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Theoretical Investigation of the Enzymatic Phosphoryl Transfer of ?-phosphoglucomutase: Revisiting Both Steps of the Catalytic Cycle  

SciTech Connect

Enzyme catalyzed phosphate transfer is a part of almost all metabolic processes. Such reactions are of central importance for the energy balance in all organisms and play important roles in cellular control at all levels. Mutases transfer a phosphoryl group while nucleases cleave the phosphodiester linkages between two nucleotides. The subject of our present study is the Lactococcus lactis ?-phosphoglucomutase (?-PGM), which effectively catalyzes the interconversion of ?-D-glucose-1-phosphate (?-G1P) to ?- D-glucose-6-phosphate (?-G6P) and vice versa via stabile intermediate ?-D-glucose-1,6-(bis)phosphate (?-G1,6diP) in the presence of Mg2+. In this paper we revisited the reaction mechanism of the phosphoryl transfer starting from the bisphosphate ?-G1,6diP in both directions (toward ?-G1P and ?-G6P) combining docking techniques and QM/MM theoretical method at the DFT/PBE0 level of theory. In addition we performed NEB (nudged elastic band) and free energy calculations to optimize the path and to identify the transition states and the energies involved in the catalytic cycle. Our calculations reveal that both steps proceed via dissociative pentacoordinated phosphorane, which is not a stabile intermediate but rather a transition state. In addition to the Mg2+ ion, Ser114 and Lys145 also play important roles in stabilizing the large negative charge on the phosphate through strong coordination with the phosphate oxygens and guiding the phosphate group throughout the catalytic process. The calculated energy barrier of the reaction for the ?-G1P to ?-G1,6diP step is only slightly higher than for the ?-G1,6diP to ?-G6P step (16.10 kcal mol-1 versus 15.10 kcal mol-1) and is in excellent agreement with experimental findings (14.65 kcal mol-1).

Elsasser, Brigitta M.; Dohmeier-Fischer, Silvia; Fels, Gregor



Arginine Coordination in Enzymatic Phosphoryl Transfer: Evaluation of the Effect of Arg166 Mutations in Escherichia Coli Alkaline Phosphatase  

SciTech Connect

Arginine residues are commonly found in the active sites of enzymes catalyzing phosphoryl transfer reactions. Numerous site-directed mutagenesis experiments establish the importance of these residues for efficient catalysis, but their role in catalysis is not clear. To examine the role of arginine residues in the phosphoryl transfer reaction, we have measured the consequences of mutations to arginine 166 in Escherichia coli alkaline phosphatase on hydrolysis of ethyl phosphate, on individual reaction steps in the hydrolysis of the covalent enzyme-phosphoryl intermediate, and on thio substitution effects. The results show that the role of the arginine side chain extends beyond its positive charge, as the Arg166Lys mutant is as compromised in activity as Arg166Ser. Through measurement of individual reaction steps, we construct a free energy profile for the hydrolysis of the enzyme-phosphate intermediate. This analysis indicates that the arginine side chain strengthens binding by {approx}3 kcal/mol and provides an additional 1-2 kcal/mol stabilization of the chemical transition state. A 2.1 {angstrom} X-ray diffraction structure of Arg166Ser AP is presented, which shows little difference in enzyme structure compared to the wild-type enzyme but shows a significant reorientation of the bound phosphate. Altogether, these results support a model in which the arginine contributes to catalysis through binding interactions and through additional transition state stabilization that may arise from complementarity of the guanidinum group to the geometry of the trigonal bipyramidal transition state.

O'Brien, P.J.; Lassila, J.K.; Fenn, T.D.; Zalatan, J.G.; Herschlag, D.



Mapping Sites of Protein Phosphorylation by Mass Spectrometry Utilizing a Chemical-Enzymatic Approach: Characterization of Products from ?-S1Casein Phosphopeptides  

PubMed Central

A novel chemical-enzymatic approach was developed to facilitate identification of phosphorylation sites in isolated phosphoproteins. ESI–TOF mass spectrometry was used to characterize products from the chemical-enzymatic cleavage of specific phosphorylation sites in bovine ?-S1 casein and synthetic phosphopeptides containing substitutions at a single phosphorylation site. Further refinements to this approach for identification of protein phosphorylation sites and its utility for the quantification of phosphopeptides by isotope-dilution mass spectrometry are presented. PMID:15822919

McCormick, Daniel J.; Holmes, Michael W.; Muddiman, David C.; Madden, Benjamin J.



Intramolecular domaindomain association/ dissociation and phosphoryl transfer in the mannitol  

E-print Network

of Health, Bethesda, MD 20892-0520 Edited by Alan R. Fersht, University of Cambridge, Cambridge, United- tramolecular (from the A to the B domain and from the B domain to the incoming sugar bound to the C domain to ensure effective phosphoryl transfer along the pathway. We have inves- tigated the rate of intramolecular

Clore, G. Marius


Phase-Transfer Catalysis: Phosphorylation of ?-Dicarbonyl Compounds  

Microsoft Academic Search

Although we have found that diethyl phosphorochloridate and diethyl phosphorochloridothionate react exothermically with hydroxide ions under phase-transfer conditions to yield the corresponding pyrophosphates (I-II), we now report that the phase-transfer catalysed phosphorylation of pentan-2, 4-dione and of ethyl acetoacetate produces the enol phosphates (III and IV). The yields are generally better than those obtained using the sodium or copper salts

R. Alan Jones; Santckh Singh Badesha



Mechanism of phosphoryl transfer catalyzed by shikimate kinase from Mycobacterium tuberculosis.  


The structural mechanism of the catalytic functioning of shikimate kinase from Mycobacterium tuberculosis was investigated on the basis of a series of high-resolution crystal structures corresponding to individual steps in the enzymatic reaction. The catalytic turnover of shikimate and ATP into the products shikimate-3-phosphate and ADP, followed by release of ADP, was studied in the crystalline environment. Based on a comparison of the structural states before initiation of the reaction and immediately after the catalytic step, we derived a structural model of the transition state that suggests that phosphoryl transfer proceeds with inversion by an in-line associative mechanism. The random sequential binding of shikimate and nucleotides is associated with domain movements. We identified a synergic mechanism by which binding of the first substrate may enhance the affinity for the second substrate. PMID:17020768

Hartmann, Marcus D; Bourenkov, Gleb P; Oberschall, Attila; Strizhov, Nicolai; Bartunik, Hans D



Enzymic phosphoryl transfer to carbon and oxygen acceptors: An investigation of the biosynthesis of 2-aminoethylphosphonic acid in Tetrahymena pyriformis W. and the kinetic mechanism and cofactor controlled substrate specificity of yeast inorganic pyrophosphatase  

Microsoft Academic Search

This research is concerned with the study of two enzymatic systems which catalyze phosphoryl transfer reactions to carbon and oxygen acceptors. The first portion of this study is concerned with the elucidation of the T. pyriformis 2-aminoethylphosphonate (AEP) biosynthetic pathway. The de novo formation of AEP from exogenously added precursors in Tetrahymena cell-free preparations was evaluated by using radioisotopic techniques



Continuous monitoring of enzymatic activity within native electrophoresis gels: Application to mitochondrial oxidative phosphorylation complexes  

PubMed Central

Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction media recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase where catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed. PMID:22975200

Covian, Raul; Chess, David; Balaban, Robert S.



Phosphoryl transfer by a concerted reaction mechanism in UMP/CMP-kinase.  

PubMed Central

The reaction mechanism of phosphoryl transfer catalyzed by UMP/CMP-kinase from Dictyostelium discoideum was investigated by semiempirical AM1 molecular orbital computations of an active site model system derived from crystal structures that contain a transition state analog or a bisubstrate inhibitor. The computational results suggest that the nucleoside monophosphate must be protonated for the forward reaction while it is unprotonated in the presence of aluminium fluoride, a popular transition state analog for phosphoryl transfer reactions. Furthermore, a compactification of the active site model system during the reaction and for the corresponding complex containing AlF3 was observed. For the active site residues that are part of the LID domain, conformational flexibility during the reaction proved to be crucial. On the basis of the calculations, a concerted phosphoryl transfer mechanism is suggested that involves the synchronous shift of a proton from the monophosphate to the transferred PO3-group. The proposed mechanism is thus analogous to the phosphoryl transfer mechanism in cAMP-dependent protein kinase that phosphorylates the hydroxyl groups of serine residues. PMID:11152133

Hutter, M. C.; Helms, V.



Parkin-catalyzed Ubiquitin-Ester Transfer Is Triggered by PINK1-dependent Phosphorylation*  

PubMed Central

PINK1 and PARKIN are causal genes for autosomal recessive familial Parkinsonism. PINK1 is a mitochondrial Ser/Thr kinase, whereas Parkin functions as an E3 ubiquitin ligase. Under steady-state conditions, Parkin localizes to the cytoplasm where its E3 activity is repressed. A decrease in mitochondrial membrane potential triggers Parkin E3 activity and recruits it to depolarized mitochondria for ubiquitylation of mitochondrial substrates. The molecular basis for how the E3 activity of Parkin is re-established by mitochondrial damage has yet to be determined. Here we provide in vitro biochemical evidence for ubiquitin-thioester formation on Cys-431 of recombinant Parkin. We also report that Parkin forms a ubiquitin-ester following a decrease in mitochondrial membrane potential in cells, and that this event is essential for substrate ubiquitylation. Importantly, the Parkin RING2 domain acts as a transthiolation or acyl-transferring domain rather than an E2-recruiting domain. Furthermore, formation of the ubiquitin-ester depends on PINK1 phosphorylation of Parkin Ser-65. A phosphorylation-deficient mutation completely inhibited formation of the Parkin ubiquitin-ester intermediate, whereas phosphorylation mimics, such as Ser to Glu substitution, enabled partial formation of the intermediate irrespective of Ser-65 phosphorylation. We propose that PINK1-dependent phosphorylation of Parkin leads to the ubiquitin-ester transfer reaction of the RING2 domain, and that this is an essential step in Parkin activation. PMID:23754282

Iguchi, Masahiro; Kujuro, Yuki; Okatsu, Kei; Koyano, Fumika; Kosako, Hidetaka; Kimura, Mayumi; Suzuki, Norihiro; Uchiyama, Shinichiro; Tanaka, Keiji; Matsuda, Noriyuki



Inhibition of ALK enzymatic activity in T-cell lymphoma cells induces apoptosis and suppresses proliferation and STAT3 phosphorylation independently of Jak3.  


Aberrant expression of the ALK tyrosine kinase as a chimeric protein with nucleophosmin (NPM) and other partners plays a key role in malignant cell transformation of T-lymphocytes and other cells. Here we report that two small-molecule, structurally related, quinazoline-type compounds, WHI-131 and WHI-154, directly inhibit enzymatic activity of NPM/ALK as demonstrated by in vitro kinase assays using a synthetic tyrosine-rich oligopeptide and the kinase itself as the substrates. The inhibition of NPM/ALK activity resulted in malignant T cells in suppression of their growth, induction of apoptosis and inhibition of tyrosine phosphorylation of STAT3, the key effector of the NPM/ALK-induced oncogenesis. We also show that the STAT3 tyrosine phosphorylation is mediated in the malignant T cells by NPM/ALK independently of Jak3 kinase as evidenced by the presence of STAT3 phosphorylation in the NPM/ALK-transfected BaF3 cells that do not express detectable Jak3 and in the NPM/ALK-positive malignant T cells with either Jak3 activity impaired by a pan-Jak or Jak3-selective inhibitor or Jak3 expression abrogated by Jak3 siRNA. The above results represent the 'proof-of-principle' experiments with regard to the ALK enzymatic activity as an attractive therapeutic target in T-cell lymphomas and other malignancies that express the kinase in an active form. PMID:16170336

Marzec, Michal; Kasprzycka, Monika; Ptasznik, Andrzej; Wlodarski, Pawel; Zhang, Qian; Odum, Niels; Wasik, Mariusz A



Conserved phosphoryl transfer mechanisms within kinase families and the role of the C8 proton of ATP in the activation of phosphoryl transfer  

PubMed Central

Background The kinome is made up of a large number of functionally diverse enzymes, with the classification indicating very little about the extent of the conserved kinetic mechanisms associated with phosphoryl transfer. It has been demonstrated that C8-H of ATP plays a critical role in the activity of a range of kinase and synthetase enzymes. Results A number of conserved mechanisms within the prescribed kinase fold families have been identified directly utilizing the C8-H of ATP in the initiation of phosphoryl transfer. These mechanisms are based on structurally conserved amino acid residues that are within hydrogen bonding distance of a co-crystallized nucleotide. On the basis of these conserved mechanisms, the role of the nucleotide C8-H in initiating the formation of a pentavalent intermediate between the ?-phosphate of the ATP and the substrate nucleophile is defined. All reactions can be clustered into two mechanisms by which the C8-H is induced to be labile via the coordination of a backbone carbonyl to C6-NH2 of the adenyl moiety, namely a "push" mechanism, and a "pull" mechanism, based on the protonation of N7. Associated with the "push" mechanism and "pull" mechanisms are a series of proton transfer cascades, initiated from C8-H, via the tri-phosphate backbone, culminating in the formation of the pentavalent transition state between the ?-phosphate of the ATP and the substrate nucleophile. Conclusions The "push" mechanism and a "pull" mechanism are responsible for inducing the C8-H of adenyl moiety to become more labile. These mechanisms and the associated proton transfer cascades achieve the proton transfer via different family-specific conserved sets of amino acids. Each of these mechanisms would allow for the regulation of the rate of formation of the pentavalent intermediate between the ATP and the substrate nucleophile. Phosphoryl transfer within kinases is therefore a specific event mediated and regulated via the coordination of the adenyl moiety of ATP and the C8-H of the adenyl moiety. PMID:22397702



Unblocking the sink: improved CID-based analysis of phosphorylated peptides by enzymatic removal of the basic C-terminal residue.  


A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H3PO4), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H3PO4 elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H3PO4 upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available. PMID:24297471

Lanucara, Francesco; Lee, Dave Chi Hoo; Eyers, Claire E



A redox beginning: Which came first phosphoryl, acyl, or electron transfer ?. [Abstract only  

NASA Technical Reports Server (NTRS)

Thermodynamic and kinetic information available on the synthesis of prebiotic monomers and polymers will be examined in order to illuminate the prebiotic plausibility of polymer syntheses based on (a) phosphoryl transfer that yields phosphodiester polymers, (b) acyl transfer that gives polyamides, and (c) electron transfer that produces polydisulfide or poly(thio)ester polymers. New experimental results on the oxidative polymerization of 2,3-dimercaptopropanol by ferric ions on the surface of ferric hydroxide oxide will be discussed as a chemical model of polymerization by electron transfer. This redox polymerization that yields polymers with a polydisulfide backbone was found to give oligomers up to the 15-mer from 1 mM of 2,3-dimercaptopropanol after one day at 25 C. High pressure liquid chromatography (HPLC) analysis of the oligomers was carried out on an Alltech OH-100 column eluted with acetonitrile-water.

Weber, Arthur L.



Pentavalent Organo-Vanadates as Transition State Analogues for Phosphoryl Transfer Reactions  

PubMed Central

Pentavalent organo-vanadates have been put forth as transition state analogues for a variety of phosphoryl transfer reactions. In particular, uridine 2?,3?-cyclic vanadate (U>v) has been proposed to resemble the transition state during catalysis by ribonuclease A (RNase A). Here, this hypothesis is tested. Lys41 of RNase A is known to donate a hydrogen bond to a nonbridging phosphoryl oxygen in the transition state during catalysis. Site-directed mutagenesis and semisynthesis were used to create enzymes with natural and nonnatural amino acid residues at position 41. These variants differ by 105-fold in their kcat/Km values for catalysis, but <40-fold in their Ki values for inhibition of catalysis by U>v. Plots of logKi vs log(Km/kcat) for three distinct substrates [poly(cytidylic acid), uridine 3?-(p-nitrophenyl phosphate), and cytidine 2?,3?-cyclic phosphate] have slopes that range from 0.25 and 0.36. These plots would have a slope of unity if U>v were a perfect transition state analogue. Values of Ki for U>v correlate weakly with the equilibrium dissociation constant for the enzymic complexes with substrate or product, indicating that U>v bears some resemblance to the substrate and product as well as the transition state. Thus, U>v is a transition state analogue for RNase A, but only a marginal one. This finding indicates that a pentavalent organo-vanadate cannot necessarily be the basis for a rigorous analysis of the transition state for a phosphoryl transfer reaction. PMID:21423825

Messmore, June M.; Raines, Ronald T.



Application of Electron Transfer Dissociation Mass Spectrometry in Analyses of Non-enzymatically Glycated Peptides  

PubMed Central

Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the context of development of diabetic complications. The fragmentation behavior of glycated peptides produced from reaction of D-glucose with lysine residues was investigated by electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. It was found that high abundance ions corresponding to various degrees of neutral water losses, as well as furylium ion production, dominate the CID spectra, and that the sequence informative b and y ions were rarely observed when Amadori-modified peptides were fragmented. Contrary to what was observed under CID conditions, ions corresponding to neutral losses of water or furylium ion production were not observed in the ETD spectra. Instead, abundant and almost complete series of c and z type ions were observed regardless of whether the modification site was located in the middle of the sequence or close to the N-terminus, greatly facilitating the peptide sequencing. This study strongly suggests that ETD is a better technique for proteomics studies of non-enzymatically glycated peptides and proteins. PMID:17279487

Zhang, Qibin; Frolov, Andrej; Tang, Ning; Hoffmann, Ralf; van de Goor, Tom; Metz, Thomas O.; Smith, Richard D.



Metal-free cAMP-dependent protein kinase can catalyze phosphoryl transfer.  


X-ray structures of several ternary product complexes of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have been determined with no bound metal ions and with Na(+) or K(+) coordinated at two metal-binding sites. The metal-free PKAc and the enzyme with alkali metals were able to facilitate the phosphoryl transfer reaction. In all studied complexes, the ATP and the substrate peptide (SP20) were modified into the products ADP and the phosphorylated peptide. The products of the phosphotransfer reaction were also found when ATP-?S, a nonhydrolyzable ATP analogue, reacted with SP20 in the PKAc active site containing no metals. Single turnover enzyme kinetics measurements utilizing (32)P-labeled ATP confirmed the phosphotransferase activity of the enzyme in the absence of metal ions and in the presence of alkali metals. In addition, the structure of the apo-PKAc binary complex with SP20 suggests that the sequence of binding events may become ordered in a metal-free environment, with SP20 binding first to prime the enzyme for subsequent ATP binding. Comparison of these structures reveals conformational and hydrogen bonding changes that might be important for the mechanism of catalysis. PMID:24786636

Gerlits, Oksana; Das, Amit; Keshwani, Malik M; Taylor, Susan; Waltman, Mary Jo; Langan, Paul; Heller, William T; Kovalevsky, Andrey



Ground State Destabilization by Anionic Nucleophiles Contributes to the Activity of Phosphoryl Transfer Enzymes  

PubMed Central

Enzymes stabilize transition states of reactions while limiting binding to ground states, as is generally required for any catalyst. Alkaline Phosphatase (AP) and other nonspecific phosphatases are some of Nature's most impressive catalysts, achieving preferential transition state over ground state stabilization of more than 1022-fold while utilizing interactions with only the five atoms attached to the transferred phosphorus. We tested a model that AP achieves a portion of this preference by destabilizing ground state binding via charge repulsion between the anionic active site nucleophile, Ser102, and the negatively charged phosphate monoester substrate. Removal of the Ser102 alkoxide by mutation to glycine or alanine increases the observed Pi affinity by orders of magnitude at pH 8.0. To allow precise and quantitative comparisons, the ionic form of bound Pi was determined from pH dependencies of the binding of Pi and tungstate, a Pi analog lacking titratable protons over the pH range of 5–11, and from the 31P chemical shift of bound Pi. The results show that the Pi trianion binds with an exceptionally strong femtomolar affinity in the absence of Ser102, show that its binding is destabilized by ?108-fold by the Ser102 alkoxide, and provide direct evidence for ground state destabilization. Comparisons of X-ray crystal structures of AP with and without Ser102 reveal the same active site and Pi binding geometry upon removal of Ser102, suggesting that the destabilization does not result from a major structural rearrangement upon mutation of Ser102. Analogous Pi binding measurements with a protein tyrosine phosphatase suggest the generality of this ground state destabilization mechanism. Our results have uncovered an important contribution of anionic nucleophiles to phosphoryl transfer catalysis via ground state electrostatic destabilization and an enormous capacity of the AP active site for specific and strong recognition of the phosphoryl group in the transition state. PMID:23843744

Andrews, Logan D.; Fenn, Tim D.; Herschlag, Daniel



Ground state destabilization by anionic nucleophiles contributes to the activity of phosphoryl transfer enzymes.  


Enzymes stabilize transition states of reactions while limiting binding to ground states, as is generally required for any catalyst. Alkaline Phosphatase (AP) and other nonspecific phosphatases are some of Nature's most impressive catalysts, achieving preferential transition state over ground state stabilization of more than 10²²-fold while utilizing interactions with only the five atoms attached to the transferred phosphorus. We tested a model that AP achieves a portion of this preference by destabilizing ground state binding via charge repulsion between the anionic active site nucleophile, Ser102, and the negatively charged phosphate monoester substrate. Removal of the Ser102 alkoxide by mutation to glycine or alanine increases the observed Pi affinity by orders of magnitude at pH 8.0. To allow precise and quantitative comparisons, the ionic form of bound P(i) was determined from pH dependencies of the binding of Pi and tungstate, a P(i) analog lacking titratable protons over the pH range of 5-11, and from the ³¹P chemical shift of bound P(i). The results show that the Pi trianion binds with an exceptionally strong femtomolar affinity in the absence of Ser102, show that its binding is destabilized by ?10?-fold by the Ser102 alkoxide, and provide direct evidence for ground state destabilization. Comparisons of X-ray crystal structures of AP with and without Ser102 reveal the same active site and P(i) binding geometry upon removal of Ser102, suggesting that the destabilization does not result from a major structural rearrangement upon mutation of Ser102. Analogous Pi binding measurements with a protein tyrosine phosphatase suggest the generality of this ground state destabilization mechanism. Our results have uncovered an important contribution of anionic nucleophiles to phosphoryl transfer catalysis via ground state electrostatic destabilization and an enormous capacity of the AP active site for specific and strong recognition of the phosphoryl group in the transition state. PMID:23843744

Andrews, Logan D; Fenn, Tim D; Herschlag, Daniel



Non-enzymatic synthesis of the coenzymes, uridine diphosphate glucose and cytidine diphosphate choline, and other phosphorylated metabolic intermediates  

NASA Astrophysics Data System (ADS)

The synthesis of uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P) has been accomplished under simulated prebiotic conditions using urea and cyanamide, two condensing agents considered to have been present on the primitive Earth. The synthesis of UDPG was carried out by reacting G1P and UTP at 70 °C for 24 hours in the presence of the condensing agents in an aqueous medium. CDP-choline was obtained under the same conditions by reacting choline phosphate and CTP. G1P and G6P were synthesized from glucose and inorganic phosphate at 70 °C for 16 hours. Separation and identification of the reaction products have been performed by paper chromatography, thin layer chromatography, enzymatic analysis and ion pair reverse phase high performance liquid chromatography. These results suggest that metabolic intermediates could have been synthesized on the primitive Earth from simple precursors by means of prebiotic condensing agents.

Mar, A.; Dworkin, J.; Oró, J.



Enzymatic synthesis of aroma compound xylosides using transfer reaction by Trichoderma longibrachiatum xylanase.  


