (c.1958G>A), may increase the risk for neural tube defects (NTD). To examine the biological impact of this variant on MTHFD1 function, we measured enzyme activity and stability in vitro and assessed substrate flux in transfected mammalian cells. The purified Arg653Gln enzyme has normal substrate affinity but a 36% reduction in half)life at 42 degrees C. Thermolability is reduced by magnesium adenosine triphosphate and eliminated by the substrate analog folate pentaglutamate, suggesting that folate status may modulate impact of the variant. The mutation reduces the metabolic activity of MTHFD1 within cells: formate incorporation into DNA in murine Mthfd1 knockout cells transfected with Arg653Gln is reduced by 26%+/-7.7% (P<0.05), compared to cells transfected with wild)type protein, indicating a disruption of de novo purine synthesis. We assessed the impact of the variant on risk for congenital heart defects (CHD) in a cohort of Quebec children (158 cases, 110 controls) and mothers of children with heart defects (199 cases, 105 controls). The 653QQ genotype in children is associated with increased risk for heart defects (odds ratio [OR], 2.11; 95% confidence interval [CI], 1.01-4.42), particularly Tetralogy of Fallot (OR, 3.60; 95% CI, 1.38-9.42) and aortic stenosis (OR, 3.13; 95% CI, 1.13-8.66). There was no effect of maternal genotype. Our results indicate that the Arg653Gln polymorphism decreases enzyme stability and increases risk for CHD. Further evaluation of this polymorphism in folate)related disorders and its potential interaction with folate status is warranted.

Role of Divalent Metal Cations in ATP Hydrolysis Catalyzed by the Hepatitis C Virus NS3 Helicase: Magnesium Provides a Bridge for ATP to Fuel Unwinding

This study investigates the role of magnesium ions in coupling ATP hydrolysis to the nucleic acid unwinding catalyzed by the NS3 protein encoded by the hepatitis C virus (HCV). Analyses of steady-state ATP hydrolysis rates at various RNA and magnesium concentrations were used to determine values for the 15 dissociation constants describing the formation of a productive enzyme–metal–ATP–RNA complex and the four rate constants describing hydrolysis of ATP by the possible enzyme–ATP complexes. These values coupled with direct binding studies, specificity studies and analyses of site-directed mutants reveal only one ATP binding site on HCV helicase centered on the catalytic base Glu291. An adjacent residue, Asp290, binds a magnesium ion that forms a bridge to ATP, reorienting the nucleotide in the active site. RNA stimulates hydrolysis while decreasing the affinity of the enzyme for ATP, magnesium, and MgATP. The binding scheme described here explains the unusual regulation of the enzyme by ATP that has been reported previously. Binding of either free magnesium or free ATP to HCV helicase competes with MgATP, the true fuel for helicase movements, and leads to slower hydrolysis and nucleic acid unwinding.

Activating Mutations in theABCC8Gene in Neonatal Diabetes Mellitus

The ATP-sensitive potassium (K(ATP)) channel, composed of the beta-cell proteins sulfonylurea receptor (SUR1) and inward-rectifying potassium channel subunit Kir6.2, is a key regulator of insulin release. It is inhibited by the binding of adenine nucleotides to subunit Kir6.2, which closes the channel, and activated by nucleotide binding or hydrolysis on SUR1, which opens the channel. The balance of these opposing actions determines the low open-channel probability, P(O), which controls the excitability of pancreatic beta cells. We hypothesized that activating mutations in ABCC8, which encodes SUR1, cause neonatal diabetes. We screened the 39 exons of ABCC8 in 34 patients with permanent or transient neonatal diabetes of unknown origin. We assayed the electrophysiologic activity of mutant and wild-type K(ATP) channels. We identified seven missense mutations in nine patients. Four mutations were familial and showed vertical transmission with neonatal and adult-onset diabetes; the remaining mutations were not transmitted and not found in more than 300 patients without diabetes or with early-onset diabetes of similar genetic background. Mutant channels in intact cells and in physiologic concentrations of magnesium ATP had a markedly higher P(O) than did wild-type channels. These overactive channels remained sensitive to sulfonylurea, and treatment with sulfonylureas resulted in euglycemia. Dominant mutations in ABCC8 accounted for 12 percent of cases of neonatal diabetes in the study group. Diabetes results from a newly discovered mechanism whereby the basal magnesium-nucleotide-dependent stimulatory action of SUR1 on the Kir pore is elevated and blockade by sulfonylureas is preserved.