Enzymatic synthesis of aroma compound xylosides was performed by Trichoderma longibrachiatum xylanase. Information concerning the nature of xylosides present in the reaction medium was obtained by GC-EI-MS, by GC-NCI-MS of TFA derivatives, and by positive FAB-MS of the reaction mixtures. Moreover, the structures of isolated benzyl beta-D-xylopyranoside and 4-O-beta-xylopyranosyl-beta-D-xylopyranoside were established by (1)H and (13)C NMR and heteronuclear two-dimensional ((1)H-(13)C) chemical shift correlation. The results obtained for hexyl and benzyl alcohol xylosides indicated that a reaction implying a transfer of one to two or three xylose units from xylan was involved. The enzyme was able to recognize xylobiose, xylotriose, and xylan as xylose donors. Benzyl xyloside, produced independently of xylobioside and xylotrioside, was found as the major kinetic product of the reaction. Benzyl xyloside was produced in higher quantities and at a higher rate than that obtained for the di- and trixyloside derivatives. The maximum production for benzyl xyloside, 1.29 g/L, was obtained in the presence of hexane (50%) used as cosolvent. Xylosides and xylobiosides of several aroma compounds, (Z)-hex-3-en-1-ol, heptan-2-ol, geraniol, nerol, and citronellol, were synthesized in different amounts, from 850 mg/L for (Z)-hex-3-en-1-yl xylosides to 1.5 mg/L for citronellyl xylosides. No synthesis occurred when menthol, linalool, and eugenol were used as acceptors. PMID:12236678

Kadi, Nadia; Belloy, Laurence; Chalier, Pascale; Crouzet, Jean Claude



Aluminum coordination chemistry and the inhibition of phosphoryl-transferring enzymes  

SciTech Connect

Aluminium ion is a potent inhibitor of the enzymes hexokinase (K/sub i/ = 0.16 and glycerokinase (K/sub i/ = 4.0 It has been shown that aluminum forms a complex with ATP that is 80 times more stable than the magnesium complex with ATP which is the normal substrate for phosphoryl-transferring enzymes. Kinetic studies performed on several kinases at pH 7.0 have shown that Al-ATP is a competitive inhibitor vs. Mg-ATP with moderate K/sub i/ values (0.1-0.5 mM) for creatine kinase(CK) and myokinase(MK), and weakly competitive (K/sub i/ > 0.5 mM) with acetate, galactose, arginine and gluconate kinases. Equilibrium dialysis binding studies indicate no significant binding of aluminum ion by the enzymes, while the interaction of aluminum ion with ADP and ATP has been characterized by /sup 13/C, /sup 27/Al, and /sup 31/P NMR spectroscopy. It appears that the inhibition by aluminum is as the Al-nucleotide complex rather than direct binding of free aluminum ion by the enzyme. Kinetic studies indicate that Al/sup 3 +/ inhibition of CK and MK is pH dependent with decreased values of K/sub i/ at lower pH. At pH 6.1 K/sub i/ = 25 for MK (160 at pH 7.0) and 53 for CK (240 at pH 7.0). This may be due to an increased effective concentration of aluminum ion at lower pH.

Furumo, N.C.; Viola, R.E.



Physical Properties and Enzymatic Digestibility of Phosphorylated ae, wx , and Normal Maize Starch Prepared at Different pH Levels 1  

Microsoft Academic Search

Cereal Chem. 76(6):938-943 Phosphorylated starches were prepared with sodium tripolyphosphate (STPP) at pH 6, 8, and 10 from waxy (wx, 3.3% amylose), normal (22.4% amylose), and two high-amylose (ae, 47 and 66% amylose) maize starches. After phosphorylation, the gelatinization peak temperature ( Tp) decreased and pasting peak viscosity (PV) increased for all the starches except wx, which showed a slight

Huijun Liu; Lawrence Ramsden; Harold Corke



Cardiac mitochondrial matrix and respiratory complex protein phosphorylation  

PubMed Central

It has become appreciated over the last several years that protein phosphorylation within the cardiac mitochondrial matrix and respiratory complexes is extensive. Given the importance of oxidative phosphorylation and the balance of energy metabolism in the heart, the potential regulatory effect of these classical signaling events on mitochondrial function is of interest. However, the functional impact of protein phosphorylation and the kinase/phosphatase system responsible for it are relatively unknown. Exceptions include the well-characterized pyruvate dehydrogenase and branched chain ?-ketoacid dehydrogenase regulatory system. The first task of this review is to update the current status of protein phosphorylation detection primarily in the matrix and evaluate evidence linking these events with enzymatic function or protein processing. To manage the scope of this effort, we have focused on the pathways involved in energy metabolism. The high sensitivity of modern methods of detecting protein phosphorylation and the low specificity of many kinases suggests that detection of protein phosphorylation sites without information on the mole fraction of phosphorylation is difficult to interpret, especially in metabolic enzymes, and is likely irrelevant to function. However, several systems including protein translocation, adenine nucleotide translocase, cytochrome c, and complex IV protein phosphorylation have been well correlated with enzymatic function along with the classical dehydrogenase systems. The second task is to review the current understanding of the kinase/phosphatase system within the matrix. Though it is clear that protein phosphorylation occurs within the matrix, based on 32P incorporation and quantitative mass spectrometry measures, the kinase/phosphatase system responsible for this process is ill-defined. An argument is presented that remnants of the much more labile bacterial protein phosphoryl transfer system may be present in the matrix and that the evaluation of this possibility will require the application of approaches developed for bacterial cell signaling to the mitochondria. PMID:22886415

Covian, Raul



Enzymatic catalysis and transfers in solution. I. Theory and computations, a unified view  

NASA Astrophysics Data System (ADS)

The transfer of hydride, proton, or H atom between substrate and cofactor in enzymes has been extensively studied for many systems, both experimentally and computationally. A simple equation for the reaction rate, an analog of an equation obtained earlier for electron transfer rates, is obtained, but now containing an approximate analytic expression for the bond rupture-bond forming feature of these H transfers. A ``symmetrization,'' of the potential energy surfaces is again introduced [R. A. Marcus, J. Chem. Phys. 43, 679 (1965); J. Phys. Chem. 72, 891 (1968)], together with Gaussian fluctuations of the remaining coordinates of the enzyme and solution needed for reaching the transition state. Combining the two expressions for the changes in the difference of the two bond lengths of the substrate-cofactor subsystem and in the fluctuation coordinates of the protein leading to the transition state, an expression is obtained for the free energy barrier. To this end a two-dimensional reaction space (m,n) is used that contains the relative coordinates of the H in the reactants, the heavy atoms to which it is bonded, and the protein/solution reorganization coordinate, all leading to the transition state. The resulting expression may serve to characterize in terms of specific parameters (two ``reorganization'' terms, thermodynamics, and work terms), experimental and computational data for different enzymes, and different cofactor-substrate systems. A related characterization was used for electron transfers. To isolate these factors from nuclear tunneling, when the H-tunneling effect is large, use of deuterium and tritium transfers is of course helpful, although tunneling has frequently and understandably dominated the discussions. A functional form is suggested for the dependence of the deuterium kinetic isotope effect (KIE) on ?G° and a different form for the 13C KIE. Pressure effects on deuterium and 13C KIEs are also discussed. Although formulated for a one-step transfer of a light particle in an enzyme, the results would also apply to single-step transfers of other atoms and groups in enzymes and in solution.

Marcus, R. A.



Lewis acid catalysis of phosphoryl transfer from a copper(II)-NTP complex in a kinase ribozyme.  


The chemical strategies used by ribozymes to enhance reaction rates are revealed in part from their metal ion and pH requirements. We find that kinase ribozyme K28(1-77)C, in contrast with previously characterized kinase ribozymes, requires Cu(2+) for optimal catalysis of thiophosphoryl transfer from GTP?S. Phosphoryl transfer from GTP is greatly reduced in the absence of Cu(2+), indicating a specific catalytic role independent of any potential interactions with the GTP?S thiophosphoryl group. In-line probing and ATP?S competition both argue against direct Cu(2+) binding by RNA; rather, these data establish that Cu(2+) enters the active site within a Cu(2+)•GTP?S or Cu(2+)•GTP chelation complex, and that Cu(2+)•nucleobase interactions further enforce Cu(2+) selectivity and position the metal ion for Lewis acid catalysis. Replacing Mg(2+) with [Co(NH3)6](3+) significantly reduced product yield, but not kobs, indicating that the role of inner-sphere Mg(2+) coordination is structural rather than catalytic. Replacing Mg(2+) with alkaline earths of increasing ionic radii (Ca(2+), Sr(2+) and Ba(2+)) gave lower yields and approximately linear rates of product accumulation. Finally, we observe that reaction rates increased with pH in log-linear fashion with an apparent pKa = 8.0 ± 0.1, indicating deprotonation in the rate-limiting step. PMID:23358821

Biondi, Elisa; Poudyal, Raghav R; Forgy, Joshua C; Sawyer, Andrew W; Maxwell, Adam W R; Burke, Donald H



Lewis acid catalysis of phosphoryl transfer from a copper(II)-NTP complex in a kinase ribozyme  

PubMed Central

The chemical strategies used by ribozymes to enhance reaction rates are revealed in part from their metal ion and pH requirements. We find that kinase ribozyme K28(1-77)C, in contrast with previously characterized kinase ribozymes, requires Cu2+ for optimal catalysis of thiophosphoryl transfer from GTP?S. Phosphoryl transfer from GTP is greatly reduced in the absence of Cu2+, indicating a specific catalytic role independent of any potential interactions with the GTP?S thiophosphoryl group. In-line probing and ATP?S competition both argue against direct Cu2+ binding by RNA; rather, these data establish that Cu2+ enters the active site within a Cu2+•GTP?S or Cu2+•GTP chelation complex, and that Cu2+•nucleobase interactions further enforce Cu2+ selectivity and position the metal ion for Lewis acid catalysis. Replacing Mg2+ with [Co(NH3)6]3+ significantly reduced product yield, but not kobs, indicating that the role of inner-sphere Mg2+ coordination is structural rather than catalytic. Replacing Mg2+ with alkaline earths of increasing ionic radii (Ca2+, Sr2+ and Ba2+) gave lower yields and approximately linear rates of product accumulation. Finally, we observe that reaction rates increased with pH in log-linear fashion with an apparent pKa = 8.0 ± 0.1, indicating deprotonation in the rate-limiting step. PMID:23358821

Biondi, Elisa; Poudyal, Raghav R.; Forgy, Joshua C.; Sawyer, Andrew W.; Maxwell, Adam W. R.; Burke, Donald H.



An enzymatic bridge between carbohydrate and amino acid metabolism: regulation of glutamate dehydrogenase by reversible phosphorylation in a severe hypoxia-tolerant crayfish.  


Glutamate dehydrogenase (GDH) (EC is a crucial enzyme involved in bridging two metabolic pathways, gating the use of glutamate for either amino acid metabolism, or carbohydrate metabolism. The present study investigated GDH from tail muscle of the freshwater crayfish Orconectes virilis exploring changes to kinetic properties, phosphorylation levels and structural stability between two forms of the enzyme (aerobic control and 20-h severe hypoxic). Evidence indicated that GDH was converted to a high phosphate form under oxygen limitation. ProQ Diamond phosphoprotein staining showed a 42% higher bound phosphate content on GDH from muscle of severely hypoxic crayfish compared with the aerobic form, and treatment of this GDH with commercial phosphatase (alkaline phosphatase), and treatments that stimulated the activities of different endogenous protein phosphatases (stimulating PP1 + PP2A, PP2B, and PP2C) yielded significant increases in the fold activation by ADP of GDH from both control and severe hypoxic conditions. By contrast, stimulation of the activities of endogenous protein kinases (AMPK, PKA or CaMK) significantly reduced the ADP fold activation from control animals. The physiological consequence of severe hypoxia-induced GDH phosphorylation may be to suppress GDH activity under low oxygen, shutting off this critical bridge point between two metabolic pathways. PMID:22076534

Dawson, Neal J; Storey, Kenneth B



The role of the C8 proton of ATP in the regulation of phosphoryl transfer within kinases and synthetases  

PubMed Central

Background The kinome comprises functionally diverse enzymes, with the current classification indicating very little about the extent of conserved regulatory mechanisms associated with phosphoryl transfer. The apparent Km of the kinases ranges from less than 0.4 ?M to in excess of 1000 ?M for ATP. It is not known how this diverse range of enzymes mechanistically achieves the regulation of catalysis via an affinity range for ATP varying by three-orders of magnitude. Results We have demonstrated a previously undiscovered mechanism in kinase and synthetase enzymes where the overall rate of reaction is regulated via the C8-H of ATP. Using ATP deuterated at the C8 position (C8D-ATP) as a molecular probe it was shown that the C8-H plays a direct role in the regulation of the overall rate of reaction in a range of kinase and synthetase enzymes. Using comparative studies on the effect of the concentration of ATP and C8D-ATP on the activity of the enzymes we demonstrated that not only did C8D-ATP give a kinetic isotope effect (KIE) but the KIE's obtained are clearly not secondary KIE effects as the magnitude of the KIE in all cases was at least 2 fold and in most cases in excess of 7 fold. Conclusions Kinase and synthetase enzymes utilise C8D-ATP in preference to non-deuterated ATP. The KIE obtained at low ATP concentrations is clearly a primary KIE demonstrating strong evidence that the bond to the isotopically substituted hydrogen is being broken. The effect of the ATP concentration profile on the KIE was used to develop a model whereby the C8H of ATP plays a role in the overall regulation of phosphoryl transfer. This role of the C8H of ATP in the regulation of substrate binding appears to have been conserved in all kinase and synthetase enzymes as one of the mechanisms associated with binding of ATP. The induction of the C8H to be labile by active site residues coordinated to the ATP purine ring may play a significant role in explaining the broad range of Km associated with kinase enzymes. PMID:21749731



Photosystem II Core Phosphorylation Heterogeneity, Differential Herbicide Binding, and Regulation of Electron Transfer in Photosystem II Preparations from Spinach 1  

PubMed Central

The effect of photosystem II core phosphorylation on the secondary quinone acceptor of photosystem II (QB) domain environment was analyzed by comparative herbicide-binding studies with photosystem II preparations from spinach (Spinacia oleracea L.). It was found that phosphorylation reduces the binding affinity for most photosynthetic herbicides. The binding of synthetic quinones and of the electron acceptor 2,6-dichlorophenolindophenol is also reduced by photosystem II phosphorylation. Four photosystem II core populations isolated from membranes showed different extents of phosphorylation as well as different degrees of affinity for photosynthetic herbicides. These findings support the idea that heterogeneity of photosystem II observed in vivo could be, in part, due to phosphorylation. Images Figure 1 PMID:16653222

Giardi, Maria T.; Rigoni, Fernanda; Barbato, Roberto



Acylation of phosphoryl- and thiophosphorylacetonitriles under phase transfer catalysis conditions and the keto-enol tautomerism of phosphorus-substituted acylacetonitriles  

Microsoft Academic Search

A procedure was developed for acylation of phosphoryl- and thiophosphorylacetonitriles under phase transfer catalysis conditions.\\u000a The reaction in the solid KOH\\/MeCN system affordedC-acylation products in high yields. In the individual state and in aprotic solvents, these products exist in the enol form\\u000a (Z isomers) stabilizedvia a strong intramolecular hydrogen bond. In hydroxyl-containing media and in aprotic bipolar solvents, these compounds

T. A. Mastryukova; I. L. Odinets; O. I. Artyushin; R. M. Kalyanova; K. A. Lyssenko; A. G. Matveeva; P. V. Petrovskii; M. I. Kabachnik



Insights into the Phosphoryl Transfer Mechanism of Cyclin-dependent Protein Kinases from Ab Initio QM/MM Free-energy Studies  

PubMed Central

Phosphorylation reactions catalyzed by kinases and phosphatases play an indispensible role in cellular signaling, and their malfunctioning is implicated in many diseases. A better understanding of the catalytic mechanism will help design novel and effective mechanism-based inhibitors of these enzymes. In this work, ab initio quantum mechanical/molecular mechanical studies are reported for the phosphoryl transfer reaction catalyzed by a cyclin-dependent kinase, CDK2. Our results suggest that an active-site Asp residue, rather than ATP as previously proposed, serves as the general base to activate the Ser nucleophile. The corresponding transition state features a dissociative, metaphosphate-like structure, stabilized by the Mg2+ ion and several hydrogen bonds. The calculated free-energy barrier is consistent with experimental values. Implications of our results in this and other protein kinases are discussed. PMID:21999515

Smith, Gregory K.; Ke, Zhihong; Hengge, Alvan C.



Enzymatic regeneration of adenosine triphosphate cofactor  

NASA Technical Reports Server (NTRS)

Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

Marshall, D. L.



Modifications on the hydrogen bond network by mutations of Escherichia coli copper efflux oxidase affect the process of proton transfer to dioxygen leading to alterations of enzymatic activities  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer Proton transfer pathway to dioxygen in CueO was identified. Black-Right-Pointing-Pointer Glu506 is the key amino acid to transport proton. Black-Right-Pointing-Pointer The Ala mutation at Glu506 formed a compensatory proton transfer pathway. Black-Right-Pointing-Pointer The Ile mutation at Glu506 shut down the hydrogen bond network. -- Abstract: CueO has a branched hydrogen bond network leading from the exterior of the protein molecule to the trinuclear copper center. This network transports protons in the four-electron reduction of dioxygen. We replaced the acidic Glu506 and Asp507 residues with the charged and uncharged amino acid residues. Peculiar changes in the enzyme activity of the mutants relative to the native enzyme indicate that an acidic amino acid residue at position 506 is essential for effective proton transport. The Ala mutation resulted in the formation of a compensatory hydrogen bond network with one or two extra water molecules. On the other hand, the Ile mutation resulted in the complete shutdown of the hydrogen bond network leading to loss of enzymatic activities of CueO. In contrast, the hydrogen bond network without the proton transport function was constructed by the Gln mutation. These results exerted on the hydrogen bond network in CueO are discussed in comparison with proton transfers in cytochrome oxidase.

Kajikawa, Takao; Kataoka, Kunishige [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan)] [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan); Sakurai, Takeshi, E-mail: [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan)] [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan)



The crystal structure of Mycobacterium tuberculosis adenylate kinase in complex with two molecules of ADP and Mg2+ supports an associative mechanism for phosphoryl transfer.  


The crystal structure of Mycobacterium tuberculosis adenylate kinase (MtAK) in complex with two ADP molecules and Mg2+ has been determined at 1.9 A resolution. Comparison with the solution structure of the enzyme, obtained in the absence of substrates, shows significant conformational changes of the LID and NMP-binding domains upon substrate binding. The ternary complex represents the state of the enzyme at the start of the backward reaction (ATP synthesis). The structure is consistent with a direct nucleophilic attack of a terminal oxygen from the acceptor ADP molecule on the beta-phosphate from the donor substrate, and both the geometry and the distribution of positive charge in the active site support the hypothesis of an associative mechanism for phosphoryl transfer. PMID:16672241

Bellinzoni, Marco; Haouz, Ahmed; Graña, Martin; Munier-Lehmann, Hélène; Shepard, William; Alzari, Pedro M



Control of pRB phosphorylation  

Microsoft Academic Search

Two opposing enzymatic reactions control the activity of the retinoblastoma tumour suppressor protein, pRB. Phosphorylation inactivates pRB's ability to sequester miscellaneous cellular proteins, mostly involved in regulating gene transcription, whereas pRB dephosphorylation restores this ability. For some time now it has been suspected that members of the cyclin\\/cyclin-dependent kinase (cyclin\\/cdk) family mediate pRB inactivation. Recent results indicate that pRB phosphorylation

Sibylle Mittnacht



Insights into the Phosphoryl Transfer Catalyzed by cAMP-Dependent Protein Kinase: An X-ray Crystallographic Study of Complexes with Various Metals and Peptide Substrate SP20  

PubMed Central

X-ray structures of several ternary substrate and product complexes of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have been determined with different bound metal ions. In the PKAc complexes, Mg2+, Ca2+, Sr2+, and Ba2+ metal ions could bind to the active site and facilitate the phosphoryl transfer reaction. ATP and a substrate peptide (SP20) were modified, and the reaction products ADP and the phosphorylated peptide were found trapped in the enzyme active site. Finally, we determined the structure of a pseudo-Michaelis complex containing Mg2+, nonhydrolyzable AMP-PCP (?,?-methyleneadenosine 5?-triphosphate) and SP20. The product structures together with the pseudo-Michaelis complex provide snapshots of different stages of the phosphorylation reaction. Comparison of these structures reveals conformational, coordination, and hydrogen bonding changes that might occur during the reaction and shed new light on its mechanism, roles of metals, and active site residues. PMID:23672593



Insights into the phosphoryl transfer catalyzed by cAMP-dependent protein kinase: an X-ray crystallographic study of complexes with various metals and peptide substrate SP20.  


X-ray structures of several ternary substrate and product complexes of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have been determined with different bound metal ions. In the PKAc complexes, Mg(2+), Ca(2+), Sr(2+), and Ba(2+) metal ions could bind to the active site and facilitate the phosphoryl transfer reaction. ATP and a substrate peptide (SP20) were modified, and the reaction products ADP and the phosphorylated peptide were found trapped in the enzyme active site. Finally, we determined the structure of a pseudo-Michaelis complex containing Mg(2+), nonhydrolyzable AMP-PCP (?,?-methyleneadenosine 5'-triphosphate) and SP20. The product structures together with the pseudo-Michaelis complex provide snapshots of different stages of the phosphorylation reaction. Comparison of these structures reveals conformational, coordination, and hydrogen bonding changes that might occur during the reaction and shed new light on its mechanism, roles of metals, and active site residues. PMID:23672593

Gerlits, Oksana; Waltman, Mary Jo; Taylor, Susan; Langan, Paul; Kovalevsky, Andrey



Biological Phosphoryl-Transfer Reactions  

E-print Network

The Annual Review of Biochemistry is online at This article's doi: 10.1146/annurev-biochem-060409-092741 Copyright c 2011 by Annual Reviews. All rights reserved 0066StanfordUniversity-MainCampus-RobertCrownLawLibraryon12/12/11.Forpersonaluseonly. Click here for quick links to Annual Reviews content online, including

Herschlag, Dan


Structural Characterizations of Glycerol Kinase: Unraveling Phosphorylation-Induced Long-Range Activation  

SciTech Connect

Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft. We reported earlier that some mutations of His232 altered enzyme activities; we present here the crystal structures of these mutant GlpK enzymes. The structure of a mutant enzyme with enhanced enzymatic activity, His232Arg, reveals that residues at the catalytic cleft are more optimally aligned to bind ATP and mediate phosphoryl transfer. Specifically, the position of Arg18 in His232Arg shifts by approximately 1 A when compared to its position in wild-type (WT), His232Ala, and His232Glu enzymes. This new conformation of Arg18 is more optimally positioned at the presumed gamma-phosphate location of ATP, close to the glycerol substrate. In addition to structural changes exhibited at the active site, the conformational stability of the activation loop is decreased, as reflected by an approximately 35% increase in B factors ('thermal factors') in a mutant enzyme displaying diminished activity, His232Glu. Correlating conformational changes to alteration of enzymatic activities in the mutant enzymes identifies distinct localized regions that can have profound effects on intramolecular signal transduction. Alterations in pairwise interactions across the dimer interface can communicate phosphorylation states over 25 A from the activation loop to the catalytic cleft, positioning Arg18 to form favorable interactions at the beta,gamma-bridging position with ATP. This would offset loss of the hydrogen bonds at the gamma-phosphate of ATP during phosphoryl transfer to glycerol, suggesting that appropriate alignment of the second substrate of glycerol kinase, the ATP molecule, may largely determine the rate of glycerol 3-phosphate production.

Yeh, Joanne I.; Kettering, Regina; Saxl, Ruth; Bourand, Alexa; Darbon, Emmanuelle; Joly, Nathalie; Briozzo, Pierre; Deutscher, Josef; (Pitt); (CNRS-CRMD)



An insert-based enzymatic cell culture system to rapidly and reversibly induce hypoxia: investigations of hypoxia-induced cell damage, protein expression and phosphorylation in neuronal IMR-32 cells  

PubMed Central

SUMMARY Ischemia-reperfusion injury and tissue hypoxia are of high clinical relevance because they are associated with various pathophysiological conditions such as myocardial infarction and stroke. Nevertheless, the underlying mechanisms causing cell damage are still not fully understood, which is at least partially due to the lack of cell culture systems for the induction of rapid and transient hypoxic conditions. The aim of the study was to establish a model that is suitable for the investigation of cellular and molecular effects associated with transient and long-term hypoxia and to gain insights into hypoxia-mediated mechanisms employing a neuronal culture system. A semipermeable membrane insert system in combination with the hypoxia-inducing enzymes glucose oxidase and catalase was employed to rapidly and reversibly generate hypoxic conditions in the culture medium. Hydrogen peroxide assays, glucose measurements and western blotting were performed to validate the system and to evaluate the effects of the generated hypoxia on neuronal IMR-32 cells. Using the insert-based two-enzyme model, hypoxic conditions were rapidly induced in the culture medium. Glucose concentrations gradually decreased, whereas levels of hydrogen peroxide were not altered. Moreover, a rapid and reversible (onoff) generation of hypoxia could be performed by the addition and subsequent removal of the enzyme-containing inserts. Employing neuronal IMR-32 cells, we showed that 3 hours of hypoxia led to morphological signs of cellular damage and significantly increased levels of lactate dehydrogenase (a biochemical marker of cell damage). Hypoxic conditions also increased the amounts of cellular procaspase-3 and catalase as well as phosphorylation of the pro-survival kinase Akt, but not Erk1/2 or STAT5. In summary, we present a novel framework for investigating hypoxia-mediated mechanisms at the cellular level. We claim that the model, the first of its kind, enables researchers to rapidly and reversibly induce hypoxic conditions in vitro without unwanted interference of the hypoxia-inducing agent on the cultured cells. The system could help to further unravel hypoxia-associated mechanisms that are clinically relevant in various tissues and organs. PMID:24046359

Huang, Ying; Zitta, Karina; Bein, Berthold; Steinfath, Markus; Albrecht, Martin



Enzymatic depolymerization of coal  

SciTech Connect

This patent describes a method for enzymatically depolymerizing polymeric substituents of coal. It comprises: combining in an aqueous solution, in which a lignin peroxidase is enzymatically active, an aqueous soluble polymeric coal substrate, the lignin peroxidase, hydrogen peroxide and oxygen.

Wood, W.A.; Wondrack, L.M.



Activating PER Repressor through a DBT-Directed Phosphorylation Switch  

PubMed Central

Protein phosphorylation plays an essential role in the generation of circadian rhythms, regulating the stability, activity, and subcellular localization of certain proteins that constitute the biological clock. This study examines the role of the protein kinase Doubletime (DBT), a Drosophila ortholog of human casein kinase I (CKI)?/?. An enzymatically active DBT protein is shown to directly phosphorylate the Drosophila clock protein Period (PER). DBT-dependent phosphorylation sites are identified within PER, and their functional significance is assessed in a cultured cell system and in vivo. The perS mutation, which is associated with short-period (19-h) circadian rhythms, alters a key phosphorylation target within PER. Inspection of this and neighboring sequence variants indicates that several DBT-directed phosphorylations regulate PER activity in an integrated fashion: Alternative phosphorylations of two adjoining sequence motifs appear to be associated with switch-like changes in PER stability and repressor function. PMID:18666831

Kivimae, Saul; Saez, Lino; Young, Michael W



Biofuel cells: enhanced enzymatic bioelectrocatalysis.  


Enzymatic biofuel cells represent an emerging technology that can create electrical energy from biologically renewable catalysts and fuels. A wide variety of redox enzymes have been employed to create unique biofuel cells that can be used in applications such as implantable power sources, energy sources for small electronic devices, self-powered sensors, and bioelectrocatalytic logic gates. This review addresses the fundamental concepts necessary to understand the operating principles of biofuel cells, as well as recent advances in mediated electron transfer- and direct electron transfer-based biofuel cells, which have been developed to create bioelectrical devices that can produce significant power and remain stable for long periods. PMID:22524222

Meredith, Matthew T; Minteer, Shelley D



Biofuel Cells: Enhanced Enzymatic Bioelectrocatalysis  

NASA Astrophysics Data System (ADS)

Enzymatic biofuel cells represent an emerging technology that can create electrical energy from biologically renewable catalysts and fuels. A wide variety of redox enzymes have been employed to create unique biofuel cells that can be used in applications such as implantable power sources, energy sources for small electronic devices, self-powered sensors, and bioelectrocatalytic logic gates. This review addresses the fundamental concepts necessary to understand the operating principles of biofuel cells, as well as recent advances in mediated electron transfer- and direct electron transfer-based biofuel cells, which have been developed to create bioelectrical devices that can produce significant power and remain stable for long periods.

Meredith, Matthew T.; Minteer, Shelley D.



Catalytic and regulatory roles of divalent metal cations on the phosphoryl-transfer mechanism of ADP-dependent sugar kinases from hyperthermophilic archaea.  


In some archaea, glucose degradation proceeds through a modified version of the Embden-Meyerhof pathway where glucose and fructose-6-P phosphorylation is carried out by kinases that use ADP as the phosphoryl donor. Unlike their ATP-dependent counterparts these enzymes have been reported as non-regulated. Based on the three dimensional structure determination of several ADP-dependent kinases they can be classified as members of the ribokinase superfamily. In this work, we have studied the role of divalent metal cations on the catalysis and regulation of ADP-dependent glucokinases and phosphofructokinase from hyperthermophilic archaea by means of initial velocity assays as well as molecular dynamics simulations. The results show that a divalent cation is strictly necessary for the activity of these enzymes and they strongly suggest that the true substrate is the metal-nucleotide complex. Also, these enzymes are promiscuous in relation to their metal usage where the only considerations for metal assisted catalysis seem to be related to the ionic radii and coordination geometry of the cations. Molecular dynamics simulations strongly suggest that this metal is bound to the highly conserved NXXE motif, which constitutes one of the signatures of the ribokinase superfamily. Although free ADP cannot act as a phosphoryl donor it still can bind to these enzymes with a reduced affinity, stressing the importance of the metal in the proper binding of the nucleotide at the active site. Also, data show that the binding of a second metal to these enzymes produces a complex with a reduced catalytic constant. On the basis of these findings and considering evolutionary information for the ribokinase superfamily, we propose that the regulatory metal acts by modulating the energy difference between the protein-substrates complex and the reaction transition state, which could constitute a general mechanism for the metal regulation of the enzymes that belong this superfamily. PMID:21906652

Merino, Felipe; Rivas-Pardo, Jaime Andrés; Caniuguir, Andrés; García, Ivonne; Guixé, Victoria



Text S14. Evidence against electrostatic repulsion in phosphoryl transfer transition states Because of the extreme resilience of phosphate esters to hydrolysis, early on it was  

E-print Network

phosphorylated 4- methyl pyridine fall on the same correlation line versus nucleophile pKa [35-37], providingO �HPOSerO �HPO- 4 2-2- pKa inactiveinactive Kd SerO �POSerO �PO- 4 3-3- C 1 10 10 10 104 105 6 7 8 9 10 11 100 #12;0.83 0.68 0.45 0.26 0.09 2 2.5 3 3.5 4 fractionPi bound ppm A B 5 4 3 2 1 0 WT AP fraction Pi

Herschlag, Dan


Suppression of Akt1 phosphorylation by adenoviral transfer of the PTEN gene inhibits hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells  

SciTech Connect

Recent findings identify the role of proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. Phosphoinositide 3 kinase (PI3K) and serine/threonine kinase (Akt) proteins are expressed in vascular smooth muscle cells. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been identified as a negative regulator of cytokine signaling that inhibits the PI3K-Akt pathway. However, little is known about the role of PTEN/Akt signaling in hypoxia-associated vascular remodeling. In this study, we found that hypoxia-induced the expression of Akt1 mRNA and phosphorylated protein by at least twofold in rat PASMCs. Phospho-PTEN significantly decreased in the nuclei of PASMCs after hypoxic stimulation. After forcing over-expression of PTEN by adenovirus-mediated PTEN (Ad-PTEN) transfection, the expression of phospho-Akt1 was significantly suppressed in PASMCs at all time-points measured. Additionally, we showed here that hypoxia increased proliferation of PASMCs by nearly twofold and over-expression of PTEN significantly inhibited hypoxia-induced PASMCs proliferation. These findings suggest that phospho-PTEN loss in the nuclei of PASMCs under hypoxic conditions may be the major cause of aberrant activation of Akt1 and may, therefore, play an important role in hypoxia-associated pulmonary arterial remodeling. Finally, the fact that transfection with Ad-PTEN inhibits the phosphorylation of Akt1 in PASMCs suggests a potential therapeutic effect on hypoxia-associated pulmonary arterial remodeling.

Luo, Chunxia [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)] [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Yi, Bin, E-mail: [Department of Anesthesia, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China) [Department of Anesthesia, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Bai, Li [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China)] [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Xia, Yongzhi [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)] [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Wang, Guansong; Qian, Guisheng [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China)] [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Feng, Hua [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)] [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)



Chemo-Enzymatic synthesis of bioactive glycopeptide using microbial endoglycosidase  

Microsoft Academic Search

The chemo-enzymatic synthesis of a glycopeptide, which involves the chemical synthesis of N-acetylglucosaminyl peptide and the enzymatic transfer of oligosaccharide, is described. The first step of the chemo-enzymatic method is the chemical synthesis of N-acetylglucosaminyl peptide with an N-acetylglucosamine moiety bound to the asparaginyl residue of the peptide by a solid-phase method. The second step is transglycosylation of a complex-type

Kenji Yamamoto



The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu  

PubMed Central

Fic proteins are ubiquitous in all domains of life and play critical roles in multiple cellular processes through AMPylation of (transfer of AMP to) target proteins. Doc from the doc/phd toxin/antitoxin module is a member of the Fic family and inhibits bacterial translation by an unknown mechanism. Here we show that, in contrast to the predicted AMPylating activity, Doc is a new type of kinase that inhibits bacterial translation by phosphorylating the conserved threonine (Thr382) of the translation elongation factor EF-Tu, rendering it unable to bind aminoacylated tRNAs. We provide evidence that EF-Tu phosphorylation diverged from AMPylation by antiparallel binding of the NTP relative to the catalytic residues of the conserved Fic catalytic core of Doc. The results bring insights into the mechanism and role of phosphorylation of EF-Tu in bacterial physiology as well as represent an example of catalytic plasticity of enzymes and a mechanism for the evolution of new enzymatic activities. PMID:24141193

De Gieter, Steven; van Nuland, Nico A.J.; Loris, Remy; Zenkin, Nikolay



Substrate-dependent control of MAPK phosphorylation in vivo  

PubMed Central

Phosphorylation of the mitogen-activated protein kinase (MAPK) is essential for its enzymatic activity and ability to control multiple substrates inside a cell. According to the current models, control of MAPK phosphorylation is independent of its substrates, which are viewed as mere sensors of MAPK activity. Contrary to this modular view of MAPK signaling, our studies in the Drosophila embryo demonstrate that substrates can regulate the level of MAPK phosphorylation in vivo. We demonstrate that a twofold change in the gene dosage of a single substrate can induce a significant change in the phosphorylation level of MAPK and in the conversion of other substrates. Our results support a model where substrates of MAPK counteract its dephosphorylation by phosphatases. Substrate-dependent control of MAPK phosphorylation is a manifestation of a more general retroactive effect that should be intrinsic to all networks with covalent modification cycles. PMID:21283143

Kim, Yoosik; Paroush, Ze'ev; Nairz, Knud; Hafen, Ernst; Jiménez, Gerardo; Shvartsman, Stanislav Y



Enzymatic synthesis of beta-lactam antibiotics and N-fatty-acylated amino compounds by the acyl-transfer reaction catalyzed by penicillin V acylase from Streptomyces mobaraensis.  


Penicillin V acylase from Streptomyces mobaraensis (Sm-PVA) showed high acyl-transfer activity in reactions using methyl esters of carboxylic acid (acyl donor) and amino compounds (nucleophile), to produce the corresponding amides. Moreover, Sm-PVA had broad substrate specificity, as indicated by the fact that it catalyzed the efficient synthesis of beta-lactam antibiotics, capsaicin derivatives, and N-fatty-acyl-amino acid/N-fatty-acyl-peptide derivatives. PMID:17587696

Koreishi, Mayuko; Tani, Kazuha; Ise, Yuuichi; Imanaka, Hiroyuki; Imamura, Koreyoshi; Nakanishi, Kazuhiro



Differences in Phosphorylated Histone H2AX Foci Formation and Removal of Cells Exposed to Low and High Linear Energy Transfer Radiation  

PubMed Central

The use of particle ion beams in cancer radiotherapy has a long history. Today, beams of protons or heavy ions, predominantly carbon ions, can be accelerated to precisely calculated energies which can be accurately targeted to tumors. This particle therapy works by damaging the DNA of tissue cells, ultimately causing their death. Among the different types of DNA lesions, the formation of DNA double strand breaks is considered to be the most relevant of deleterious damages of ionizing radiation in cells. It is well-known that the extremely large localized energy deposition can lead to complex types of DNA double strand breaks. These effects can lead to cell death, mutations, genomic instability, or carcinogenesis. Complex double strand breaks can increase the probability of mis-rejoining by NHEJ. As a consequence differences in the repair kinetics following high and low LET irradiation qualities are attributed mainly to quantitative differences in their contributions of the fast and slow repair component. In general, there is a higher contribution of the slow component of DNA double strand repair after exposure to high LET radiation, which is thought to reflect the increased amount of complex DNA double strand breaks. These can be accurately measured by the ?-H2AX assay, because the number of phosphorylated H2AX foci correlates well with the number of double strand breaks induced by low or / and high LET radiation. PMID:23450137

Schmid, Thomas Ernst; Zlobinskaya, Olga; Multhoff, Gabriele



Reversible phosphorylation in mitochondria.  

E-print Network

??Mitochondria represent an underappreciated site of regulation by reversible phosphorylation. Our work has focused on the identification of proteins involved in regulating mitochondria by reversible… (more)

Rardin, Matthew James



Regulation of protein phosphorylation in oat mitochondria  

SciTech Connect

We sought to identify phosphorylated proteins in isolated oat mitocchondria and to characterize the enzymatic and regulatory properties of the protein kinase(s). Mitochondria from oats (Avena sativa L. cv. Garry) were purified on Percoll gradients. Mitochondria were incubated with {sup 32}P-{gamma}-ATP; proteins were separated by SDS-PAGE. A small number of bands was detected on autoradiograms, most prominently at 70 kD and 42 kD; the latter band has been tentatively identified as a subunit of the pyruvate dehydrogenase complex, a well-known phosphoprotein. The protein kinase(s) could also phosphorylate casein, but not histone. Spermine enhanced the phosphorylation of casein and inhibited the phosphorylation of the 42 kD band. These studies were carried out on both intact and burst mitochondria. Control by calcium and other ions was investigated. The question of the action of regulators on protein kinase or protein phosphatase was studied by the use of {sup 35}S-adenosine thiotriphosphate.

Pike, C.; Kopeck, K.; Sceppa, E. (Franklin and Marshall College, Lancaster, PA (USA))



Enzymatic desulfurization of coal  

SciTech Connect

Our current efforts to develop clean coal technology emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes or commercially available enzymes. Our work is focused on the treatment of model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix.

Marquis, J.K. (Boston Univ., MA (United States). School of Medicine); Kitchell, J.P. (Holometrix, Inc., Cambridge, MA (United States))



Enzymatic desulfurization of coal  

SciTech Connect

Our current efforts to develop clean coal technology, emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes or commercially available enzymes. Our work is focused on the treatment of model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix.

Marquis, J.K. (Boston Univ., MA (United States). School of Medicine); Kitchell, J.P. (Holometrix, Inc., Cambridge, MA (United States))



Enzymatic desulfurization of coal  

SciTech Connect

Our current efforts to develop clean coal technology emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes as well as commercially available enzymes. Our work is focused on the treatment of model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix.

Boyer, Y.N.; Crooker, S.C.; Kitchell, J.P.; Nochur, S.V. (Holometrix, Inc., Cambridge, MA (United States)); Marquis, J.K. (Boston Univ., MA (United States). School of Medicine)



Protein Scaffolds Can Enhance the Bistability of Multisite Phosphorylation Systems  

PubMed Central

The phosphorylation of a substrate at multiple sites is a common protein modification that can give rise to important structural and electrostatic changes. Scaffold proteins can enhance protein phosphorylation by facilitating an interaction between a protein kinase enzyme and its target substrate. In this work we consider a simple mathematical model of a scaffold protein and show that under specific conditions, the presence of the scaffold can substantially raise the likelihood that the resulting system will exhibit bistable behavior. This phenomenon is especially pronounced when the enzymatic reactions have sufficiently large KM, compared to the concentration of the target substrate. We also find for a closely related model that bistable systems tend to have a specific kinetic conformation. Using deficiency theory and other methods, we provide a number of necessary conditions for bistability, such as the presence of multiple phosphorylation sites and the dependence of the scaffold binding/unbinding rates on the number of phosphorylated sites. PMID:22737061

Chan, Carlo; Liu, Xinfeng; Wang, Liming; Bardwell, Lee



Site-specifically phosphorylated lysine peptides.  


Protein phosphorylation controls major processes in cells. Although phosphorylation of serine, threonine, and tyrosine and also recently histidine and arginine are well-established, the extent and biological significance of lysine phosphorylation has remained elusive. Research in this area has been particularly limited by the inaccessibility of peptides and proteins that are phosphorylated at specific lysine residues, which are incompatible with solid-phase peptide synthesis (SPPS) due to the intrinsic acid lability of the P(?O)-N phosphoramidate bond. To address this issue, we have developed a new synthetic route for the synthesis of site-specifically phospholysine (pLys)-containing peptides by employing the chemoselectivity of the Staudinger-phosphite reaction. Our synthetic approach relies on the SPPS of unprotected ?-azido lysine-containing peptides and their subsequent reaction to phosphoramidates with phosphite esters before they are converted into the natural modification via UV irradiation or basic deprotection. With these peptides in hand, we demonstrate that electron-transfer dissociation tandem mass spectrometry can be used for unambiguous assignment of phosphorylated-lysine residues within histone peptides and that these peptides can be detected in cell lysates using a bottom-up proteomic approach. This new tagging method is expected to be an essential tool for evaluating the biological relevance of lysine phosphorylation. PMID:25196693

Bertran-Vicente, Jordi; Serwa, Remigiusz A; Schümann, Michael; Schmieder, Peter; Krause, Eberhard; Hackenberger, Christian P R



Enzymatic DNA molecules  

NASA Technical Reports Server (NTRS)

The present invention discloses deoxyribonucleic acid enzymes--catalytic or enzymatic DNA molecules--capable of cleaving nucleic acid sequences or molecules, particularly RNA, in a site-specific manner, as well as compositions including same. Methods of making and using the disclosed enzymes and compositions are also disclosed.

Joyce, Gerald F. (Inventor); Breaker, Ronald R. (Inventor)



Enzymatic desulfurization of coal  

SciTech Connect

The overall objective of this program was to investigate the feasibility of an enzymatic desulfurization process specifically intended for organic sulfur removal from coal. Toward that end, a series of specific objectives were defined: (1) establish the feasibility of (bio)oxidative pretreatment followed by biochemical sulfate cleavage for representative sulfur-containing model compounds and coals using commercially-available enzymes; (2) investigate the potential for the isolation and selective use of enzyme preparations from coal-utilizing microbial systems for desulfurization of sulfur-containing model compounds and coals; and (3) develop a conceptual design and economic analysis of a process for enzymatic removal of organic sulfur from coal. Within the scope of this program, it was proposed to carry out a portion of each of these efforts concurrently. (VC)

Boyer, Y.N.; Crooker, S.C.; Kitchell, J.P.; Nochur, S.V.



Protein phosphorylation and photorespiration.  


Photorespiration allows the recycling of carbon atoms of 2-phosphoglycolate produced by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenase activity, as well as the removal of potentially toxic metabolites. The photorespiratory pathway takes place in the light, encompasses four cellular compartments and interacts with several other metabolic pathways and functions. Therefore, the regulation of this cycle is probably of paramount importance to plant metabolism, however, our current knowledge is poor. To rapidly respond to changing conditions, proteins undergo a number of different post-translational modifications that include acetylation, methylation and ubiquitylation, but protein phosphorylation is probably the most common. The reversible covalent addition of a phosphate group to a specific amino acid residue allows the modulation of protein function, such as activity, subcellular localisation, capacity to interact with other proteins and stability. Recent data indicate that many photorespiratory enzymes can be phosphorylated, and thus it seems that the photorespiratory cycle is, in part, regulated by protein phosphorylation. In this review, the known phosphorylation sites of each Arabidopsis thaliana photorespiratory enzyme and several photorespiratory-associated proteins are described and discussed. A brief account of phosphoproteomic protocols is also given since the published data compiled in this review are the fruit of this approach. PMID:23506267

Hodges, M; Jossier, M; Boex-Fontvieille, E; Tcherkez, G



The enzymatic synthesis of membrane glucolipids in Acholeplasma laidlawii.  


In membranes of the prokaryote Acholeplasma laidlawii, the physiological regulation of the two major membrane lipids, monoglucosyldiacylglycerol (MGlcDAG) and diglucosyldiacylglycerol (DGlcDAG), is governed by factors affecting the equilibria between lamellar and non-lamellar phases of the membrane lipids. The synthesis of the glucolipids is considered to be a two-step glucosylation: (i) DAG+UDP-Glc----MGlcDAG+UDP; and (ii) MGlcDAG+UDP-Glc----DGlcDAG+UPD. This was corroborated by in vivo pulse labelling experiments showing turnover of MGlcDAG but not DGlcDAG. The enzymatic synthesis of MGlcDAG was localized to fresh or freeze-dried membranes in vitro. Synthesis of DGlcDAG was minor in such membranes but of substantial magnitude in intact cells. Synthesis of MGlcDAG was stimulated by small amounts of SDS but completely inhibited upon solubilization of the membranes by a variety of detergents. The inhibitory effect of several UDP-Glc analogs on glucolipid synthesis demonstrated the importance of UDP-Glc as the sugar donor. Synthesis of both glucolipids was lost in freeze-dried plus lipid-extracted cells but restored when lipids were transferred back to the extracted cell membrane. By selectively adding specific lipids, a strong dependence on the acceptor lipid DAG, as well as the need for general matrix lipids for enzyme activity, was established. In addition, the anionic phosphatidylglycerol (PG), but not the other phospholipids, had a strong stimulatory effect. The presence of different phosphorylating agents stimulated the synthesis of DGlcDAG and partially inhibited that of MGlcDAG. This, together with the lipid dependency, may constitute mechanisms for the regulation of the enzyme activities in vivo. PMID:1533160

Dahlqvist, A; Andersson, S; Wieslander, A



Enzymatic temperature change indicator  


A temperature change indicator is described which is composed of an enzyme and a substrate for that enzyme suspended in a solid organic solvent or mixture of solvents as a support medium. The organic solvent or solvents are chosen so as to melt at a specific temperature or in a specific temperature range. When the temperature of the indicator is elevated above the chosen, or critical temperature, the solid organic solvent support will melt, and the enzymatic reaction will occur, producing a visually detectable product which is stable to further temperature variation.

Klibanov, Alexander M. (Newton, MA); Dordick, Jonathan S. (Iowa City, IA)



Phosphorylation of Polyacrolein Oximes  

Microsoft Academic Search

Esterification of polyacrolein oximes with phosphorus pentoxide in DMF at 100°C for 4 h gives rise to phosphoric acid esters. Solid-phase 31P NMR spectroscopy, IR spectroscopy and potentiometric titration were used to establish that the phosphorylation proceeds by the hydroxy group of N-hydroxypiperidine rings incorporated into the polymeric macromolecule. Formation of acrylonitrile links is not observed. The influence of water

L. M. Antonik; A. G. Khabibulina



Determining in vivo Phosphorylation Sites using Mass Spectrometry  

PubMed Central

Phosphorylation is the most studied protein post-translational modification (PTM) in biological systems since it controls cell growth, proliferation, survival, etc. High resolution/high mass accuracy mass spectrometers are used to identify protein phosphorylation sites due to their speed, sensitivity, selectivity and throughput. The protocol described here focuses on two common strategies: 1) Identifying phosphorylation sites from individual proteins and small protein complexes, and 2) Identifying global phosphorylation sites from whole cell and tissue extracts. For the first, endogenous or epitope tagged proteins are typically immunopurified (IP) from cell lysates, purified via gel electrophoresis or precipitation and enzymatically digested into peptides. Samples can be optionally enriched for phosphopeptides using immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO2) and then analyzed by microcapillary liquid chromatography/tandem mass spectrometry (LC-MS/MS). Global phosphorylation site analyses that capture pSer/pThr/pTyr sites from biological sources sites are more resource and time-consuming and involve digesting the whole cell lysate, followed by peptide fractionation by strong cation exchange chromatography (SCX), phosphopeptide enrichment by IMAC or TiO2 and LC-MS/MS. Alternatively, one can fractionate the protein lysate by SDS-PAGE, followed by digestion, phosphopeptide enrichment and LC-MS/MS. One can also IP only phospho-tyrosine peptides using a pTyr antibody followed by LC-MS/MS. PMID:22470061

Breitkopf, Susanne B.; Asara, John M.