Mutations in the Amino Terminus of the Cystic Fibrosis Transmembrane Conductance Regulator Enhance Endocytosis

Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator (CFTR) is mediated by a tyrosine-based internalization signal in the CFTR carboxyl-terminal tail 1424YDSI1427. In the present studies, two naturally occurring cystic fibrosis mutations in the amino terminus of CFTR, R31C, and R31L were examined. To determine the defect that these mutations cause, the Arg-31 mutants were expressed in COS-7 cells and their biogenesis and trafficking to the cell surface tested in metabolic pulse-chase and surface biotinylation assays, respectively. The results indicated that both Arg-31 mutants were processed to band C at approximately 50% the efficiency of the wild-type protein. However, once processed and delivered to the cell surface, their half-lives were the same as wild-type protein. Interestingly, indirect immunofluorescence and cell surface biotinylation indicated that the surface pool was much smaller than could be accounted for based on the biogenesis defect alone. Therefore, the Arg-31 mutants were tested in internalization assays and found to be internalized at 2x the rate of the wild-type protein. Patch clamp and 6-methoxy-N-(3-sulfopropyl)quinolinium analysis confirmed reduced amounts of functional Arg-31 channels at the cell surface. Together, the results suggest that both R31C and R31L mutations compromise biogenesis and enhance internalization of CFTR. These two additive effects contribute to the loss of surface expression and the associated defect in chloride conductance that is consistent with a disease phenotype.

Kinetic mechanism for p38 MAP kinase alpha. A partial rapid-equilibrium random-order ternary-complex mechanism for the phosphorylation of a protein substrate

p38 Mitogen-activated protein kinase alpha (p38 MAPKalpha) is a member of the MAPK family. It is activated by cellular stresses and has a number of cellular substrates whose coordinated regulation mediates inflammatory responses. In addition, it is a useful anti-inflammatory drug target that has a high specificity for Ser-Pro or Thr-Pro motifs in proteins and contains a number of transcription factors as well as protein kinases in its catalog of known substrates. Fundamental to signal transduction research is the understanding of the kinetic mechanisms of protein kinases and other protein modifying enzymes. To achieve this end, because peptides often make only a subset of the full range of interactions made by proteins, protein substrates must be utilized to fully elucidate kinetic mechanisms. We show using an untagged highly active form of p38 MAPKalpha, expressed and purified from Escherichia coli[Szafranska AE, Luo X & Dalby KN (2005) Anal Biochem336, 1-10) that at pH 7.5, 10 mm Mg2+ and 27 degrees C p38 MAPKalpha phosphorylates ATF2Delta115 through a partial rapid-equilibrium random-order ternary-complex mechanism. This mechanism is supported by a combination of steady-state substrate and inhibition kinetics, as well as microcalorimetry and published structural studies. The steady-state kinetic experiments suggest that magnesium adenosine triphosphate (MgATP), adenylyl (beta,gamma-methylene) diphosphonic acid (MgAMP-PCP) and magnesium adenosine diphosphate (MgADP) bind p38 MAPKalpha with dissociation constants of KA = 360 microm, KI = 240 microm, and KI > 2000 microm, respectively. Calorimetry experiments suggest that MgAMP-PCP and MgADP bind the p38 MAPKalpha-ATF2Delta115 binary complex slightly more tightly than they do the free enzyme, with a dissociation constant of Kd approximately 70 microm. Interestingly, MgAMP-PCP exhibits a mixed inhibition pattern with respect to ATF2Delta115, whereas MgADP exhibits an uncompetitive-like pattern. This discrepancy occurs because MgADP, unlike MgAMP-PCP, binds the free enzyme weakly. Intriguingly, no inhibition by 2 mm adenine or 2 mm MgAMP was detected, suggesting that the presence of a beta-phosphate is essential for significant binding of an ATP analog to the enzyme. Surprisingly, we found that inhibition by the well-known p38 MAPKalpha inhibitor SB 203580 does not follow classical linear inhibition kinetics at concentrations > 100 nm, as previously suggested, demonstrating that caution must be used when interpreting kinetic experiments using this inhibitor.

A novel method to reduce prolonged warm ischemia injury in amputated limbs

INTRODUCTION: In the US it is estimated that more than 1.2 million partial or complete limb amputations per year cannot be saved because of excessive ischemia time. The purpose of this study was to determine whether perfusion with a novel fusogenic lipid vesicle (FLV) that delivers magnesium adenosine triphosphate (Mg-ATP) directly into cells could maintain amputated limbs viable after prolonged warm ischemia.