The enzymatic function of tafazzin.  


Tafazzin is a putative enzyme that is involved in cardiolipin metabolism, it may carry mutations responsible for Barth syndrome. To identify the biochemical reaction catalyzed by tafazzin, we expressed the full-length isoform of Drosophila melanogaster tafazzin in a baculovirus-Sf9 insect cell system. Tafazzin expression induced a new enzymatic function in Sf9 cell mitochondria, namely 1-palmitoyl-2-[14C]linoleoyl-phosphatidylcholine:monolysocardiolipin linoleoyltransferase. We also found evidence for the reverse reaction, because tafazzin expression caused transfer of acyl groups from phospholipids to 1-[14C]palmitoyl-2-lyso-phosphatidylcholine. An affinity-purified tafazzin construct, tagged with the maltose-binding protein, catalyzed both forward and reverse transacylations between cardiolipin and phosphatidylcholine, but was unable to utilize CoA or acyl-CoA as substrates. Whereas tafazzin supported transacylations between various phospholipid-lysophospholipid pairs, it showed the highest rate for the phosphatidylcholine-cardiolipin transacylation. Transacylation activities were about 10-fold higher for linoleoyl groups than for oleoyl groups, and they were negligible for arachidonoyl groups. The data show that Drosophila tafazzin is a CoA-independent, acyl-specific phospholipid transacylase with substrate preference for cardiolipin and phosphatidylcholine. PMID:17082194

Xu, Yang; Malhotra, Ashim; Ren, Mindong; Schlame, Michael



Optimization of cell conditions for enzymatic fuel cell using statistical analysis  

Microsoft Academic Search

Enzyme has been an attractive alternative to metal catalyst in biofuel cells. Enzymatic fuel cell (EFC) has the possibility of lower electric properties as well as the benefits of renewability and low temperature operation. Efficiency of the enzymatic fuel cell was related to cell conditions such as the component concentrations (substrate, enzyme cofactors, and electron transfer mediator), pH, and reaction

Seung Woo Jeon; Jin Young Lee; Jong Ho Lee; Seong Woo Kang; Chul Hwan Park; Seung Wook Kim



Phosphorylation by PINK1 Releases the UBL Domain and Initializes the Conformational Opening of the E3 Ubiquitin Ligase Parkin.  


Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design. PMID:25375667

Caulfield, Thomas R; Fiesel, Fabienne C; Moussaud-Lamodière, Elisabeth L; Dourado, Daniel F A R; Flores, Samuel C; Springer, Wolfdieter



Phosphorylation by PINK1 Releases the UBL Domain and Initializes the Conformational Opening of the E3 Ubiquitin Ligase Parkin  

PubMed Central

Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design. PMID:25375667

Moussaud-Lamodière, Elisabeth L.; Dourado, Daniel F. A. R.; Flores, Samuel C.; Springer, Wolfdieter



Vibrationally Enhanced Hydrogen Tunneling in Enzymatic Reactions  

NASA Astrophysics Data System (ADS)

Evidence for tunneling in enzymatic hydrogen transfer reactions has recently been observed. We argue that such reactions are likely to proceed via ground-state tunneling through a barrier which has been greatly shortened by thermally activated vibrational fluctuations. These fluctuations enhance the tunneling rate, cause it to depend on temperature and lead to a modest, temperature dependent "kinetic isotope effect" (KIE), which is defined to be the factor by which the reaction slows down due to isotopic substitution of the transferred hydrogen. We present a quantitative model for this mechanism which leads to a simple expression for the KIE in terms of two parameters: the tunneling action, S, and the ratio of its derivatives, S^ {'2}/S^{ ''}. This expression is used to fit the four KIE quantities measured in the bovine serum amine oxidase (BSAO) system: the hydrogen/tritium and hydrogen/deuterium KIE's and their Arrhenius slopes.

Bruno, William James



An Unbiased Proteomic Screen Reveals Caspase Cleavage Is Positively and Negatively Regulated by Substrate Phosphorylation*  

PubMed Central

Post-translational modifications of proteins regulate diverse cellular functions, with mounting evidence suggesting that hierarchical cross-talk between distinct modifications may fine-tune cellular responses. For example, in apoptosis, caspases promote cell death via cleavage of key structural and enzymatic proteins that in some instances is inhibited by phosphorylation near the scissile bond. In this study, we systematically investigated how protein phosphorylation affects susceptibility to caspase cleavage using an N-terminomic strategy, namely, a modified terminal amino isotopic labeling of substrates (TAILS) workflow, to identify proteins for which caspase-catalyzed cleavage is modulated by phosphatase treatment. We validated the effects of phosphorylation on three of the identified proteins and found that Yap1 and Golgin-160 exhibit decreased cleavage when phosphorylated, whereas cleavage of MST3 was promoted by phosphorylation. Furthermore, using synthetic peptides we systematically examined the influence of phosphoserine throughout the entirety of caspase-3, -7, and -8 recognition motifs and observed a general inhibitory effect of phosphorylation even at residues considered outside the classical consensus motif. Overall, our work demonstrates a role for phosphorylation in controlling caspase-mediated cleavage and shows that N-terminomic strategies can be tailored to study cross-talk between phosphorylation and proteolysis. PMID:24556848

Turowec, Jacob P.; Zukowski, Stephanie A.; Knight, James D. R.; Smalley, David M.; Graves, Lee M.; Johnson, Gary L.; Li, Shawn S. C.; Lajoie, Gilles A.; Litchfield, David W.



Global Identification and Characterization of Both O-GlcNAcylation and Phosphorylation at the Murine Synapse*  

PubMed Central

O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic, reversible monosaccharide modifier of serine and threonine residues on intracellular protein domains. Crosstalk between O-GlcNAcylation and phosphorylation has been hypothesized. Here, we identified over 1750 and 16,500 sites of O-GlcNAcylation and phosphorylation from murine synaptosomes, respectively. In total, 135 (7%) of all O-GlcNAcylation sites were also found to be sites of phosphorylation. Although many proteins were extensively phosphorylated and minimally O-GlcNAcylated, proteins found to be extensively O-GlcNAcylated were almost always phosphorylated to a similar or greater extent, indicating the O-GlcNAcylation system is specifically targeting a subset of the proteome that is also phosphorylated. Both PTMs usually occur on disordered regions of protein structure, within which, the location of O-GlcNAcylation and phosphorylation is virtually random with respect to each other, suggesting that negative crosstalk at the structural level is not a common phenomenon. As a class, protein kinases are found to be more extensively O-GlcNAcylated than proteins in general, indicating the potential for crosstalk of phosphorylation with O-GlcNAcylation via regulation of enzymatic activity. PMID:22645316

Trinidad, Jonathan C.; Barkan, David T.; Gulledge, Brittany F.; Thalhammer, Agnes; Sali, Andrej; Schoepfer, Ralf; Burlingame, Alma L.



Enolisation of the phosphoryl group  

Microsoft Academic Search

The potential ability of the phosphoryl group to undergo enolisation is demonstrated. The conditions for enolisation of phosphoryl and thiophosphoryl groups are examined. In order to determine the acidifying capacity of groups attached to the alpha-carbon atom, constants that depend on the number of substituents on this atom have been used. The limiting values of the totals of the constants

Tatyana A. Mastryukova; Martin I. Kabachnik



How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria†  

PubMed Central

The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens. PMID:17158705

Deutscher, Josef; Francke, Christof; Postma, Pieter W.



Mutational analysis of phosphorylation sites in the Dictyostelium myosin II tail: disruption of myosin function by a single charge change  

E-print Network

Mutational analysis of phosphorylation sites in the Dictyostelium myosin II tail: disruption of myosin function by a single charge change Ste¡en Nock, Wenchuan Liang, Hans M. Warrick, James A. Spudich/disassembly of non-muscle myosin II filaments is critical for the regulation of enzymatic activities and localization

Spudich, James A.


Electron transfer and protein engineering studies of the soluble methane monooxygenase from Methylococcus capsulatus (Bath)  

E-print Network

Chapter 1. Introduction: Electron Transfer in Biological Systems In many biological processes, including oxidative phosphorylation and photosynthesis, electron transfer reactions play vital roles. Electrons must be transported ...

Blazyk, Jessica L. (Jessica Lee), 1974-



Enzymatic Hydrolysis of Cellulosic Biomass  

SciTech Connect

Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs, and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components are not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of polymerization, and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric xylan, lignin, etc.) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability, and activity of individual enzymes and their synergistic effects in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification, and over-expression of selected enzymes and modifications in lignocellulosic biomass are also discussed.

Yang, Bin; Dai, Ziyu; Ding, Shi-You; Wyman, Charles E.



Enzymatic Synthesis of TDP-deoxysugars  

PubMed Central

Many biologically active bacterial natural products contain highly modified deoxysugar residues that are often critical for the activity of the parent compounds. Most of these deoxysugars are secondary metabolites that are biosynthesized in the form of nucleotide diphosphate (NDP) sugars prior to their transfer to natural product aglycones by glycosyltransferases. Over the past decade, many biosynthetic pathways that lead to the formation of these unusual sugars have been unraveled, and the mechanisms of many key enzymatic transformations involved in these pathways have been elucidated. However, obtaining workable quantities of NDP-deoxysugars for in vitro studies is often a difficult task. This limitation has hindered an in-depth investigation of the substrate specificity of deoxysugar biosynthetic enzymes, many of which are promiscuous with respect to their NDP-sugar substrates and are, thus, potentially useful catalysts for natural product glycoengineering. Presented in this review are procedures for the enzymatic synthesis and purification of a variety of NDP-deoxysugars, including some early intermediates in NDP-deoxysugar biosynthetic pathways, and highly modified NDP-deoxysugars that are late intermediates in their respective biosynthetic pathways. The procedures described herein could be used as general guidelines for the development of specific protocols for the synthesis of other NDP-deoxysugars. PMID:19362653

White-Phillip, Jessica; Thibodeaux, Christopher J.; Liu, Hung-wen



Phosphorylation regulates human OCT4.  


The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography-MS to identify 14 localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency. PMID:22474382

Brumbaugh, Justin; Hou, Zhonggang; Russell, Jason D; Howden, Sara E; Yu, Pengzhi; Ledvina, Aaron R; Coon, Joshua J; Thomson, James A



Interactions of a new 2-styrylchromone with mitochondrial oxidative phosphorylation.  


Many chromones, especially those having 2-substituents, manifest a remarkable variety of biological activities, such as the important cytotoxicity against human leukaemia cells, antiallergic, anticancer activities; unfortunately chromones normally disturb mitochondrial bioenergetics. A new 2-styrylchromone has been synthesized by the Baker-Venkataraman method and a classical approach has been used to assess the effects of 2-styrylchromone (3'-allyl-4',5,7-trimethoxy-2-styrylchromone) on rat liver mitochondrial bioenergetic. Mitochondrial respiratory rate and transmembrane potential were measured polarographically using a Clark oxygen electrode and with a selective electrode, respectively. All the disturbance induced by 2-styrylchromone on the enzymatic activities (succinate dehydrogenase, succinate cytochrome c reductase, and cytochrome c oxidase) and in the mitochondrial osmotic volume were determined spectrophotometrically. State 4, state 3, and uncoupled (presence of carbonylcyanide p-trifluoromethoxyphenylhydrazone) respiration rates were decreased by 2-styrylchromone in a concentration-dependent manner. Depression of respiratory activity promoted by 2-styrylchromone is essentially mediated through partial inhibition of succinate cytochrome c reductase. Phosphorylation capacity was strongly depressed as a result of an inhibition on the enzymatic complex (F(0)F(1)-ATPase) and also because of a deleterious effect on the integrity of the mitochondrial membrane, which uncoupled the respiration-generated proton gradient with the proton-driven phosphorylation. The structural integrity of the outside membrane is severely affected since cytochrome c can be released. 2-Styrylchromone uncouples oxidative phosphorylation by an inhibitory action on the redox chain and ATP synthase activity. Additionally, it can release cytochrome c. Cell death can probably result due to the induction of procaspase-9 and other procaspases and by a strong decrease of the available ATP. PMID:12439863

Peixoto, Francisco; Barros, Ana I R N A; Silva, Artur M S



Raf Activation Is Regulated by Tyrosine 510 Phosphorylation in Drosophila  

PubMed Central

The proto-oncoprotein Raf is pivotal for mitogen-activated protein kinase (MAPK) signaling, and its aberrant activation has been implicated in multiple human cancers. However, the precise molecular mechanism of Raf activation, especially for B-Raf, remains unresolved. By genetic and biochemical studies, we demonstrate that phosphorylation of tyrosine 510 is essential for activation of Drosophila Raf (Draf), which is an ortholog of mammalian B-Raf. Y510 of Draf is phosphorylated by the c-src homolog Src64B. Acidic substitution of Y510 promotes and phenylalanine substitution impairs Draf activation without affecting its enzymatic activity, suggesting that Y510 plays a purely regulatory role. We further show that Y510 regulates Draf activation by affecting the autoinhibitory interaction between the N- and C-terminal fragments of the protein. Finally, we show that Src64B is required for Draf activation in several developmental processes. Together, these results suggest a novel mechanism of Raf activation via Src-mediated tyrosine phosphorylation. Since Y510 is a conserved residue in the kinase domain of all Raf proteins, this mechanism is likely evolutionarily conserved. PMID:18494562

Xia, Fan; Li, Jinghong; Hickey, Gavin W; Tsurumi, Amy; Larson, Kimberly; Guo, Dongdong; Yan, Shian-Jang; Silver-Morse, Louis; Li, Willis X



Filamentous smooth muscle myosin is regulated by phosphorylation  

SciTech Connect

The enzymatic activity of filamentous dephosphorylated smooth muscle myosin has been difficult to determine because the polymer disassembles to the folded conformation in the presence of MgATP. Monoclonal antirod antibodies were used here to fix dephosphorylated myosin in the filamentous state. The steady-state actin-activated ATPase of phosphorylated filaments was 30-100-fold higher than that of antibody-stabilized dephosphorylated filaments, suggesting that phosphorylation can activate ATPase activity independent of changes in assembly. The degree of regulation may exceed 100-fold, because steady-state measurements slightly overestimate the rate of product release from dephosphorylated filaments. Single-turnover experiments in the absence of actin showed that although dephosphorylated folded myosin released products at the low rate of 0.0005 s-1. The addition of actin did not increase this rate to any appreciable extent. Dephosphorylated filaments and dephosphorylated heavy meromyosin thus have similar low rates of phosphate release both in the presence and absence of actin. These results show that light chain phosphorylation alone, without invoking other mechanisms, is an effective switch for regulating the activity of smooth muscle myosin filaments.

Trybus, K.M. (Brandeis Univ., Waltham, MA (USA))



Phosphorylation Regulates myo-Inositol-3-phosphate Synthase  

PubMed Central

myo-Inositol-3-phosphate synthase (MIPS) plays a crucial role in inositol homeostasis. Transcription of the coding gene INO1 is highly regulated. However, regulation of the enzyme is not well defined. We previously showed that MIPS is indirectly inhibited by valproate, suggesting that the enzyme is post-translationally regulated. Using 32Pi labeling and phosphoamino acid analysis, we show that yeast MIPS is a phosphoprotein. Mass spectrometry analysis identified five phosphosites, three of which are conserved in the human MIPS. Analysis of phosphorylation-deficient and phosphomimetic site mutants indicated that the three conserved sites in yeast (Ser-184, Ser-296, and Ser-374) and humans (Ser-177, Ser-279, and Ser-357) affect MIPS activity. Both S296A and S296D yeast mutants and S177A and S177D human mutants exhibited decreased enzymatic activity, suggesting that a serine residue is critical at that location. The phosphomimetic mutations S184D (human S279D) and S374D (human S357D) but not the phosphodeficient mutations decreased activity, suggesting that phosphorylation of these two sites is inhibitory. The double mutation S184A/S374A caused an increase in MIPS activity, conferred a growth advantage, and partially rescued sensitivity to valproate. Our findings identify a novel mechanism of regulation of inositol synthesis by phosphorylation of MIPS. PMID:23902760

Deranieh, Rania M.; He, Quan; Caruso, Joseph A.; Greenberg, Miriam L.



Metal-histidine-glutamate as a regulator of enzymatic cycles: a case study of carbonic anhydrasew  

E-print Network

Metal-histidine-glutamate as a regulator of enzymatic cycles: a case study of carbonic anhydrasew be tuned, in turn, by an easy proton transfer to a nearby glutamate. We study this situation in Human with a glutamate residue (Glu117). Proton transfer from His119 to Glu117 has been hypothesized in the past, however

Boyer, Edmond


Herpes simplex virus 2 VP22 phosphorylation induced by cellular and viral kinases does not influence intracellular localization  

SciTech Connect

Phosphorylation of the herpes simplex virus (HSV) VP22 protein is regulated by cellular kinases and the UL13 viral kinase, but the sites at which these enzymes induce phosphorylation of HSV-2 VP22 are not known. Using serine-to-alanine mutants to map phosphorylation sites on HSV-2 VP22 in cells, we made three major observations. First, phosphorylation by a cellular kinase mapped to serines 70, 71, and/or 72 within CKII consensus sites analogous to previously identified phosphorylation sites in HSV-1 VP22. Second, we mapped UL13-mediated phosphorylation of HSV-2 VP22 to serines 28 and 34, describing for the first time UL13-dependent phosphorylation sites on VP22. Third, previously identified VP22-associated cellular kinase sites in HSV-1 VP22 (serines 292 and 294) were not phosphorylated in HSV-2 VP22 (serines 291 and 293). VP22 expressed alone accumulated in the cytoplasm and to a lesser extent in the nucleus. Phosphorylation by endogenous cellular kinase(s) did not alter the localization of VP22. Co-expression of HSV-2 VP22 with active UL13, but not with enzymatically inactive UL13, resulted in nuclear accumulation of VP22 and altered nuclear morphology. Surprisingly, redistribution of VP22 to the nucleus occurred independently of UL13-induced phosphorylation of VP22. The altered nuclear morphology of UL13-expressing cells was not due to apoptosis. These results demonstrate that phosphorylation of HSV-2 VP22 at multiple serine residues is induced by UL13 and cellular kinase(s), and that the nuclear/cytoplasmic distribution of VP22 is independent of its phosphorylation status but is controlled indirectly by UL13 kinase activity.

Geiss, Brian J. [Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO 63104-1083 (United States); Cano, Gina L. [Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO 63104-1083 (United States); Tavis, John E. [Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO 63104-1083 (United States); Morrison, Lynda A. [Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO 63104-1083 (United States)]. E-mail:



Interphase phosphorylation of lamin A.  


Nuclear lamins form the major structural elements that comprise the nuclear lamina. Loss of nuclear structural integrity has been implicated as a key factor in the lamin A/C gene mutations that cause laminopathies, whereas the normal regulation of lamin A assembly and organization in interphase cells is still undefined. We assumed phosphorylation to be a major determinant, identifying 20 prime interphase phosphorylation sites, of which eight were high-turnover sites. We examined the roles of these latter sites by site-directed mutagenesis, followed by detailed microscopic analysis - including fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and nuclear extraction techniques. The results reveal three phosphorylation regions, each with dominant sites, together controlling lamin A structure and dynamics. Interestingly, two of these interphase sites are hyper-phosphorylated in mitotic cells and one of these sites is within the sequence that is missing in progerin of the Hutchinson-Gilford progeria syndrome. We present a model where different phosphorylation combinations yield markedly different effects on the assembly, subunit turnover and the mobility of lamin A between, and within, the lamina, the nucleoplasm and the cytoplasm of interphase cells. PMID:24741066

Kochin, Vitaly; Shimi, Takeshi; Torvaldson, Elin; Adam, Stephen A; Goldman, Anne; Pack, Chan-Gi; Melo-Cardenas, Johanna; Imanishi, Susumu Y; Goldman, Robert D; Eriksson, John E



Probing phosphorylation-dependent protein interactions within functional domains of histone deacetylase 5 (HDAC5).  


Class IIa histone deacetylases (HDACs) are critical transcriptional regulators, shuttling between nuclear and cytoplasmic cellular compartments. Within the nucleus, these HDACs repress transcription as components of multiprotein complexes, such as the nuclear corepressor and beclin-6 corepressor (BCoR) complexes. Cytoplasmic relocalization relieves this transcriptional repressive function. Class IIa HDAC shuttling is controlled, in part, by phosphorylations flanking the nuclear localization signal (NLS). Furthermore, we have reported that phosphorylation within the NLS by the kinase Aurora B modulates the localization and function of the class IIa HDAC5 during mitosis. While we identified numerous additional HDAC5 phosphorylations, their regulatory functions remain unknown. Here, we studied phosphorylation sites within functional HDAC5 domains, including the deacetylation domain (DAC, Ser755), nuclear export signal (NES, Ser1108), and an acidic domain (AD, Ser611). We have generated phosphomutant cell lines to investigate how absence of phosphorylation at these sites impacts HDAC5 localization, enzymatic activity, and protein interactions. Combining molecular biology and quantitative MS, we have defined the interactions and HDAC5-containing complexes mediated by site-specific phosphorylation and quantified selected changes using parallel reaction monitoring. These results expand the current understanding of HDAC regulation, and the functions of this critical family of proteins within human cells. PMID:24920159

Guise, Amanda J; Mathias, Rommel A; Rowland, Elizabeth A; Yu, Fang; Cristea, Ileana M



Mitochondrial c-Src regulates cell survival through phosphorylation of respiratory chain components  

PubMed Central

Mitochondrial protein tyrosine phosphorylation is an important mechanism for the modulation of mitochondrial functions. In the present study, we have identified novel substrates of c-Src in mitochondria and investigated their function in the regulation of oxidative phosphorylation. The Src family kinase inhibitor PP2 {amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4d] pyrimidine} exhibits significant reduction of respiration. Similar results were obtained from cells expressing kinase-dead c-Src, which harbours a mitochondrial-targeting sequence. Phosphorylation-site analysis selects c-Src targets, including NDUFV2 (NADH dehydrogenase [ubiquinone] flavoprotein 2) at Tyr193 of respiratory complex I and SDHA (succinate dehydrogenase A) at Tyr215 of complex II. The phosphorylation of these sites by c-Src is supported by an in vivo assay using cells expressing their phosphorylation-defective mutants. Comparison of cells expressing wild-type proteins and their mutants reveals that NDUFV2 phosphorylation is required for NADH dehydrogenase activity, affecting respiration activity and cellular ATP content. SDHA phosphorylation shows no effect on enzyme activity, but perturbed electron transfer, which induces reactive oxygen species. Loss of viability is observed in T98G cells and the primary neurons expressing these mutants. These results suggest that mitochondrial c-Src regulates the oxidative phosphorylation system by phosphorylating respiratory components and that c-Src activity is essential for cell viability. PMID:22823520

Ogura, Masato; Yamaki, Junko; Homma, Miwako K.; Homma, Yoshimi



Nucleoside phosphorylation by phosphate minerals.  


In the presence of formamide, crystal phosphate minerals may act as phosphate donors to nucleosides, yielding both 5'- and, to a lesser extent, 3'-phosphorylated forms. With the mineral Libethenite the formation of 5'-AMP can be as high as 6% of the adenosine input and last for at least 10(3) h. At high concentrations, soluble non-mineral phosphate donors (KH(2)PO(4) or 5'-CMP) afford 2'- and 2':3'-cyclic AMP in addition to 5'-and 3'-AMP. The phosphate minerals analyzed were Herderite Ca[BePO(4)F], Hureaulite Mn(2+)(5)(PO(3)(OH)(2)(PO(4))(2)(H(2)O)(4), Libethenite Cu(2+)(2)(PO(4))(OH), Pyromorphite Pb(5)(PO(4))(3)Cl, Turquoise Cu(2+)Al(6)(PO(4))(4)(OH)(8)(H(2)O)(4), Fluorapatite Ca(5)(PO(4))(3)F, Hydroxylapatite Ca(5)(PO(4))(3)OH, Vivianite Fe(2+)(3)(PO(4))(2)(H(2)O)(8), Cornetite Cu(2+)(3)(PO(4))(OH)(3), Pseudomalachite Cu(2+)(5)(PO(4))(2)(OH)(4), Reichenbachite Cu(2+)(5)(PO(4))(2)(OH)(4), and Ludjibaite Cu(2+)(5)(PO(4))(2)(OH)(4)). Based on their behavior in the formamide-driven nucleoside phosphorylation reaction, these minerals can be characterized as: 1) inactive, 2) low level phosphorylating agents, or 3) active phosphorylating agents. Instances were detected (Libethenite and Hydroxylapatite) in which phosphorylation occurs on the mineral surface, followed by release of the phosphorylated compounds. Libethenite and Cornetite markedly protect the beta-glycosidic bond. Thus, activated nucleic monomers can form in a liquid non-aqueous environment in conditions compatible with the thermodynamics of polymerization, providing a solution to the standard-state Gibbs free energy change (DeltaG degrees ') problem, the major obstacle for polymerizations in the liquid phase in plausible prebiotic scenarios. PMID:17412692

Costanzo, Giovanna; Saladino, Raffaele; Crestini, Claudia; Ciciriello, Fabiana; Di Mauro, Ernesto



Enzymatic reactions on immobilised substrates.  


This review gives an overview of enzymatic reactions that have been conducted on substrates attached to solid surfaces. Such biochemical reactions have become more important with the drive to miniaturisation and automation in chemistry, biology and medicine. Technical aspects such as choice of solid surface and analytical methods are discussed and examples of enzyme reactions that have been successful on these surfaces are provided. PMID:23579870

Gray, Christopher J; Weissenborn, Martin J; Eyers, Claire E; Flitsch, Sabine L



The Enzymatic Oxidation of Graphene Oxide  

PubMed Central

Two-dimensional graphitic carbon is a new material with many emerging applications, and studying its chemical properties is an important goal. Here, we reported a new phenomenon – the enzymatic oxidation of a single layer of graphitic carbon by horseradish peroxidase (HRP). In the presence of low concentrations of hydrogen peroxide (~40 µM), HRP catalyzed the oxidation of graphene oxide, which resulted in the formation of holes on its basal plane. During the same period of analysis, HRP failed to oxidize chemically reduced graphene oxide (RGO). The enzymatic oxidation was characterized by Raman, UV-Vis, EPR and FT-IR spectroscopy, TEM, AFM, SDS-PAGE, and GC-MS. Computational docking studies indicated that HRP was preferentially bound to the basal plane rather than the edge for both graphene oxide and RGO. Due to the more dynamic nature of HRP on graphene oxide, the heme active site of HRP was in closer proximity to graphene oxide compared to RGO, thereby facilitating the oxidation of the basal plane of graphene oxide. We also studied the electronic properties of the reduced intermediate product, holey reduced graphene oxide (hRGO), using field-effect transistor (FET) measurements. While RGO exhibited a V-shaped transfer characteristic similar to a single layer of graphene that was attributed to its zero band gap, hRGO demonstrated a p-type semiconducting behavior with a positive shift in the Dirac points. This p-type behavior rendered hRGO, which can be conceptualized as interconnected graphene nanoribbons, as a potentially attractive material for FET sensors. PMID:21344859

Kotchey, Gregg P.; Allen, Brett L.; Vedala, Harindra; Yanamala, Naveena; Kapralov, Alexander A.; Tyurina, Yulia Y.; Klein-Seetharaman, Judith; Kagan, Valerian E.; Star, Alexander



Oxidative Phosphorylation at the fin de siècle  

NSDL National Science Digital Library

Mitochondria produce most of the energy in animal cells by a process called oxidative phosphorylation. Electrons are passed along a series of respiratory enzyme complexes located in the inner mitochondrial membrane, and the energy released by this electron transfer is used to pump protons across the membrane. The resultant electrochemical gradient enables another complex, adenosine 5â²-triphosphate (ATP) synthase, to synthesize the energy carrier ATP.

Matti Saraste (European Molecular Biology Laboratory;)



Electrochemical enzymatic biosensors using carbon nanofiber nanoelectrode arrays  

NASA Astrophysics Data System (ADS)

The reduction of electrode size down to nanometers could dramatically enhance detection sensitivity and temporal resolution. Nanoelectrode arrays (NEAs) are of particular interest for ultrasensitive biosensors. Here we report the study of two types of biosensors for measuring enzyme activities using NEAs fabricated with vertically aligned carbon nanofibers (VACNFs). VACNFs of ~100 nm in average diameter and 3-5 ?m in length were grown on conductive substrates as uniform vertical arrays which were then encapsulated in SiO2 matrix leaving only the tips exposed. We demonstrate that such VACNF NEAs can be used in profiling enzyme activities through monitoring the change in electrochemical signals induced by enzymatic reactions to the peptides attached to the VACNF tip. The cleavage of the tetrapeptide with a ferrocene tag by a cancerrelated protease (legumain) was monitored with AC voltammetry. Real-time electrochemical impedance spectroscopy (REIS) was used for fast label-free detection of two reversible processes, i.e. phosphorylation by c-Src tyrosine kinase and dephosphorylation by protein tyrosine phosphatase 1B (PTP1B). The REIS data of phosphorylation were slow and unreliable, but those of dephosphorylation showed large and fast exponential decay due to much higher activity of phosphatase PTP1B. The kinetic data were analyzed with a heterogeneous Michaelis-Menten model to derive the "specificity constant" kcat/Km, which is 8.2x103 M-1s-1 for legumain and (2.1 ± 0.1) x 107 M-1s-1 for phosphatase (PTP1B), well consistent with literature. It is promising to develop VACNF NEA based electrochemical enzymatic biosensors as portable multiplex electronic techniques for rapid cancer diagnosis and treatment monitoring.

Li, Jun; Li, Yi-fen; Swisher, Luxi Z.; Syed, Lateef U.; Prior, Allan M.; Nguyen, Thu A.; Hua, Duy H.


Non-Enzymatic DNA Cleavage Reaction Induced by 5-Ethynyluracil in Methylamine Aqueous Solution and Application to DNA Concatenation  

PubMed Central

DNA can be concatenated by hybridization of DNA fragments with protruding single-stranded termini. DNA cleavage occurring at a nucleotide containing a DNA base analogue is a useful method to obtain DNA with designed protruding termini. Here, we report a novel non-enzymatic DNA cleavage reaction for DNA concatenation. We found that DNA is cleaved at a nucleotide containing 5-ethynyluracil in a methylamine aqueous solution to generate 5?-phosphorylated DNA fragment as a cleavage product. We demonstrated that the reaction can be applied to DNA concatenation of PCR-amplified DNA fragments. This novel non-enzymatic DNA cleavage reaction is a simple practical approach for DNA concatenation. PMID:24647759

Ikeda, Shuji; Tainaka, Kazuki; Matsumoto, Katsuhiko; Shinohara, Yuta; Ode, Koji L.; Susaki, Etsuo A.; Ueda, Hiroki R.



Human and Quail Aromatase Activity Is Rapidly and Reversibly Inhibited by Phosphorylating Conditions  

PubMed Central

Besides their slow genomic actions, estrogens also induce rapid physiological responses. To be functionally relevant, these effects must be associated with rapid changes in local concentrations of estrogens. Rapid changes in aromatase activity (AA) controlled by calcium-dependent phosphorylations of the enzyme can alter in a rapid manner local estrogen concentrations, but so far this mechanism was identified only in the avian (quail) brain. We show here that AA is also rapidly down-regulated by phosphorylating conditions in quail ovary homogenates and in various cell lines transfected with human aromatase (HEK 293, Neuro2A, and C6). Enzymatic activity was also rapidly inhibited after depolarization of aromatase-expressing HEK 293 cells with 100 mm KCl, and activity was fully restored when cells returned to control conditions. Western blot analysis demonstrated that the reduction of enzymatic activity is not due to protein degradation. We next investigated by site-directed mutagenesis the potential implication in the control of AA of specific aromatase residues identified by bioinformatic analysis. Mutation of the amino acids S118, S247, S267, T462, T493, or S497 to alanine, alone or in combination, did not block the rapid inhibition of enzymatic activity induced by phosphorylating conditions, but basal AA was markedly decreased in the S118A mutant. Altogether, these results demonstrate that the rapid inhibition of AA is a widespread and fully reversible process and that phosphorylation of specific residues modulate AA. These processes provide a new general mechanism by which local estrogen concentration can be rapidly altered in the brain and other tissues. PMID:21914772

Harada, Nobuhiro; Balthazart, Jacques; Cornil, Charlotte A.



Global analysis of protein phosphorylation in yeast  

Microsoft Academic Search

Protein phosphorylation is estimated to affect 30% of the proteome and is a major regulatory mechanism that controls many basic cellular processes. Until recently, our biochemical understanding of protein phosphorylation on a global scale has been extremely limited; only one half of the yeast kinases have known in vivo substrates and the phosphorylating kinase is known for less than 160

Jason Ptacek; Geeta Devgan; Gregory Michaud; Heng Zhu; Xiaowei Zhu; Joseph Fasolo; Hong Guo; Ghil Jona; Ashton Breitkreutz; Richelle Sopko; Rhonda R. McCartney; Martin C. Schmidt; Najma Rachidi; Soo-Jung Lee; Angie S. Mah; Lihao Meng; Michael J. R. Stark; David F. Stern; Claudio de Virgilio; Mike Tyers; Brenda Andrews; Mark Gerstein; Barry Schweitzer; Paul F. Predki; Michael Snyder



Spin biochemistry: magnetic 24Mg-25Mg-26Mg isotope effect in mitochondrial ADP phosphorylation.  


The rates of adenosine triphosphate (ATP) production by isolated mitochondria and mitochondrial creatine kinase incubated in isotopically pure media containing, separately, (24)Mg(2+), (25)Mg(2+), and (26)Mg(2+) ions were shown to be strongly dependent on the magnesium nuclear spin and magnetic moment. The rate of adenosine 5'-diphosphate phosphorylation in mitochondria with magnetic nuclei (25)Mg is about twice higher than that with the spinless, nonmagnetic nuclei (24,26)Mg. When mitochondrial oxidative phosphorylation was selectively blocked by treatment with 1-methylnicotine amide, (25)Mg(2+) ions were shown to be nearly four times more active in mitochondrial ATP synthesis than (24,26)Mg(2+) ions. The rate of ATP production associated with creatine kinase is twice higher for (25)Mg(2+) than for (24,26)Mg and does not depend on the blockade of oxidative phosphorylation. There is no difference between (24)Mg(2+) and (26)Mg(2+) effects in both oxidative and substrate phosphorylation. These observations demonstrate that the enzymatic phosphorylation is a nuclear spin selective process controlled by magnetic isotope effect. The reaction mechanism proposed includes a participation of intermediate ion-radical pairs with Mg(+) cation as a radical partner. Therefore, the key mitochondrial phosphotransferases work as a magnesium nuclear spin mediated molecular machines. PMID:16049349

Buchachenko, Anatoly L; Kouznetsov, Dmitri A; Arkhangelsky, Stanislav E; Orlova, Marina A; Markarian, Artyom A



Comprehensive Characterization of Heat Shock Protein 27 Phosphorylation in Human Endothelial Cells Stimulated by the Microbial Dithiole Thiolutin  

PubMed Central

Thiolutin is a sulfur-based microbial compound with known activity as an angiogenesis inhibitor. Relative to previously studied angiogenesis inhibitors, thiolutin is a remarkably potent inducer of heat shock protein 27 (Hsp27) phosphorylation. This phosphorylation requires p38 kinase but is independent of increased p38 phosphorylation. To elucidate how thiolutin regulates Hsp27 phosphorylation and ultimately angiogenesis, Hsp27 was immunoprecipitated using nonphosphorylated and phospho-Ser78 specific antibodies from lysates of thiolutin treated and untreated human umbilical vein endothelial cells and analyzed by LC–MS. Separate LC–MS analyses of Lys-C, Lys-C plus trypsin, and Lys-C plus Glu-C digests provided 100% sequence coverage, including the identification of a very large 13 kDa Lys-C fragment using a special sample handling procedure (4 M guanidine HCl) prior to the LC–MS analysis to improve the large peptide recovery. The analysis revealed a novel post-translational modification of Hsp27 involving truncation of the N-terminal Met and acetylation of the penultimate Thr. Analysis of a Glu-C fragment containing two phosphorylation sites, Ser78 and Ser82, and a tryptic fragment containing the other phosphorylation site, Ser15, enabled quantitative stoichiometry of Hsp27 phosphorylation by LC–MS. The strategy revealed details of Hsp27 phosphorylation, including significant di-phosphorylation at both Ser78 and Ser82, that would be difficult to obtain by traditional approaches because oligomerization of the hydrophobic N-terminal region of the molecule prevents efficient enzymatic cleavage. The combination of Western blotting, immunoprecipation, and LC–MS provides a quantitative analysis of thiolutin-stimulated Hsp27 phosphorylation and further defines the role of Hsp27 in the antiangiogenic activities of thiolutin and related dithiolethiones. PMID:18720982

Dai, Shujia; Jia, Yifeng; Wu, Shiaw-Lin; Isenberg, Jeff S.; Ridnour, Lisa A.; Bandle, Russell W.; Wink, David A.; Roberts, David D.; Karger, Barry L.



Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro  

SciTech Connect

DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purified fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, (..gamma..-/sup 32/P)ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme-substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. The authors conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, they speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity.

Kaiserman, H.B.; Ingebritsen, T.S.; Benbow, R.M.



Photoinitiated proton-coupled electron transfer and radical transport kinetics in class la ribonucleotide reductase  

E-print Network

Proton-coupled electron transfer (PCET) is a critical mechanism in biology, underpinning key processes such as radical transport, energy transduction, and enzymatic substrate activation. Ribonucleotide reductases (RNRs) ...

Pizano, Arturo A. (Arturo Alejandro)



Oxidative Phosphorylation at the fin de siècle  

NSDL National Science Digital Library

Mitochondria produce most of the energy in animal cells by a process called oxidative phosphorylation. Electrons are passed along a series of respiratory enzyme complexes located in the inner mitochondrial membrane, and the energy released by this electron transfer is used to pump protons across the membrane. The resultant electrochemical gradient enables another complex, adenosine 5â²-triphosphate (ATP) synthase, to synthesize the energy carrier ATP. Important new mechanistic insights into oxidative phosphorylation have emerged from recent three-dimensional structural analyses of ATP synthase and two of the respiratory enzyme complexes, cytochrome bc1 and cytochrome c oxidase. This work, and new enzymological studies of ATP synthase's unusual catalytic mechanism, are reviewed here.

Matti Saraste (European Molecular Biology Laboratory;)



Phosphorylated nano-diamond/ Polyimide Nanocomposites  

NASA Astrophysics Data System (ADS)

In this study, a novel route to synthesize polyimide (PI)/phosphorylated nanodiamond films with improved thermal and mechanical properties was developed. Surface phosphorylation of nano-diamond was performed in dichloromethane. Phosphorylation dramatically enhanced the thermal stability of nano-diamond. Poly(amic acid) (PAA), which is the precursor of PI, was successfully synthesized with 3,3',4,4'-Benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydianiline (4,4'-ODA) in the solution of N,N- dimethylformamide (DMF). Pure BTDA-ODA polyimide films and phosphorylated nanodiamond containing BTDA-ODA PI films were prepared. The PAA displayed good compatibility with phosphorylated nano-diamond. The morphology of the polyimide (PI)/phosphorylated nano-diamond was characterized by scanning electron microscopy (SEM). Chemical structure of polyimide and polyimide (PI)/phosphorylated nano-diamond was characterized by FTIR. SEM and FTIR results showed that the phosphorylated nano-diamond was successfully prepared. Thermal properties of the polyimide (PI)/phosphorylated nanodiamond was characterized by thermogravimetric analysis (TGA). TGA results showed that the thermal stability of (PI)/phosphorylated nano-diamond film was increased.

Beyler-Çi?il, Asli; Çakmakçi, Emrah; Vezir Kahraman, Memet



Enzymatic production of L-tryptophan in a reverse micelle reactor  

Microsoft Academic Search

The enzymatic production of tryptophan from indole and serine was investigated in a micellar solution of the surfactant Brij 56 in cyclohexane. An anion exchanger was employed to facilitate the transfer of tryptophan and serine between the water pool of the reverse micelle and the bulk organic phase. The influence of potassium ion, water content, pH, and co-surfactant on enzyme

D. K. Eggers; H. W. Blanch



Protein phosphorylations in poliovirus infected cells.  


In vivo phosphorylation of proteins that are associated with polysomes of poliovirus-infected VERO (African green monkey kidney) and HeLa (Henrietta Lacks) cells differed from phosphorylations observed with uninfected cells that were fed fresh medium. With both types of cells infection stimulated phosphorylation of proteins with molecular weights of 40 000-41 000, 39 000, 34 000, 32 000, and 24 000. Similarities of phosphorylations in VERO and HeLa cells suggest that they are a specific consequence of infection and might serve a regulatory function during protein synthesis. PMID:6260321

James, L A; Tershak, D R



Applying the brakes to multisite SR protein phosphorylation: substrate-induced effects on the splicing kinase SRPK1.  


To investigate how a protein kinase interacts with its protein substrate during extended, multisite phosphorylation, the kinetic mechanism of a protein kinase involved in mRNA splicing control was investigated using rapid quench flow techniques. The protein kinase SRPK1 phosphorylates ~10 serines in the arginine--serine-rich domain (RS domain) of the SR protein SRSF1 in a C- to N-terminal direction, a modification that directs this essential splicing factor from the cytoplasm to the nucleus. Transient-state kinetic experiments illustrate that the first phosphate is added rapidly onto the RS domain of SRSF1 (t(1/2) = 0.1 s) followed by slower, multisite phosphorylation at the remaining serines (t(1/2) = 15 s). Mutagenesis experiments suggest that efficient phosphorylation rates are maintained by an extensive hydrogen bonding and electrostatic network between the RS domain of the SR protein and the active site and docking groove of the kinase. Catalytic trapping and viscosometric experiments demonstrate that while the phosphoryl transfer step is fast, ADP release limits multisite phosphorylation. By studying phosphate incorporation into selectively pre-phosphorylated forms of the enzyme-substrate complex, the kinetic mechanism for site-specific phosphorylation along the reaction coordinate was assessed. The binding affinity of the SR protein, the phosphoryl transfer rate, and ADP exchange rate were found to decline significantly as a function of progressive phosphorylation in the RS domain. These findings indicate that the protein substrate actively modulates initiation, extension, and termination events associated with prolonged, multisite phosphorylation. PMID:21728354

Aubol, Brandon E; Adams, Joseph A



Regulation of brain-type creatine kinase by AMP-activated protein kinase: interaction, phosphorylation and ER localization.  


AMP-activated protein kinase (AMPK) and cytosolic brain-type creatine kinase (BCK) cooperate under energy stress to compensate for loss of adenosine triphosphate (ATP) by either stimulating ATP-generating and inhibiting ATP-consuming pathways, or by direct ATP regeneration from phosphocreatine, respectively. Here we report on AMPK-dependent phosphorylation of BCK from different species identified by in vitro screening for AMPK substrates in mouse brain. Mass spectrometry, protein sequencing, and site-directed mutagenesis identified Ser6 as a relevant residue with one site phosphorylated per BCK dimer. Yeast two-hybrid analysis revealed interaction of active AMPK specifically with non-phosphorylated BCK. Pharmacological activation of AMPK mimicking energy stress led to BCK phosphorylation in astrocytes and fibroblasts, as evidenced with a highly specific phospho-Ser6 antibody. BCK phosphorylation at Ser6 did not affect its enzymatic activity, but led to the appearance of the phosphorylated enzyme at the endoplasmic reticulum (ER), close to the ER calcium pump, a location known for muscle-type cytosolic creatine kinase (CK) to support Ca²?-pumping. PMID:24727412

Ramírez Ríos, Sacnicte; Lamarche, Frédéric; Cottet-Rousselle, Cécile; Klaus, Anna; Tuerk, Roland; Thali, Ramon; Auchli, Yolanda; Brunisholz, René; Neumann, Dietbert; Barret, Luc; Tokarska-Schlattner, Malgorzata; Schlattner, Uwe



Correlation between persistent forms of zeaxanthin-dependent energy dissipation and thylakoid protein phosphorylation.  


High light stress induced not only a sustained form of xanthophyll cycle-dependent energy dissipation but also sustained thylakoid protein phosphorylation. The effect of protein phosphatase inhibitors (fluoride and molybdate ions) on recovery from a 1-h exposure to a high PFD was examined in leaf discs of Parthenocissus quinquefolia (Virginia creeper). Inhibition of protein dephosphorylation induced zeaxanthin retention and sustained energy dissipation (NPQ) upon return to low PFD for recovery, but had no significant effects on pigment and Chl fluorescence characteristics under high light exposure. In addition, whole plants of Monstera deliciosa and spinach grown at low to moderate PFDs were transferred to high PFDs, and thylakoid protein phosphorylation pattern (assessed with anti-phosphothreonine antibody) as well as pigment and Chl fluorescence characteristics were examined over several days. A correlation was obtained between dark-sustained D1/D2 phosphorylation and dark-sustained zeaxanthin retention and maintenance of PS II in a state primed for energy dissipation in both species. The degree of these dark-sustained phenomena was more pronounced in M. deliciosa compared with spinach. Moreover, M. deliciosa but not spinach plants showed unusual phosphorylation patterns of Lhcb proteins with pronounced dark-sustained Lhcb phosphorylation even under low PFD growth conditions. Subsequent to the transfer to a high PFD, dark-sustained Lhcb protein phosphorylation was further enhanced. Thus, phosphorylation patterns of D1/D2 and Lhcb proteins differed from each other as well as among plant species. The results presented here suggest an association between dark-sustained D1/D2 phosphorylation and sustained retention of zeaxanthin and energy dissipation (NPQ) in light-stressed, and particularly 'photoinhibited', leaves. Functional implications of these observations are discussed. PMID:16228317

Ebbert, V; Demmig-Adams, B; Adams, W W; Mueh, K E; Staehelin, L A



Preparation of casein phosphorylated peptides and casein non-phosphorylated peptides using alcalase  

Microsoft Academic Search

Casein was digested with a cheaper enzyme, alcalase, to produce casein phosphorylated peptides and casein non-phosphorylated\\u000a peptides concurrently. The casein hydrolyzates were separated to the two kinds of peptides by using combined treatment of\\u000a CaCl2 and ethanol. Casein phosphorylated peptides and non-phosphorylated peptides constitute some peptides with molecular weight\\u000a lower than 2509 Da and 2254 Da respectively as determined using size exclusion

Li Zhao; Zhang Wang; Shi ying Xu



Recent Advances in Carbon Nanotube-Based Enzymatic Fuel Cells  

PubMed Central

This review summarizes recent trends in the field of enzymatic fuel cells. Thanks to the high specificity of enzymes, biofuel cells can generate electrical energy by oxidation of a targeted fuel (sugars, alcohols, or hydrogen) at the anode and reduction of oxidants (O2, H2O2) at the cathode in complex media. The combination of carbon nanotubes (CNT), enzymes and redox mediators was widely exploited to develop biofuel cells since the electrons involved in the bio-electrocatalytic processes can be efficiently transferred from or to an external circuit. Original approaches to construct electron transfer based CNT-bioelectrodes and impressive biofuel cell performances are reported as well as biomedical applications. PMID:25386555

Cosnier, Serge; Holzinger, Michael; Le Goff, Alan



Extracellular Phosphorylation and Phosphorylated Proteins: Not Just Curiosities But Physiologically Important  

NSDL National Science Digital Library

With 3 figures, 1 table, and 221 references plus 2 supplementary figures and 1 supplementary table, this Review describes the evidence supporting physiological and pathological functions of phosphorylated extracellular proteins. In addition, evidence for extracellular phosphorylation in various contexts and tissues is also presented. To facilitate the study of the extracellular phosphoproteome, annotation of the phosphorylated proteins found in proteomic databases is supplied.