Direct energy delivery improves tissue perfusion after resuscitated shock

Conventional resuscitation (CR) from hemorrhagic shock (HS) does not restore intestinal blood flow. Indicators of anaerobic metabolism suggest that cellular energy production also is compromised. We hypothesize that the direct intravenous delivery of lipid-encapsulated high-energy phosphates to cells improves intestinal perfusion during HS and resuscitation (RES). MAP (MAP) was monitored in male rats (200 g), terminal ileum microvessel diameters were measured by in vivo videomicroscopy, and blood flow (Doppler velocimetry) was calculated. Cellular energy delivery was accomplished by intravenous infusion during RES of fusogenic unilamellar lipid vesicles that contain adenosine triphosphate (ATP; VitaSol). Our protocol was HS to 50% baseline MAP for 60 minutes, 30 minutes of RES, and continued microscopy observation for 120 minutes. Experimental groups (n=8 each) were HS+CR (group I); HS+CR+ VitaSol (group II); HS+CR+Vehicle, Vehicle is the phospholipid vesicles without magnesium ATP, (group III); HS+ VitaSol (group IV); sham-operated control+VitaSol (group V); and a time-matched sham-operated control (group VI). The survival outcome and total tissue water from wet weight/dry weight ratio as a function of adjunct VitaSol resuscitation were evaluated in separate intact animal experiments. HS caused a selective vasoconstriction of the intestinal inflow arterioles (100 microm), which was not seen in the smaller intestinal premucosal arterioles (7-15 microm). CR, which restored baseline hemodynamics, resulted in an initial restoration of intestinal microvascular diameters at all arteriolar levels. However, this was followed by a progressive vasoconstriction and hypoperfusion in premucosal vessels at 120 minutes after RES (-20.48% +/- 2.95% from baseline diameters). In contrast, VitaSol with CR caused enhanced premucosal dilation (+34.27% +/- 4.62%) and augmented flow (+20.50% +/- 10.70%) above prehemorrhage baseline. Vesicles alone had no effect, and VitaSol alone caused only a modest dilation. CR of moderate HS (40% of baseline MAP for 60 minutes, n=10) caused 20% mortality, whereas adjunct VitaSol resuscitation had a 100% survival and less tissue water content. Our data confirms that CR causes progressive intestinal hypoperfusion. Cellular resuscitation with direct intravenous energy delivery improves intestinal perfusion after CR and results in improved survival and less tissue edema.

Identification, characterization of levoglucosan kinase, and cloning and expression of levoglucosan kinase cDNA from Aspergillus niger CBX-209 in Escherichia coli

The first enzyme responsible for assimilating levoglucosan in Aspergillus niger CBX-209 was corroborated to be levoglucosan kinase that catalyzes the transfer of a phosphate group from ATP to levoglucosan to yield a glucose 6-phosphate in the presence of magnesium ion and ATP by FAB-mass spectrometric method combined with previous observations from HPLC and enzymological experiments. Levoglucosan kinase was purified to apparent homogeneity by using a combination of seven purification steps. SDS–PAGE revealed a single protein band of 56 KDa. It is a monomeric enzyme and maximal enzyme activity was measured at pH 9.3 and 30 °C. This kinase is stable below 20 °C at a quite broad pHs ranging from 6 to 10 and levoglucosan could protect the enzyme from thermal inactivation. Exclusive substrate specificity for levoglucosan suggested that not only the structure of the intramolecular glucosidic linkage but also the configuration of the pyranose frame would be specific for recognition by levoglucosan kinase. The K m values of this enzyme were 71.2 mM for levoglucosan and 0.25 mM for ATP, determined by double reciprocal plottings and ADP inhibited on the enzyme activity competitively with a Ki value of 0.20 mM. A cDNA library from A. niger was constructed in Escherichia coli DH5α. The library was screened for levoglucosan kinase gene on NCE selective medium and three positive recombinants were selected after a five day culture. Detection of activities of levoglucosan kinase in the cell extracts indicated that levoglucosan kinase gene ( lgk) was expressed by the recombinant strain of E. coli DH5α.

Characterization of the fomA and fomB gene products from Streptomyces wedmorensis, which confer fosfomycin resistance on Escherichia coli.

Together, the fomA and fomB genes in the fosfomycin biosynthetic gene cluster of Streptomyces wedmorensis confer high-level fosfomycin resistance on Escherichia coli. To elucidate their functions, the fomA and fomB genes were overexpressed in E. coli and the gene products were characterized. The recombinant FomA protein converted fosfomycin to fosfomycin monophosphate, which was inactive on E. coli, in the presence of a magnesium ion and ATP. On the other hand, the recombinant FomB protein did not inactivate fosfomycin. However, a reaction mixture containing FomA and FomB proteins converted fosfomycin to fosfomycin monophosphate and fosfomycin diphosphate in the presence of ATP and a magnesium ion, indicating that FomA and FomB catalyzed phosphorylations of fosfomycin and fosfomycin monophosphate, respectively. These results suggest that the self-resistance mechanism of the fosfomycin-producing organism S. wedmorensis is mono- and diphosphorylation of the phosphonate function of fosfomycin catalyzed by FomA and FomB.

Development of an improved assay for purine nucleoside kinase activity in cell extracts and detection of inosine kinase activity in Brevibacterium acetylicum ATCC 953, related species, and Corynebacterium flaccumfaciens ATCC 6887.

An improved assay was developed to detect direct purine nucleoside phosphorylating activity in cell-free extracts. Direct inosine phosphorylating activity was detected in 2 of 70 species tested. Both activities, which depended on magnesium ion and ATP, phosphorylated a hydroxyl group at the 5' position of inosine. The new assay was shown to be useful for screening of direct purine nucleoside phosphorylating activity and have the potential to detect inosine kinase in the presence of a background of nucleoside phosphorylase and purine phosphoribosyltransferase activities. Previously, the latter two activities made it difficult to correctly detect direct phosphorylation of inosine by inosine kinase.