Garif Yalak (ETH Zurich;Department of Health Sciences and Technology REV); Viola Vogel (ETH Zurich;Department of Health Sciences and Technology REV)



Relationships between histone phosphorylation and cell proliferation  

SciTech Connect

From studies with various Peromyscus cell lines, correlations were made which led to the proposal that H2A phosphorylation is most active in constitutive heterochromatin. Recent studies on the two H2A variants found in these cells have revealed that the high level of H2A phosphorylation associated with heterochromatin is not the result of an increase in H2A phosphorylation rate or an increase in the number of phosphorylation sites, but rather, is due to an increase in the proportion of one of the H2A variants which is more highly phosphorylated than the other. If H2A phosphorylation is necessary for the constitutive heterochromatin state, it is reasonable that the cell would accomplish the generation of this structure by permanently installing a more highly phosphorylated H2A in the heterochromatin nucleosome rather than by trying to modulate the phosphorylation rate in such a condensed structure. The proposal that histone phosphorylation is involved with the condensed structures of chromatin is based primarily on correlations between histone phosphorylation measurements and cellular phenomena. One proof that this concept is correct ultimately rests in the ability to demonstrate these correlations in isolated chromosomes and chromatin fractions. This demonstration is presently limited by the excessive dephosphorylation of histones which occurs during the isolation of chromosomes and chromatin fractions. Thus, the demonstration of an effective inhibitor of histone dephosphorylation which is compatible with the isolation of nuclear structures and chromatin fractions having native morphologies is essential for future studies on the biological function of histone phosphorylation. (ERB)

Gurley, L.R.; D'Anna, J.A.; Halleck, M.S.; Barham, S.S.; Walters, R.A.; Jett, J.H.; Tobey, R.A.



Activation of Escherichia coli antiterminator BglG requires its phosphorylation  

PubMed Central

Transcriptional antiterminator proteins of the BglG family control the expression of enzyme II (EII) carbohydrate transporters of the bacterial phosphotransferase system (PTS). In the PTS, phosphoryl groups are transferred from phosphoenolpyruvate (PEP) via the phosphotransferases enzyme I (EI) and HPr to the EIIs, which phosphorylate their substrates during transport. Activity of the antiterminators is negatively controlled by reversible phosphorylation catalyzed by the cognate EIIs in response to substrate availability and positively controlled by the PTS. For the Escherichia coli BglG antiterminator, two different mechanisms for activation by the PTS were proposed. According to the first model, BglG is activated by HPr-catalyzed phosphorylation at a site distinct from the EII-dependent phosphorylation site. According to the second model, BglG is not activated by phosphorylation, but solely through interaction with EI and HPr, which are localized at the cell pole. Subsequently BglG is released from the cell pole to the cytoplasm as an active dimer. Here we addressed this discrepancy and found that activation of BglG requires phosphorylatable HPr or the HPr homolog FruB in vivo. Further, we uniquely demonstrate that purified BglG protein becomes phosphorylated by FruB as well as by HPr in vitro. Histidine residue 208 in BglG is essential for this phosphorylation. These data suggest that BglG is in fact activated by phosphorylation and that there is no principal difference between the PTS-exerted mechanisms controlling the activities of BglG family proteins in Gram-positive and Gram-negative bacteria. PMID:22984181

Rothe, Fabian M.; Bahr, Thomas; Stulke, Jorg; Rak, Bodo; Gorke, Boris



Enzymatic modification of flaxseed fibers.  


Flaxseed (Linum usitatissimum L.) fibers were modified by oxidoreductive and cellulolytic enzymes. The lignin amount and intrinsic plant peroxidase activity was evaluated by histochemical and spectrophotometric assays. Peroxidase activity was not found from bast fibers. The flaxseed fibers were further separated and treated with laccase to conjugate the model compounds, that is, the hydrophobic gallate molecules on fiber surfaces. Laccase was able to slowly oxidize fiber-conjugated phenolics, but no fundamental changes in fiber cell surface structure or notable coupling of the applied hydrophobic gallate molecules onto the fibers occurred, as revealed by Fourier transform infrared spectroscopy. The reactivity of the mature fibers was further investigated using cellulolytic enzymes. Cellobiohydrolase (CBH) and endoglucanase (EG)-rich enzyme preparations were applied to reach a hydrolysis degree of 1-6% (of the fiber dry matter) using a standard enzyme dosage. The CBH mixture altered the fiber surface morphology distinctly, and SEM images illustrated fibers in which the cellulose fibrils seemed to be loosened and partially hydrolyzed. In contrast, the effect of the EG-rich preparation without CBH activity was notable on the fiber surface, polishing the surfaces. The cellulolytic treatments were potentially interesting for specific enzymatic modifications of flax fiber surfaces, whereas the approach to use oxidoreductive enzyme treatments on mature linseed fibers offered little potential, obviously due to the low lignin content of the fibers. PMID:23098092

Maijala, Pekka; Mäkinen, Marliina; Galkin, Sari; Fagerstedt, Kurt; Härkäsalmi, Tiina; Viikari, Liisa



Enzymatic degradation of thiolated chitosan.  


The objective of this study was to evaluate the biodegradability of thiolated chitosans in comparison to unmodified chitosan. Mediated by carbodiimide, thioglycolic acid (TGA) and mercaptonicotinic acid (MNA) were covalently attached to chitosan via formation an amide bond. Applying two different concentrations of carbodiimide 50 and 100?mM, two chitosan TGA conjugates (TGA A and TGA B) were obtained. According to chitosan solution (3% m/v) thiomer solutions were prepared and chitosanolytic enzyme solutions were added. Lysozyme, pectinase and cellulase were examined in chitosan degrading activity. The enzymatic degradability of these thiomers was investigated by viscosity measurements with a plate-plate viscometer. The obtained chitosan TGA conjugate A displayed 267.7 µmol and conjugate B displayed 116.3 µmol of immobilized thiol groups. With 325.4 µmol immobilized thiol groups, chitosan MNA conjugate displayed the most content of thiol groups. In rheological studies subsequently the modification proved that chitosan TGA conjugates with a higher coupling rate of thiol groups were not only degraded to a lesser extent by 20.9-26.4% but also more slowly. Chitosan mercaptonicotinic acid was degraded by 31.4-50.1% depending the investigated enzyme and even faster than unmodified chitosan. According to these results the biodegradability can be influenced by various modifications of the polymer which showed in particular that the rate of biodegradation is increased when MNA is the ligand, whereas the degradation is hampered when TGA is used as ligand for chitosan. PMID:23057506

Laffleur, Flavia; Hintzen, Fabian; Rahmat, Deni; Shahnaz, Gul; Millotti, Gioconda; Bernkop-Schnürch, Andreas



Chaperon-like Activation of Serum-Inducible Tryptophanyl-tRNA Synthetase Phosphorylation through Refolding as a Tool for Analysis of Clinical Samples1  

PubMed Central

Tryptophanyl-tRNA synthetase (TrpRS) expression alters in colorectal (CRC), pancreatic (PC), and cervical (CC) cancers. Here, phosphorylation of unfolded TrpRS and its fragments is stimulated by human cancer sera (CS; n = 13) and serum of rabbit tumor induced by Rous sarcoma virus, unaffected by donor sera (NS; 11/15) and abolished by alkaline phosphatase. At 20 years of follow-up, serum-inducible TrpRS phosphorylation found years before healthy donors (3/15) diagnosed with PC, CRC, or leukemia. I have examined a specificity of serum-inducible TrpRS phosphorylation and found, surprisingly, that serine phosphorylation of unfolded TrpRS is stimulated by anti-TrpRS rabbit antisera but is unaffected by rabbit nonimmune sera and antisera to other antigens. Anti-TrpRS immunoglobulin G (IgG) inhibits phosphorylation of full-length TrpRS and stimulates phosphorylation of its 20-kDa fragment. Phosphorylation of this fragment is stimulated also by CS but not NS. 2-Mercaptoethanol and cyclic AMP exerted synergistic inhibitory effect on TrpRS phosphorylation. Anti-TrpRS sera and casein act as chaperones increasing TrpRS phosphorylation through refolding. Histone-specific protein kinase activity in CS (n = 44) and anti-TrpRS sera was lower than that in NS (n = 11), rabbit nonimmune sera and antisera to other antigens. TrpRS inhibitors, tryptamine, and tryptophanol stimulate in vivo accumulation of enzymatically inactive, nonphosphorylated, aggregated and anti-TrpRS IgG refoldable TrpRS. Phosphorylation of postsurgical tissues (n = 18) reveals TrpRS in ovarian cancer (OVC) and CC but not in normal placenta and liver. In OVC, TrpRS phosphorylation increase correlates with elevated tryptophan-dependent ATP-inorganic pyrophosphate exchange. Although not inducing cancer, TrpRS triggers signaling concomitant with cancer. PMID:22191002

Paley, Elena L



Enzymatic hydrolysis of low substituted carboxymethyl cellulose  

E-print Network

cellulase from Trichoderma viride at specified optimum conditions of 50 C and pH 4. 8. 2. Identify products from the enzymatic hydrolysis of low DS CMC and measure amounts of glucose and total reducing sugars as a function of time during enzymatic... cellulase enzyme from Trichoderma viride (Boehringer Mannheim, Indianapolis, IN). Additional experiments were carried out using commercial CMC with an average DS of 0, 5 and 0. 73, courtesy of Hercules Inc. (Wilmington, DE). The following procedure...

Chanona Dominquez, Guadalupe



Volume 200, number 1 FEBS 3625 May 1986 Protein phosphorylation as a control for excitation energy  

E-print Network

transfer in Rhodospirillum rubrum Nigel G. Holmes and John F. Allen Department of Plant Sciences. They correlate these changes with phosphorylation of the B880ir subunit of the light-harvesting pigment, fluorescence yield at time, t; LHC-II, light-harvesting chlorophyll a/b pigment-protein complex 1 observations

Allen, John F.


Modulation of P1798 lymphosarcoma proliferation by protein phosphorylation  

SciTech Connect

The role of protein kinases in modulating cell proliferation was examined. Studies characterized the regulation of cell proliferation by adenosine 3':5'-monophosphate-dependent protein kinase (cA-Pk). Calcium/calmodulin-dependent myosin light chain kinase (MLCK) was isolated and examined as a potential substrate regulated by cA-PK in the rapidly proliferating P1798 lymphosarcoma. Modulation of cell proliferation by cA-PK was characterized by quantitating cell division by (methyl-/sup 3/H) thymidine ((/sup 3/H)-dT) incorporation into DNA, cAMP accumulations, and activation of cA-PK using P1798 lymphosarcoma cells. Epinephrine and prostaglandin E/sub 1/ (PGE/sub 1/) were demonstrated to suppress (/sup 3/H)-dT incorporation into DNA, to stimulate cAMP accumulation, and to activate cA-PK with dose-dependency. Calcium/calmodulin-dependent MLCK was partially purified from P1798 lymphosarcoma. P1798 MLCK phosphorylated myosin regulatory light chains (P-LC) from thymus, cardiac and skeletal muscles. One mol (/sup 32/Pi) was transferred into one mol cardiac or skeletal P-LC by P1798 MLCK. Apparent Km values of 65 and 51 were determined for ATP and cardiac P-LC, respectively. The apparent molecular weight of P1798 MLCK was 135,000. P1798 MLCK was phosphorylated by cA-PK. Phosphorylated MLCK showed a 41% decrease in calcium-dependent activity. Two additional protein kinases from P1798 lymphosarcoma phosphorylated cardiac and skeletal light chains (MLC).

Michnoff, C.A.H.



The impact of N-terminal phosphorylation on LHCII conformation in state transition  

NASA Astrophysics Data System (ADS)

State transition is an important protection mechanism of plants for maintaining optimal efficiency through redistributing unbalanced excitation energy between photo-system II (PSII) and photosystem I (PSI). This process depends on the reversible phosphorylation/dephosphorylation of the major light-harvesting complex II (LHCII) and its bi-directional migration between PSII and PSI. But it remains unclear how phosphorylation/dephosphorylation modulates the LHCII conformation and further regulates its reversible migration. Here molecular dynamics simulations (MDS) were employed to elucidate the impact of phosphorylation on LHCII conformation. The results indicated that N-terminal phosphorylation loosened LHCII trimer with decreased hydrogen bond (H-bond) interactions and extended the distances between neighboring monomers, which stemmed from the conformational adjustment of each monomer itself. Global conformational change of LHCII monomer started from its stromal Nterminal (including the phosphorylation sites) by enhancing its interaction to lipid membrane and by adjusting the interaction network with surrounded inter-monomer and intra-monomer transmembrane helixes of B, C, and A, and finally triggered the reorientation of transmembrane helixes and transferred the conformational change to luminal side helixes and loops. These results further our understanding in molecular mechanism of LHCII migration during state transition from the phosphorylation-induced microstructural feature of LHCII.

Ding, Jin-Hong; Li, Ning; Wang, Man-Liu; Zhang, Yan; Lü, Shou-Qin; Long, Mian



Light-regulated phosphorylation of maize phosphoenolpyruvate carboxykinase plays a vital role in its activity.  


Phosphoenolpyruvate carboxykinase (PEPCK)-the major decarboxylase in PEPCK-type C4 plants-is also present in appreciable amounts in the bundle sheath cells of NADP-malic enzyme-type C4 plants, such as maize (Zea mays), where it plays an apparent crucial role during photosynthesis (Wingler et al., in Plant Physiol 120(2):539-546, 1999; Furumoto et al., in Plant Mol Biol 41(3):301-311, 1999). Herein, we describe the use of mass spectrometry to demonstrate phosphorylation of maize PEPCK residues Ser55, Thr58, Thr59, and Thr120. Western blotting indicated that the extent of Ser55 phosphorylation dramatically increases in the leaves of maize seedlings when the seedlings are transferred from darkness to light, and decreases in the leaves of seedlings transferred from light to darkness. The effect of light on phosphorylation of this residue is opposite that of the effect of light on PEPCK activity, with the decarboxylase activity of PEPCK being less in illuminated leaves than in leaves left in the dark. This inverse relationship between PEPCK activity and the extent of phosphorylation suggests that the suppressive effect of light on PEPCK decarboxylation activity might be mediated by reversible phosphorylation of Ser55. PMID:24435212

Chao, Qing; Liu, Xiao-Yu; Mei, Ying-Chang; Gao, Zhi-Fang; Chen, Yi-Bo; Qian, Chun-Rong; Hao, Yu-Bo; Wang, Bai-Chen



Affinity-based SDS PAGE identification of phosphorylated Arabidopsis MAPKs and substrates by acrylamide pendant Phos-Tag™.  


Protein phosphorylation is the most abundant and best studied protein posttranslational modification, dedicated to the regulation of protein function and subcellular localization as well as to protein-protein interactions. Identification and quantitation of the dynamic, conditional protein phosphorylation can be achieved by either metabolic labeling of the protein of interest with (32)P-labeled ATP followed by autoradiographic analysis, the use of specific monoclonal or polyclonal antibodies against the phosphorylated protein species and finally by phosphoproteome delineation using mass spectrometry.Hereby we present a fourth alternative which relies on the enforced-affinity-based-electrophoretic separation of phosphorylated from non-phosphorylated protein species by standard SDS-PAGE systems co-polymerized with Phos-Tag™ and Mn(2+) or Zn(2+) cations. Phosphate groups of phosphorylated Ser, Thr, and Tyr residues form complexes with Mn(2+) and Zn(2+) cations with polyacrylamide immobilized Phos-Tag™. Following appropriate treatment of the gels, separated proteins can be quantitatively transferred to PVDF or nitrocellulose membranes and probed with common-not phosphorylation state specific-antibodies and delineate the occurrence of a certain phosphoprotein species against its non-phosphorylated counterpart. PMID:24908119

Komis, George; Taká?, Tomáš; Bekešová, Slávka; Vadovi?, Pavol; Samaj, Jozef



In the beginning, there was protein phosphorylation.  


The importance of reversible protein phosphorylation to cellular regulation cannot be overstated. In eukaryotic cells, protein kinase/phosphatase signaling pathways regulate a staggering number of cellular processes, including cell proliferation, cell death (apoptosis, necroptosis, necrosis), metabolism (at both the cellular and organismal levels), behavior and neurological function, development, and pathogen resistance. Although protein phosphorylation as a mode of eukaryotic cell regulation is familiar to most biochemists, many are less familiar with protein kinase/phosphatase signaling networks that function in prokaryotes. In this thematic minireview series, we present four minireviews that cover the important field of prokaryotic protein phosphorylation. PMID:24554697

Kyriakis, John M



Structural perspective on enzymatic halogenation.  


Simple halogen substituents frequently afford key structural features that account for the potency and selectivity of natural products, including antibiotics and hormones. For example, when a single chlorine atom on the antibiotic vancomycin is replaced by hydrogen, the resulting antibacterial activity decreases by up to 70% ( Harris , C. M. ; Kannan , R. ; Kopecka , H. ; Harris , T. M. J. Am. Chem. Soc. 1985 , 107 , 6652 - 6658 ). This Account analyzes how structure underlies mechanism in halogenases, the molecular machines designed by nature to incorporate halogens into diverse substrates. Traditional synthetic methods of integrating halogens into complex molecules are often complicated by a lack of specificity and regioselectivity. Nature, however, has developed a variety of elegant mechanisms for halogenating specific substrates with both regio- and stereoselectivity. An improved understanding of the biological routes toward halogenation could lead to the development of novel synthetic methods for the creation of new compounds with enhanced functions. Already, researchers have co-opted a fluorinase from the microorganism Streptomyces cattleya to produce (18)F-labeled molecules for use in positron emission tomography (PET) ( Deng , H. ; Cobb , S. L. ; Gee , A. D. ; Lockhart , A. ; Martarello , L. ; McGlinchey , R. P. ; O'Hagan , D. ; Onega , M. Chem. Commun. 2006 , 652 - 654 ). Therefore, the discovery and characterization of naturally occurring enzymatic halogenation mechanisms has become an active area of research. The catalogue of known halogenating enzymes has expanded from the familiar haloperoxidases to include oxygen-dependent enzymes and fluorinases. Recently, the discovery of a nucleophilic halogenase that catalyzes chlorinations has expanded the repertoire of biological halogenation chemistry ( Dong , C. ; Huang , F. ; Deng , H. ; Schaffrath , C. ; Spencer , J. B. ; O'Hagan , D. ; Naismith , J. H. Nature 2004 , 427 , 561 - 565 ). Structural characterization has provided a basis toward a mechanistic understanding of the specificity and chemistry of these enzymes. In particular, the latest crystallographic snapshots of active site architecture and halide binding sites have provided key insights into enzyme catalysis. Herein is a summary of the five classes of halogenases, focusing on the three most recently discovered: flavin-dependent halogenases, non-heme iron-dependent halogenases, and nucleophilic halogenases. Further, the potential roles of halide-binding sites in determining halide selectivity are discussed, as well as whether or not binding-site composition is always a seminal factor for selectivity. Expanding our understanding of the basic chemical principles that dictate the activity of the halogenases will advance both biology and chemistry. A thorough mechanistic analysis will elucidate the biological principles that dictate specificity, and the application of those principles to new synthetic techniques will expand the utility of halogenations in small-molecule development. PMID:18774824

Blasiak, Leah C; Drennan, Catherine L



Pseudomonas aeruginosa exotoxin Y is a promiscuous cyclase that increases endothelial tau phosphorylation and permeability.  


Exotoxin Y (ExoY) is a type III secretion system effector found in ~ 90% of the Pseudomonas aeruginosa isolates. Although it is known that ExoY causes inter-endothelial gaps and vascular leak, the mechanisms by which this occurs are poorly understood. Using both a bacteria-delivered and a codon-optimized conditionally expressed ExoY, we report that this toxin is a dual soluble adenylyl and guanylyl cyclase that results in intracellular cAMP and cGMP accumulation. The enzymatic activity of ExoY caused phosphorylation of endothelial Tau serine 214, accumulation of insoluble Tau, inter-endothelial cell gap formation, and increased macromolecular permeability. To discern whether the cAMP or cGMP signal was responsible for Tau phosphorylation and barrier disruption, pulmonary microvascular endothelial cells were engineered for the conditional expression of either wild-type guanylyl cyclase, which synthesizes cGMP, or a mutated guanylyl cyclase, which synthesizes cAMP. Sodium nitroprusside stimulation of the cGMP-generating cyclase resulted in transient Tau serine 214 phosphorylation and gap formation, whereas stimulation of the cAMP-generating cyclase induced a robust increase in Tau serine 214 phosphorylation, gap formation, and macromolecular permeability. These results indicate that the cAMP signal is the dominant stimulus for Tau phosphorylation. Hence, ExoY is a promiscuous cyclase and edema factor that uses cAMP and, to some extent, cGMP to induce the hyperphosphorylation and insolubility of endothelial Tau. Because hyperphosphorylated and insoluble Tau are hallmarks in neurodegenerative tauopathies such as Alzheimer disease, acute Pseudomonas infections cause a pathophysiological sequela in endothelium previously recognized only in chronic neurodegenerative diseases. PMID:22637478

Ochoa, Cristhiaan D; Alexeyev, Mikhail; Pastukh, Viktoriya; Balczon, Ron; Stevens, Troy



Phosphorylation based insulation devices design and implementation  

E-print Network

This thesis presents the analysis of a phosphorylation based insulation device implemented in Saccharomyces cerevisae and the minimization of the retroactivity to the input and retroactivity to the output of a single cycle ...

Rivera Ortiz, Phillip M. (Phillip Michael)



Modification of yeast ribosomal proteins. Phosphorylation.  

PubMed Central

Two-dimensional polyacrylamide-gel electrophoretic analysis of yeast ribosomal proteins labelled in vivo with 32PO43- revealed that the proteins S2 and S10 of the 40S ribosomal subunit, and the proteins L9, L30, L44 and L45 of the 60S ribosomal subunit, are phosphorylated in vivo. Most of the phosphate groups appeared to be linked to serine residues. Teh number of phosphate groups per molecule of phosphorylated protein species ranged from 0.01 to 0.79. Since most of the phosphorylated ribosomal proteins appear to associate with the pre-ribosomal particles at a very late stage of ribosome assembly, phosphorylation is more likely to play a role in the functioning of the ribosome than in its assembly. Images Fig. 1. Fig. 2. Fig. 3. PMID:367365

Kruiswijk, T; de Hey, J T; Planta, R J



Ex vivo Enzymatic Treatment of Aged CD4 T Cells Restores Cognate T-cell Helper Function and Enhances Antibody Production in Mice  

PubMed Central

Previous in vitro studies have shown that CD4 T cells from old mice have defects in T cell receptor (TCR) signaling, immune synapse formation, activation, and proliferation. We have reported that removing a specific set of surface glycoproteins by ex vivo treatment with O-sialoglycoprotein endopeptidase (OSGE) can reverse many aspects of the age-related decline in CD4 T cell function. However, the specific mechanism by which this process occurs remains unclear, and it is unknown whether this enzymatic treatment can also restore important aspects of adaptive immunity in vivo. By using an in vivo model of the immune response based on adoptive transfer of CD4 T cells from pigeon cytochrome C (PCC)-specific transgenic H-2(k/k) TCR-V?11V?3 CD4+ mice to syngeneic hosts, we now demonstrat that aging diminishes CD28 costimulatory signals in CD4 T cells. These age-associated defects include changes in phosphorylation of AKT and expression of glucose transporter type I, inducible T-cell costimulatory molecule, and CD40 ligand, suggesting that the lack of CD28 costimulation contributes to age-dependent loss of CD4 function. All of these deficits can be reversed by ex vivo OSGE treatment. Blocking B7-CD28 interactions on T cells prevents OSGE-mediated restoration of T cell function, suggesting that changes in surface glycosylation, including CD28, may be responsible for age-related costimulation decline. Finally, we showed that the age-related decline in CD4 cognate helper function for immunoglobin G production and long-term humoral immunity can also be restored by OSGE treatments of CD4 T cells prior to adoptive transfer. PMID:23136198

Perkey, Eric; Miller, Richard A.; Garcia, Gonzalo G.



Large-scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis  

Microsoft Academic Search

Protein phosphorylation regulates a wide range of cellular processes. Here, we report the proteome-wide mapping of in vivo phosphorylation sites in Arabidopsis by using complementary phosphopeptide enrichment techniques coupled with high-accuracy mass spectrometry. Using unfractionated whole cell lysates of Arabidopsis, we identified 2597 phosphopeptides with 2172 high-confidence, unique phosphorylation sites from 1346 proteins. The distribution of phosphoserine, phosphothreonine, and phosphotyrosine

Naoyuki Sugiyama; Hirofumi Nakagami; Keiichi Mochida; Arsalan Daudi; Masaru Tomita; Ken Shirasu; Yasushi Ishihama



Phosphorylated retinoblastoma protein stimulates DNA polymerase ?  

Microsoft Academic Search

Human retinoblastoma (Rb) protein, immunopurified from an extract of recombinant baculovirus infected cells, stimulated 10 – 100-fold the activity of DNA polymerase ? from calf thymus or human HeLa cells. Purified Rb protein is composed of two electrophoretically distinguishable forms, i.e., partially phosphorylated and under-phosphorylated forms. Dephosphorylation of Rb protein by protein phosphatase 2A largely diminished its stimulatory effect. On

Masaharu Takemura; Tomotaka Kitagawa; Shunji Izuta; Junji Wasa; Akira Takai; Tetsu Akiyama; Shonen Yoshida



Tyrosine Phosphorylation of Botulinum Neurotoxin Protease Domains  

PubMed Central

Botulinum neurotoxins are most potent of all toxins. Their N-terminal light chain domain (Lc) translocates into peripheral cholinergic neurons to exert its endoproteolytic action leading to muscle paralysis. Therapeutic development against these toxins is a major challenge due to their in vitro and in vivo structural differences. Although three-dimensional structures and reaction mechanisms are very similar, the seven serotypes designated A through G vastly vary in their intracellular catalytic stability. To investigate if protein phosphorylation could account for this difference, we employed Src-catalyzed tyrosine phosphorylation of the Lc of six serotypes namely LcA, LcB, LcC1, LcD, LcE, and LcG. Very little phosphorylation was observed with LcD and LcE but LcA, LcB, and LcG were maximally phosphorylated by Src. Phosphorylation of LcA, LcB, and LcG did not affect their secondary and tertiary structures and thermostability significantly. Phosphorylation of Y250 and Y251 made LcA resistant to autocatalysis and drastically reduced its kcat/Km for catalysis. A tyrosine residue present near the essential cysteine at the C-terminal tail of LcA, LcB, and LcG was readily phosphorylated in vitro. Inclusion of a competitive inhibitor protected Y426 of LcA from phosphorylation, shedding light on the role of the C-terminus in the enzyme’s substrate or product binding. PMID:22675300

Toth, Stephen; Brueggmann, Ernst E.; Oyler, George A.; Smith, Leonard A.; Hines, Harry B.; Ahmed, S. Ashraf



Phosphorylation of human skeletal muscle myosin  

SciTech Connect

Phosphorylation of the P-light chains (phosphorylatable light chains) in human skeletal muscle myosin was studied in vitro and in vivo under resting an d contracted conditions. biopsy samples from rested vastus lateralis muscle of male and female subjects were incubated in oxygenated physiological solution at 30/sup 0/C. Samples frozen following a quiescent period showed the presence of only unphosphorylated P-light chains designated LC2f (light chain two of fast myosin) CL2s and LC2s'(light chains two of slow myosin). Treatment with caffeine (10 mM) or direct electrical stimulation resulted in the appearance of three additional bands which were identified as the phosphorylated forms of the P-light chains i.e. LC2f-P, LC2s-P and LC2s'-P. The presence of phosphate was confirmed by prior incubation with (/sup 30/P) orthophosphate. Muscle samples rapidly frozen from resting vastus lateralis muscle revealed the presence of unphosphorylated and phosphorylated P-light chains in approximately equal ratios. Muscle samples rapidly frozen following a maximal 10 second isometric contraction showed virtually only phosphorylated fast and slow P-light chains. These results reveal that the P-light chains in human fast and slow myosin may be rapidly phosphorylated, but the basal level of phosphorylation in rested human muscle considerably exceeds that observed in animal muscles studied in vitro or in situ.

Houston, M.E.; Lingley, M.D.; Stuart, D.S.; Hoffman-Goetz, L.



Increased phospholipase A2 activity with phosphorylation of peroxiredoxin 6 requires a conformational change in the protein  

PubMed Central

We have shown previously and confirmed in the present study that the phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6) is markedly increased by phosphorylation. This report evaluated the conformation and thermodynamic stability of Prdx6 protein after phosphorylation to understand the physical basis for increased activity. Phosphorylation resulted in decreased negative far-UV CD, increased ANS binding, and lack of rigid tertiary structure, compatible with a change in conformation to that of a molten globule. The ?GDo was 3.3 ± 0.3 kcal mol-1 for Prdx6 and 1.7 ± 0.7 kcal mol-1 for pPrdx6 suggesting that phosphorylation destabilizes the protein. Phosphorylation of Prdx6 changed the conformation of the N-terminal domain exposing Trp 33, as determined by tryptophan fluorescence and NaI fluorescence quenching. The kinetics of interaction of proteins with unilamellar liposomes (DPPC/egg PC/cholesterol/PG; 50:25:15:10, mol/mol) was evaluated with tryptophan fluorescence. pPrdx6 bound to liposomes with higher affinity (Kd, 5.6 ± 1.2 ?M) in comparison to Prdx6 (Kd, 24.9 ± 4.5 ?M). By isothermal titration calorimetry, pPrdx6 bound to liposomes with a large exothermic heat loss (?H = -31.49 ± 0.22 kcal mol-1). Correlating our conformation studies with the published crystal structure of oxidized Prdx6 suggests that phosphorylation results in exposure of hydrophobic residues, thereby providing accessibility to the sites for liposome binding. Because binding of the enzyme to the phospholipid substrate interface is a requirement for PLA2 activity, these results indicate that a change in the conformation of Prdx6 upon its phosphorylation is the basis for enhancement of PLA2 enzymatic activity. PMID:22663767

Rahaman, Hamidur; Zhou, Suiping; Dodia, Chandra; Feinstein, Sheldon I.; Huang, Shaohui; Speicher, David; Fisher, Aron B.



Enzymatic aerobic ring rearrangement of optically active furylcarbinols.  


Biogenic furans are currently discussed as highly attractive alternative feedstock in a post-fossil society; thus, also the creation of sustainable furan valorization pathways appears of great importance. Here an artificial Achmatowicz monooxygenase activity for the aerobic ring expansion of furans is achieved by the combination of commercial glucose oxidase as oxygen-activating biocatalyst and wild-type chloroperoxidase as oxygen-transfer mediator, providing a biological ready-to-use solution for this truly synthetic furan rearrangement. In concert with enzymatic transformations for the enantioselective preparation of optically active furylcarbinols, purely biocatalytic reaction cascades for the stereocontrolled construction of complex pyranones are obtained, exhibiting high functional group tolerance even to oxidation-sensitive moieties. PMID:25335580

Thiel, Daniel; Dokni?, Diana; Deska, Jan



Enzymatic solubilization of nitrogenous constituents of carrots  

E-print Network

enzymatic activity are reported to be the result of pH induced ioni zation of either enzyme, substrate or enzyme-substrate complex (Reed, 1966). In selecting a proper pH for enzymat1c hydrolys1s, the activity range for each enzyme must be considered... on type of substrate (Rohm & Haas, 1966). For example, Hale (1969) when studing the activities of enzymes on the hydrolys is of fish prote1n, found that the enzyme ficin exhibited high act1v1ty fdr a shor t hydrolysis period. However, other workers...

Curry, James Cannon



IKK-i and TBK-1 are enzymatically distinct from the homologous enzyme IKK-2: comparative analysis of recombinant human IKK-i, TBK-1, and IKK-2.  


NF-kappaB is sequestered in the cytoplasm by the inhibitory IkappaB proteins. Stimulation of cells by agonists leads to the rapid phosphorylation of IkappaBs leading to their degradation that results in NF-kappaB activation. IKK-1 and IKK-2 are two direct IkappaB kinases. Two recently identified novel IKKs are IKK-i and TBK-1. We have cloned, expressed, and purified to homogeneity recombinant human (rh)IKK-i and rhTBK-1 and compared their enzymatic properties with those of rhIKK-2. We show that rhIKK-i and rhTBK-1 are enzymatically similar to each other. We demonstrate by phosphopeptide mapping and site-specific mutagenesis that rhIKK-i and rhTBK-1 are phosphorylated on serine 172 in the mitogen-activated protein kinase kinase activation loop and that this phosphorylation is necessary for kinase activity. Also, rhIKK-i and rhTBK-1 have differential peptide substrate specificities compared with rhIKK-2, the mitogen-activated protein kinase kinase activation loop of IKK-2 being a more favorable substrate than the IkappaBalpha peptide. Finally, using analogs of ATP, we demonstrate unique differences in the ATP-binding sites of rhIKK-i, rhTBK-1, and rhIKK-2. Thus, although these IKKs are structurally similar, their enzymatic properties may provide insights into their unique functions. PMID:11839743

Kishore, Nandini; Huynh, Q Khai; Mathialagan, Sumathy; Hall, Troii; Rouw, Sharon; Creely, David; Lange, Gary; Caroll, James; Reitz, Beverley; Donnelly, Ann; Boddupalli, Hymavathi; Combs, Rodney G; Kretzmer, Kuniko; Tripp, Catherine S



Visualizing an ultra-weak protein-protein interaction in phosphorylation signaling.  


Proteins interact with each other to fulfill their functions. The importance of weak protein-protein interactions has been increasingly recognized. However, owing to technical difficulties, ultra-weak interactions remain to be characterized. Phosphorylation can take place via a KD ?25?mM interaction between two bacterial enzymes. Using paramagnetic NMR spectroscopy and with the introduction of a novel Gd(III) -based probe, we determined the structure of the resulting complex to atomic resolution. The structure accounts for the mechanism of phosphoryl transfer between the two enzymes and demonstrates the physical basis for their ultra-weak interaction. Further, molecular dynamics (MD) simulations suggest that the complex has a lifetime in the micro- to millisecond regimen. Hence such interaction is termed a fleeting interaction. From mathematical modeling, we propose that an ultra-weak fleeting interaction enables rapid flux of phosphoryl signal, providing a high effective protein concentration. PMID:25131700

Xing, Qiong; Huang, Peng; Yang, Ju; Sun, Jian-Qiang; Gong, Zhou; Dong, Xu; Guo, Da-Chuan; Chen, Shao-Min; Yang, Yu-Hong; Wang, Yan; Yang, Ming-Hui; Yi, Ming; Ding, Yi-Ming; Liu, Mai-Li; Zhang, Wei-Ping; Tang, Chun



Functional dissection of the phosphorylated termini of fission yeast DNA topoisomerase II  

PubMed Central

Fission Yeast DNA topoisomerase II (165 kD) consists of an enzymatically active 125-kD core, approximately 10-kD NH2-terminal and 30-kD COOH-terminal domains. The question addressed in the present study is what is the role of the topo II termini. Although deletion of either the NH2 or the COOH terminus is viable, deletion of both termini is lethal; the termini share an essential role for viability. We show here that topo II phosphorylation sites are localized in the terminal domains, but dephosphorylated topo II is still active. The topo II terminal sequences are required for nuclear localization; topo II double terminal deletion mutants are deficient for nuclear targeting, whereas wild-type and single deletion mutant topo IIs are transported into the nucleus with different efficiencies. Functional subdomains in the NH2 terminus are further dissected; we identified a 15 amino acid nuclear localization sequence (NLS) which is essential for viability and nuclear localization when the COOH terminus is deleted. This NLS could be substituted with SV-40 large T-antigen NLS. Two other functional subdomains were found; a non-essential acidic stretch which is phosphorylated and apparently enhances the nuclear localization and an essential hydrophilic stretch of unknown function. Motifs similar to these three NH2-terminal subdomains are also found in the COOH terminus. Our results support the possibility that phosphorylation of topo II does not play an essential role in fission yeast. PMID:1332977



Covalent co-immobilization of glucose oxidase and ferrocenedicarboxylic acid for an enzymatic biofuel cell  

Microsoft Academic Search

An enzymatic biofuel cell was prepared based on covalently linked glucose oxidase (GOx) and bilirubin oxidase (BOx) as the anodic and cathodic catalyst, respectively. We performed the covalent co-immobilization of GOx and a ferrocene derivative 1,1?-ferrocenedicarboxylic acid, as an electron transfer mediator, on a graphite carbon felt electrode. In an effort to explore a new type of bioanode, each step

Joonmok Shim; Gha-Young Kim; Seung-Hyeon Moon



Enzymatic route to preparative-scale synthesis of UDP-GlcNAc/GalNAc, their analogues and GDP-fucose  

PubMed Central

Enzymatic synthesis using glycosyltransferases is a powerful approach to building polysaccharides with high efficiency and selectivity. Sugar nucleotides are fundamental donor molecules in enzymatic glycosylation reactions by Leloir-type glycosyltransferases. The applications of these donors are restricted by their limited availability. In this protocol, N-acetylglucosamine (GlcNAc)/N-acetylgalactosamine (GalNAc) are phosphorylated by N-acetylhexosamine 1-kinase (NahK) and subsequently pyrophosphorylated by N-acetylglucosamine uridyltransferase (GlmU) to give UDP–GlcNAc/GalNAc. Other UDP–GlcNAc/GalNAc analogues can also be prepared depending on the tolerance of these enzymes to the modified sugar substrates. Starting from l-fucose, GDP–fucose is constructed by one bifunctional enzyme l-fucose pyrophosphorylase (FKP) via two reactions. PMID:20224564

Zhao, Guohui; Guan, Wanyi; Cai, Li; Wang, Peng George



Original article Enzymatic equipment of Ascosphaera apis  

E-print Network

Original article Enzymatic equipment of Ascosphaera apis and the development of infection — The process of infection of honey bee brood by the fungus Ascosphaera apis was ini- tiated the fungus pierces the cuticle. Ascosphaera apis / Apis mellifera / larval cuticle / penetration / infection

Paris-Sud XI, Université de


A singular enzymatic megacomplex from Bacillus subtilis  

E-print Network

A singular enzymatic megacomplex from Bacillus subtilis Paul D. Straight*, Michael A. Fischbach% of the Bacillus subtilis genome, encodes the subunits of 2.5 megadalton active hybrid NRPS/PKS. Many copies enzymes in the context of their native producer organisms. The genome of Bacillus subtilis contains

Rudner, David


Recent development of miniatured enzymatic biofuel cell  

Microsoft Academic Search

Enzymatic biofuel cells (EBFCs) that oxidize biological fuels using enzyme-modified electrodes are considered a promising candidate for implantable power sources. However, there are still challenges to overcome before biofuel cells become competitive in any practical applications. Currently, the short lifespan of the catalytic enzymes and poor power density are the most critical issues in developing EBFCs. In this paper, we

Yin Song; Varun Penmatsa; Chunlei Wang



Frank Westheimer's Early Demonstration of Enzymatic Specificity  

ERIC Educational Resources Information Center

In this article I review one of the most significant accomplishments of Frank H. Westheimer, one of the most respected chemists of the 20th century. This accomplishment was a series of stereospecific enzymatic oxidation and reduction experiments that led chemists to recognize what we now call the enantiotopic and diastereotopic relationships of…

Ault, Addison



Synthesis of thermosensitive polymers having enzymatic degradability  

Microsoft Academic Search

.   2-Methylene-1,3,6-trioxocane and N-isopropylacrylamide were copolymerized in different feed ratios in the presence of a radical initiator. The resulting copolymers\\u000a were characterized by gel-permeation chromatography and differential scanning calorimeter. The copolymers obtained were thermosensitive\\u000a and had enzymatic degradability.

Yoichi Hiraguri; Yutaka Tokiwa



Enzymatic hydrolysis of pretreated rice straw  

Microsoft Academic Search

California rice straw is being evaluated as a feedstock for production of power and fuel. This paper examines the initial steps in the process: pretreatment of rice straw and enzymatic hydrolysis of the polysaccharides in the pretreated material to soluble sugars. Rice straw was subjected to three distinct pretreatment procedures: acid-catalyzed steam explosion (Swan Biomass Company), acid hydrolysis (U.S. DOE

E. Yu. Vlasenko; H. Ding; J. M. Labavitch; S. P. Shoemaker



The construction, fouling and enzymatic cleaning of a textile dye surface.  


The enzymatic cleaning of a rubisco protein stain bound onto Surface Plasmon Resonance (SPR) biosensor chips having a dye-bound upper layer is investigated. This novel method allowed, for the first time, a detailed kinetic study of rubisco cleanability (defined as fraction of adsorbed protein removed from a surface) from dyed surfaces (mimicking fabrics) at different enzyme concentrations. Analysis of kinetic data using an established mathematical model able to decouple enzyme transfer and reaction processes [Onaizi, He, Middelberg, Chem. Eng. Sci. 64 (2008) 3868] revealed a striking effect of dyeing on enzymatic cleaning performance. Specifically, the absolute rate constants for enzyme transfer to and from a dye-bound rubisco stain were significantly higher than reported previously for un-dyed surfaces. These increased transfer rates resulted in higher surface cleanability. Higher enzyme mobility (i.e., higher enzyme adsorption and desorption rates) at the liquid-dye interface was observed, consistent with previous suggestions that enzyme surface mobility is likely correlated with overall enzyme cleaning performance. Our results show that reaction engineering models of enzymatic action at surfaces may provide insight able to guide the design of better stain-resistant surfaces, and may also guide efforts to improve cleaning formulations. PMID:20708195

Onaizi, Sagheer A; He, Lizhong; Middelberg, Anton P J



Attempting to rewrite History: challenges with the analysis of histidine-phosphorylated peptides.  


A significant number of proteins in both eukaryotes and prokaryotes are known to be post-translationally modified by the addition of phosphate, serving as a means of rapidly regulating protein function. Phosphorylation of the amino acids serine, threonine and tyrosine are the focus of the vast majority of studies aimed at elucidating the extent and roles of such modification, yet other amino acids, including histidine and aspartate, are also phosphorylated. Although histidine phosphorylation is known to play extensive roles in signalling in eukaryotes, plants and fungi, roles for phosphohistidine are poorly defined in higher eukaryotes. Characterization of histidine phosphorylation aimed at elucidating such information is problematic due to the acid-labile nature of the phosphoramidate bond, essential for many of its biological functions. Although MS-based strategies have proven extremely useful in the analysis of other types of phosphorylated peptides, the chromatographic procedures essential for such approaches promote rapid hydrolysis of phosphohistidine-containing peptides. Phosphate transfer to non-biologically relevant aspartate residues during MS analysis further complicates the scenario. PMID:23863184

Gonzalez-Sanchez, Maria-Belen; Lanucara, Francesco; Helm, Matthew; Eyers, Claire E



Identification of CaMKII phosphorylation sites in Connexin43 by high-resolution mass spectrometry.  


Connexin43 (Cx43) is a major cardiac gap junction channel protein required for normal electrical and contractile activity. Gap junction channel assembly, function, and turnover are regulated by phosphorylation under both normal and disease conditions. The carboxyl terminus (CT) of Cx43 contains numerous amino acid residues that are phosphorylated by protein kinases. However, our knowledge of the specific residues and kinases involved is incomplete. The objective of this study was to identify amino acid residues in the Cx43-CT that are targets of the multifunctional protein kinase, Ca(2+)/calmodulin protein kinase II (CaMKII), an enzyme known to play critical roles in Ca(2+) homeostasis, transcription, apoptosis, and ischemic heart disease. We subjected fusion protein containing the Cx43-CT to phosphorylation by CaMKII in vitro, digestion with Lys-C and trypsin followed by enrichment for phosphorylated peptides using TiO(2), and analysis in an LTQ XL Orbitrap with collision-induced dissociation and electron transfer dissociation. We deduced the sites of modification by interpreting tandem spectra from these "orthogonal" methods of gas phase peptide fragmentation. We have identified 15 serine residues, including one novel site, in the Cx43-CT that are phosphorylated by CaMKII, the activity of which may be important in regulating Cx43 in normal and diseased hearts. PMID:21158428

Huang, Richard Y-C; Laing, James G; Kanter, Evelyn M; Berthoud, Viviana M; Bao, Mingwei; Rohrs, Henry W; Townsend, R Reid; Yamada, Kathryn A



Protein phosphorylation in human peripheral nerve: Altered phosphorylation of a 25-kDa glycoprotein in leprosy  

Microsoft Academic Search

Protein phosphorylation in a low speed supernatant of human peripheral nerve (tibial and sural) homogenate was investigated.\\u000a The major phosphorylated proteins had molecular mass in the range of 70, 55, 45, and 25 kDa. Mg2+ or Mn2+ was essential for maximum phosphorylation although Zn2+, Co2+, and Ca2+, could partially support phosphorylation. External protein substrates casein and histone were also phosphorylated.

Lavanya M. Suneetha; Ravi Jacob Korula; A. S. Balasubramanian



Enzymatic Bioprocessing - New Tool of Extensive Natural Fibre Source Utilization  

Microsoft Academic Search

New enzymes are going to have great potential in bast fibre processing and modification for different end uses. There are several new technologies using enzymes able to modify fiber parametres, achieve desired properties, improve processing results and ecology in the area of bast fibre processing and fabric finishing. Enzymatic retting of flax, enzymatic cottonization of bast fibre, enzymatic hemp separation,

Marek Jan; Antonov Viktor; Bjelkova Marie; Smirous Prokop; Fischer Holger; Janosik Stefan


Transfer form

10/02 Transfer Investigational Agent Form This form is to be used for an intra-institutional transfer, one transfer/form. Division of Cancer Prevention National Cancer Institute National Institutes of Health TRANSFER FROM: Investigator transferring agent:


Human acetyl-CoA carboxylase 2 expressed in silkworm Bombyx mori exhibits posttranslational biotinylation and phosphorylation.  


Biotin-dependent human acetyl-CoA carboxylases (ACCs) are integral in homeostatic lipid metabolism. By securing posttranslational biotinylation, ACCs perform coordinated catalytic functions allosterically regulated by phosphorylation/dephosphorylation and citrate. The production of authentic recombinant ACCs is heeded to provide a reliable tool for molecular studies and drug discovery. Here, we examined whether the human ACC2 (hACC2), an isoform of ACC produced using the silkworm BmNPV bacmid system, is equipped with proper posttranslational modifications to carry out catalytic functions as the silkworm harbors an inherent posttranslational modification machinery. Purified hACC2 possessed genuine biotinylation capacity probed by biotin-specific streptavidin and biotin antibodies. In addition, phosphorylated hACC2 displayed limited catalytic activity whereas dephosphorylated hACC2 revealed an enhanced enzymatic activity. Moreover, hACC2 polymerization, analyzed by native page gel analysis and atomic force microscopy imaging, was allosterically regulated by citrate and the phosphorylation/dephosphorylation modulated citrate-induced hACC2 polymerization process. Thus, the silkworm BmNPV bacmid system provides a reliable eukaryotic protein production platform for structural and functional analysis and therapeutic drug discovery applications implementing suitable posttranslational biotinylation and phosphorylation. PMID:24740690

Hwang, In-Wook; Makishima, Yu; Kato, Tatsuya; Park, Sungjo; Terzic, Andre; Park, Enoch Y



Factors impeding enzymatic wheat gluten hydrolysis at high solid concentrations.  


Enzymatic wheat gluten hydrolysis at high solid concentrations is advantageous from an environmental and economic point of view. However, increased wheat gluten concentrations result in a concentration effect with a decreased hydrolysis rate at constant enzyme-to-substrate ratios and a decreased maximum attainable degree of hydrolysis (DH%). We here identified the underlying factors causing the concentration effect. Wheat gluten was hydrolyzed at solid concentrations from 4.4% to 70%. The decreased hydrolysis rate was present at all solid concentrations and at any time of the reaction. Mass transfer limitations, enzyme inhibition and water activity were shown to not cause this hydrolysis rate limitation up to 50% solids. However, the hydrolysis rate limitation can be, at least partly, explained by a second-order enzyme inactivation process. Furthermore, mass transfer impeded the hydrolysis above 60% solids. Addition of enzyme after 24 h at high solid concentrations scarcely increased the DH%, suggesting that the maximum attainable DH% decreases at high solid concentrations. Reduced enzyme activities caused by low water activities can explain this DH% limitation. Finally, a possible influence of the plastein reaction on the DH% limitation is discussed. PMID:24474643

Hardt, N A; Janssen, A E M; Boom, R M; van der Goot, A J



A singular enzymatic megacomplex from Bacillus subtilis  

PubMed Central

Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome (?2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies ?2% of the Bacillus subtilis genome, encodes the subunits of ?2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membrane-associated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs. PMID:17190806

Straight, Paul D.; Fischbach, Michael A.; Walsh, Christopher T.; Rudner, David Z.; Kolter, Roberto



Characterization of an Arabidopsis thaliana mutant lacking a cytosolic non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase  

Microsoft Academic Search

Non-phosphorylating glyceraldehyde-\\u00093-phosphate dehydrogenase (NP-GAPDH) is a conserved cytosolic protein found in higher\\u000a plants. In photosynthetic cells, the enzyme is involved in a shuttle transfer mechanism to export NADPH from the chloroplast\\u000a to the cytosol. To investigate the role of this enzyme in plant tissues, we characterized a mutant from Arabidopsis thaliana having an insertion at the NP-GAPDH gene locus.

Sebastián P. Rius; Paula Casati; Alberto A. Iglesias; Diego F. Gomez-Casati



Lanthanide complexes with aromatic o-phosphorylated ligands: synthesis, structure elucidation and photophysical properties.  


Lanthanide complexes LnL3 (Ln = Sm, Eu, Tb, Dy, Tm, Yb, Lu) with aromatic o-phosphorylated ligands (HL(1) and HL(2)) have been synthesized and identified. Their molecular structure was proposed on the basis of a new complex approach, including DFT calculations, Sparkle/PM3 modelling, EXAFS spectroscopy and luminescent probing. The photophysical properties of all of the complexes were investigated in detail to obtain a deeper insight into the energy transfer processes. PMID:24297298

Shuvaev, Sergey; Utochnikova, Valentina; Marciniak, ?ukasz; Freidzon, Alexandra; Sinev, Ilya; Van Deun, Rik; Freire, Ricardo O; Zubavichus, Yan; Grünert, Wolfgang; Kuzmina, Natalia



Redox polymer mediation for enzymatic biofuel cells  

Microsoft Academic Search

Mediated biocatalytic cathodes prepared from the oxygen-reducing enzyme laccase and redox-conducting osmium hydrogels were characterized for use as cathodes in enzymatic biofuel cells. A series of osmium-based redox polymers was synthesized with redox potentials spanning the range from 0.11 V to 0.85 V (SHE), and the resulting biocatalytic electrodes were modeled to determine reaction kinetic constants using the current response,

Joshua Gallaway



Enzymatic determination of phenols using peanut peroxidase  

Microsoft Academic Search

The influence of phenol and its derivatives on the kinetics of oxidation of aryldiamines (indicator-substrates) catalyzed by novel plant peroxidase—cationic peanut peroxidase—was studied. The character of influence of phenols on the kinetics of enzymatic oxidation of benzidine, o-dianisidine, and 3,3?,5,5?-tetramethylbenzidine (TMB) with hydrogen peroxide was found to depend on a correlation between redox properties of phenols and the indicator-substrate of

Nailya A Bagirova; Tatyana N Shekhovtsova; Robert B van Huystee



Pretreatment and enzymatic saccharification of corn fiber  

Microsoft Academic Search

Corn fiber consists of about 20% starch, 14% cellulose, and 35% hemicellulose, and has the potential to serve as a low-cost\\u000a feedstock for production of fuel ethanol. Several pretreatments (hot water, alkali, and dilute, acid) and enzymatic saccharification\\u000a procedures were evaluated for the conversion of corn fiber starch, cellulose, and hemicellulose to monomeric sugars. Hot water\\u000a pretreatment (121°C, 1 h)

Badal C. Saha; Rodney J. Bothast



Ion channels, phosphorylation and mammalian sperm capacitation  

PubMed Central

Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies. PMID:21540868

Visconti, Pablo E; Krapf, Dario; de la Vega-Beltran, Jose Luis; Acevedo, Juan Jose; Darszon, Alberto



APP processing is regulated by cytoplasmic phosphorylation  

Microsoft Academic Search

myloid- ? peptide (A ? ) aggregate in senile plaque is a key characteristic of Alzheimer's disease (AD). Here, we show that phosphorylation of amyloid precursor protein (APP) on threonine 668 (P-APP) may play a role in APP metabolism. In AD brains, P-APP accumulates in large vesicular structures in afflicted hippocampal pyramidal neurons that costain with antibodies against endosome markers

Ming-Sum Lee; Shih-Chu Kao; Cynthia A. Lemere; Weiming Xia; Huang-Chun Tseng; Ying Zhou; Rachael Neve; Michael K. Ahlijanian; Li-Huei Tsai



Mitochondrial DNA & Oxidative Phosphorylation ATP SYNTHASE  

E-print Network

# RNAi #12;Ethidium bromide - DNA intercalating dye EtBr inserts itself between the bases of the DNAMitochondrial DNA & Oxidative Phosphorylation 2 proteins ATP SYNTHASE COMPLEX IV COMPLEX IIICOMPLEX II COMPLEX I 1 protein 3 proteins Human mitochondrial DNA 7 proteins #12;Replication Organisation

Schnaufer, Achim


Protein Phosphorylation and Long-term Synaptic  

E-print Network

Protein Phosphorylation and Long-term Synaptic Plasticity A Barria, Oregon Health Sciences University, Portland, Oregon, USA V Derkach,Oregon Health Sciences University, Portland, Oregon, USA TR Soderling,Oregon Health Sciences University, Portland, Oregon, USA Learning and memory are complex

Malinow, Roberto


Enzymatic glycoprotein synthesis: Preparation of ribonuclease glycoforms via enzymatic glycopeptide condensation and glycosylation  

SciTech Connect

In order to study the effects carbohydrates have on glycoprotein structure and funciton, it is imperative to be able to synthesize the appropriate natural and non-natural glycoprotein variants in a single form. Because the available in vivo techniques provide only heterogeneous mixtures of different glycoforms, enzymatic in vitro methodologies have been pursued. Using the N-glycoprotein RNase B as a model system, the oligosaccharide was removed leaving only the N-acetylglucosamine as a `tag` to the site of glycosylation. Glycosyltransferases were then used to build a unique carbohydrate moiety. A new RNase glycoform containing the branched oligosccharide, sialyl Lewis X or the Hg derivative, was synthesized enzymatically to demonstrate the feasibility of the method. In addition, the monoglycosylated protein was digested into several smaller pieces by subtilisin BPN`. These fragments were religated by subtilisin 8397 to the full length RNase by addition glycerol; this method points to a new chemical-enzymatic process for the synthesis of glycoproteins using synthetic peptides and glycopeptides as substrates for enzymatic ligation followed by further enzymatic glycosylations. 29 refs., 6 figs.

Witte, K.; Sears, P.; Martin, R.; Wong, C.H. [Scripps Research Inst., La Jolla, CA (United States)] [Scripps Research Inst., La Jolla, CA (United States)



Potentiometric selectivity of ion-selective electrodes for alkaline-earth elements based on podands with phosphoryl terminal groups  

Microsoft Academic Search

Dependence of the analytical characteristics (detection limits, DLs) and potentiometric selectivity Kijpot) of the ion-selective electrodes (ISEs) based on phosphoryl-containing podands on the structures was investigated. It was demonstrated that the DL was determined by complex facilitated transfer energy. It was shown that the DLs of the majority of the ionophores weakly depend on the lipophilicity of the ionophores. Ionophore

O. M. Petrukhin; A. B. Kharitonov; Yu. I. Urusov; E. V. Schipulo; N. Ye. Kruchinina; V. E. Baulin



Cold enzymatic bleaching of fluid whey.  


Chemical bleaching of fluid whey and retentate with hydrogen peroxide (HP) alone requires high concentrations (100-500 mg of HP/kg) and recent studies have demonstrated that off-flavors are generated during chemical bleaching that carry through to spray-dried whey proteins. Bleaching of fluid whey and retentate with enzymes such as naturally present lactoperoxidase or an exogenous commercial peroxidase (EP) at cold temperatures (4°C) may be a viable alternative to traditional chemical bleaching of whey. The objective of this study was to determine the optimum level of HP for enzymatic bleaching (both lactoperoxidase and EP) at 4°C and to compare bleaching efficacy and sensory characteristics to HP chemical bleaching at 4°C. Selected treatments were subsequently applied for whey protein concentrate with 80% protein (WPC80) manufacture. Fluid Cheddar whey and retentate (80% protein) were manufactured in triplicate from pasteurized whole milk. The optimum concentration of HP (0 to 250 mg/kg) to activate enzymatic bleaching at 4°C was determined by quantifying the loss of norbixin. In subsequent experiments, bleaching efficacy, descriptive sensory analysis, and volatile compounds were monitored at selected time points. A control with no bleaching was also evaluated. Enzymatic bleaching of fluid whey and retentate at 4°C resulted in faster bleaching and higher bleaching efficacy (color loss) than bleaching with HP alone at 250 mg/kg. Due to concentrated levels of naturally present lactoperoxidase, retentate bleached to completion (>80% norbixin destruction in 30 min) faster than fluid whey at 4°C (>80% norbixin destruction in 12h). In fluid whey, the addition of EP decreased bleaching time. Spray-dried WPC80 from bleached wheys, regardless of bleaching treatment, were characterized by a lack of sweet aromatic and buttery flavors, and the presence of cardboard flavor concurrent with higher relative abundance of 1-octen-3-ol and 1-octen-3-one. Among enzymatically bleached WPC80, lactoperoxidase-bleached WPC80 contained higher relative abundance of 2,3-octadienone, 2-pentyl furan, and hexanal than those bleached with added EP. Bleach times, bleaching efficacy, and flavor results suggest that enzymatic bleaching may be a viable and desirable alternative to HP bleaching of fluid whey or retentate. PMID:24140314

Campbell, R E; Drake, M A



Phosphorylation of nitrogen heterocycles. X. Azole catalysis in the phosphorylation by phosphorus-32 recoil atoms  

SciTech Connect

A specific catalytic function was found for azoles in the phosphorylation of alcohols by /sup 32/P recoil atoms due to the intermediate formation of azolido (/sup 32/P)phosphates whose phosphorylating capacity determines the direction of these reactions. The possibility of the Atherton-Todd reaction under nuclear chemical synthesis conditions upon stabilization of phosphorus-32 recoil atoms in the CCl/sub 4/-alkanol-azole system was demonstrated.

Makarov, A.M.; Matevosyan, G.L.; Zavlin, P.M.



NetworKIN: a resource for exploring cellular phosphorylation networks  

E-print Network

Protein kinases control cellular responses by phosphorylating specific substrates. Recent proteome-wide mapping of protein phosphorylation sites by mass spectrometry has discovered thousands of in vivo sites. Systematically ...

Jensen, Lars Juhl


Protein phosphorylation in isolated human adipocytes - Adrenergic control of the phosphorylation of hormone-sensitive lipase  

SciTech Connect

The effect of adrenergic agents on protein phosphorylation in human adipocytes was examined. Freshly isolated human fat cells were incubated with {sup 32}PO{sub 4} in order to label intracellular ATP, then treated with a variety of adrenergic and other pharmacologic agents. Treatment with the {beta}-adrenergic agonist isoproterenol led to a significant increase in phosphate content of at least five protein bands (M{sub r} 52, 53, 63, 67, 84 kDa). The increase in phosphorylation was partially inhibited by the {alpha}-2 agonist clonidine. Epinephrine, a combined {alpha} and {beta} agonist, was less effective at increasing phosphate content of the proteins than was isoproterenol. Neither insulin nor the {alpha}-1 agonist phenylephrine had any discernible effect on the pattern of protein phosphorylation. The 84 kDa phosphorylated peptide band appears to contain hormone-sensitive lipase, a key enzyme in the lipolytic pathway which is activated by phosphorylation. These results are somewhat different than previously reported results for rat adipocytes, and represent the first report of overall pattern and adrenergic modulation of protein phosphorylation in human adipocytes.

Smiley, R.M. (Rockefeller Univ., New York, NY (USA) Columbia Univ College of Physicians and Surgeons, New York, NY (USA)); Paul, S.; Browning, M.D.; Leibel, R.L.; Hirsch, J. (Rockefeller Univ., New York, NY (USA))



Neurofilament Phosphorylation during Development and Disease: Which Came First, the Phosphorylation or the Accumulation?  

PubMed Central

Posttranslational modification of proteins is a ubiquitous cellular mechanism for regulating protein function. Some of the most heavily modified neuronal proteins are cytoskeletal proteins of long myelinated axons referred to as neurofilaments (NFs). NFs are type IV intermediate filaments (IFs) that can be composed of four subunits, neurofilament heavy (NF-H), neurofilament medium (NF-M), neurofilament light (NF-L), and ?-internexin. Within wild type axons, NFs are responsible for mediating radial growth, a process that determines axonal diameter. NFs are phosphorylated on highly conserved lysine-serine-proline (KSP) repeats located along the C-termini of both NF-M and NF-H within myelinated axonal regions. Phosphorylation is thought to regulate aspects of NF transport and function. However, a key pathological hallmark of several neurodegenerative diseases is ectopic accumulation and phosphorylation of NFs. The goal of this review is to provide an overview of the posttranslational modifications that occur in both normal and diseased axons. We review evidence that challenges the role of KSP phosphorylation as essential for radial growth and suggests an alternative role for NF phosphorylation in myelinated axons. Furthermore, we demonstrate that regulation of NF phosphorylation dynamics may be essential to avoiding NF accumulations. PMID:22570767

Dale, Jeffrey M.; Garcia, Michael L.



Herpes Simplex Virus 1 Protein Kinase Us3 Phosphorylates Viral dUTPase and Regulates Its Catalytic Activity in Infected Cells  

PubMed Central

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). In this study, a large-scale phosphoproteomic analysis of titanium dioxide affinity chromatography-enriched phosphopeptides from HSV-1-infected cells using high-accuracy mass spectrometry (MS) and subsequent analyses showed that Us3 phosphorylated HSV-1-encoded dUTPase (vdUTPase) at serine 187 (Ser-187) in HSV-1-infected cells. Thus, the following observations were made. (i) In in vitro kinase assays, Ser-187 in the vdUTPase domain was specifically phosphorylated by Us3. (ii) Phosphorylation of vdUTPase Ser-187 in HSV-1-infected cells was detected by phosphate-affinity polyacrylamide gel electrophoresis analyses and was dependent on the kinase activity of Us3. (iii) Replacement of Ser-187 with alanine (S187A) in vdUTPase and an amino acid substitution in Us3 that inactivated its kinase activity significantly downregulated the enzymatic activity of vdUTPase in HSV-1-infected cells, whereas a phosphomimetic substitution at vdUTPase Ser-187 restored the wild-type enzymatic activity of vdUTPase. (iv) The vdUTPase S187A mutation as well as the kinase-dead mutation in Us3 significantly reduced HSV-1 replication in human neuroblastoma SK-N-SH cells at a multiplicity of infection (MOI) of 5 but not at an MOI of 0.01, whereas the phosphomimetic substitution at vdUTPase Ser-187 restored the wild-type viral replication at an MOI of 5. In contrast, these mutations had no effect on HSV-1 replication in Vero and HEp-2 cells. Collectively, our results suggested that Us3 phosphorylation of vdUTPase Ser-187 promoted HSV-1 replication in a manner dependent on cell types and MOIs by regulating optimal enzymatic activity of vdUTPase. PMID:24173231

Kato, Akihisa; Tsuda, Shumpei; Liu, Zhuoming; Kozuka-Hata, Hiroko; Oyama, Masaaki



Involvement of Cx43 phosphorylation in 5'-N-ethylcarboxamide-induced migration and proliferation of mouse embryonic stem cells.  


Despite a lot of gap junction research, the complex connection between gap junction and cell proliferation remains an exciting area of investigation. Thus, we examined the effect of connexin 43 (Cx43) on the migration and proliferation of embryonic stem (ES) cells and its related signaling pathways following stimulation with the adenosine analogue 5'-N-ethylcarboxamide (NECA). NECA increased phosphorylation of Cx43 which was blocked by caffeine, a non-selective adenosine receptor antagonist. In experiment to measure the gap junctional intercellular communication, NECA blocked transfer of Lucifer yellow to neighboring cells in a scrape loading/dye transfer (SL/DT) assay. In addition, NECA-induced phosphorylation of phosphoinositide 3-kinase (PI3K)/Akt, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and nuclear factor-kappa B (NF-kappaB) signal pathways. Inhibition of these signaling pathways reduced NECA-induced phosphorylation of Cx43. Moreover, NECA-treated cells demonstrated phosphorylation of Src, which was blocked by caffeine. In this experiment, a disruption of Cx43 using Cx43-specific small interfering RNA (siRNA) also enhanced Src phosphorylation. In a further study, phosphorylations of integrin beta1, focal adhesion kinase (FAK), and paxillin by NECA were restrained by caffeine as well as the Src blocker, PP2. Finally, we identified that NECA-stimulated cell migration and expressions of cell-cycle regulatory proteins [cyclin D1, cyclin-dependent kinase (CDK) 4, cyclin E, and CDK2]; these increases were inhibited by caffeine, or PP2. We conclude that NECA-stimulated Cx43 phosphorylation mediated by PI3K/Akt, PKC, MAPKs, and NF-kappaB, which subsequently stimulated cell migration and proliferation through Src, integrin beta1, FAK, and paxillin signal pathways. PMID:20232318

Kim, Mi Ok; Lee, Yu Jin; Han, Ho Jae



In vitro rescue study of a malignant familial hypertrophic cardiomyopathy phenotype by pseudo-phosphorylation of myosin regulatory light chain.  


Pseudo-phosphorylation of cardiac myosin regulatory light chain (RLC) has never been examined as a rescue method to alleviate a cardiomyopathy phenotype brought about by a disease causing mutation in the myosin RLC. This study focuses on the aspartic acid to valine substitution (D166V) in the myosin RLC shown to be associated with a malignant phenotype of familial hypertrophic cardiomyopathy (FHC). The mutation has also been demonstrated to cause severe functional abnormalities in transgenic mice expressing D166V in the heart. To explore this novel rescue strategy, pseudo-phosphorylation of D166V was used to determine whether the D166V-induced detrimental phenotype could be brought back to the level of wild-type (WT) RLC. The S15D substitution at the phosphorylation site of RLC was inserted into the recombinant WT and D166V mutant to mimic constitutively phosphorylated RLC proteins. Non-phosphorylatable (S15A) constructs were used as controls. A multi-faceted approach was taken to determine the effect of pseudo-phosphorylation on the ability of myosin to generate force and motion. Using mutant reconstituted porcine cardiac muscle preparations, we showed an S15D-induced rescue of both the enzymatic and binding properties of D166V-myosin to actin. A significant increase in force production capacity was noted in the in vitro motility assays for S15D-D166V vs. D166V reconstituted myosin. A similar pseudo-phosphorylation induced effect was observed on the D166V-elicited abnormal Ca(2+) sensitivity of force in porcine papillary muscle strips reconstituted with phosphomimic recombinant RLCs. Results from this study demonstrate a novel in vitro rescue strategy that could be utilized in vivo to ameliorate a malignant cardiomyopathic phenotype. We show for the first time that pseudo-RLC phosphorylation can reverse the majority of the mutation-induced phenotypes highlighting the importance of RLC phosphorylation in combating cardiac disease. PMID:24374283

Muthu, Priya; Liang, Jingsheng; Schmidt, William; Moore, Jeffrey R; Szczesna-Cordary, Danuta



Comparative performance of enzymatic and combined alkaline-enzymatic pretreatments on methane production from ensiled sorghum forage.  


This study investigated the effect of enzymatic and combined alkaline-enzymatic pretreatments on chemical composition and methane production from ensiled sorghum forage. Four commercial enzymatic preparations were tested and the two yielding the highest sugars release were added to evaluate any hydrolytic effect on both untreated and alkaline pretreated samples. In the combined alkaline-enzymatic pretreatment trials, the highest sugar release was found with Primafast and BGL preparations (added at a final concentration 0.12 and 0.20 mL/g TS, respectively), with a total monomeric content of 12 and 6.5 g/L. Fibre composition analysis confirmed that the combined alkaline-enzymatic pretreatment led to cellulose (up to 32 %) and hemicelluloses (up to 56 %) solubilisation, compared to the enzymatic pretreatment alone. BMP tests were performed on both untreated and pretreated samples, and time courses of methane production were fitted. Both enzymatic and combined alkaline-enzymatic pretreatment led to a methane production increase (304 and 362 mL CH4/g VS), compared to that of untreated sorghum (265 mL CH4/g VS), as  +15 and  +37 %, respectively. Moreover, higher specific methane production rates, compared to that of untreated sorghum (20.31 mL CH4/g VS/d), were obtained by applying the enzymatic and combined alkaline-enzymatic pretreatment (33.94 and 31.65 mL CH4/g VS/d), respectively. PMID:24962776

Rollini, Manuela; Sambusiti, Cecilia; Musatti, Alida; Ficara, Elena; Retinò, Isabella; Malpei, Francesca



Continuous enzymatic liquefaction of starch for saccharification  

SciTech Connect

A process was explored for continuous enzymatic liquefaction of corn starch at high concentration and subsequent saccharification to glucose. The process appears to be quite efficient for conversion of starch to glucose and enzymatic liquefaction and should be readily adaptable to industrial fermentation processes. Preliminary work indicated that milled corn or other cereal grains also can be suitably converted by such a process. Essentially, the process involved incorporation of a thermostable, bacterial alpha-amylase for liquefaction and, subsequently, of a glucoamylase into the continuous mixer under conditions conductive to rapid enzymatic hydrolyses. Also studied was the effect on substrate liquefaction of variables such as starch concentration (40-70%), level of alpha-amylase (0.14-0.4%, dry starch basis), temperature (70-100 degrees C), pH (5.8-7.1), and residence time (6 and 12 minutes). The degree of liquefaction was assessed by determining 1) the Brookfield viscosity, 2) the amount of reducing groups, and 3) the rate and extent of glucose formed after glucoamylase treatment. Best liquefaction processing conditions were achieved by using 50-60% starch concentration, at 95 degrees C, with 0.4% alpha-amylase, and a 6 minute residence period in the mixer. Under these conditions, rates and extents of glucose obtained after glucoamylase treatment approached those obtained in longer laboratory batch liquefactions. The amount of glucose formed in 24 hours with the use of 0.4% glucoamylase was 86% of theory after a 6-min continuous liquefaction, compared to 90% for a 30-min laboratory batch liquefaction (95 degrees C, 0.4% alpha-amylase). (Refs. 15).

Carr, M.E.; Black, L.T.; Bagby, M.O.



Solid polymer electrolyte from phosphorylated chitosan  

NASA Astrophysics Data System (ADS)

Recently, the need of secondary battery application continues to increase. The secondary battery which using a liquid electrolyte was indicated had some weakness. A solid polymer electrolyte is an alternative electrolytes membrane which developed in order to replace the liquid electrolyte type. In the present study, the effect of phosphorylation on to polymer electrolyte membrane which synthesized from chitosan and lithium perchlorate salts was investigated. The effect of the component's composition respectively on the properties of polymer electrolyte, was carried out by analyzed of it's characterization such as functional groups, ion conductivity, and thermal properties. The mechanical properties i.e tensile resistance and the morphology structure of membrane surface were determined. The phosphorylation processing of polymer electrolyte membrane of chitosan and lithium perchlorate was conducted by immersing with phosphoric acid for 2 hours, and then irradiated on a microwave for 60 seconds. The degree of deacetylation of chitosan derived from shrimp shells was obtained around 75.4%. Relative molecular mass of chitosan was obtained by viscometry method is 796,792 g/mol. The ionic conductivity of chitosan membrane was increase from 6.33 × 10-6 S/cm up to 6.01 × 10-4 S/cm after adding by 15 % solution of lithium perchlorate. After phosphorylation, the ionic conductivity of phosphorylated lithium chitosan membrane was observed 1.37 × 10-3 S/cm, while the tensile resistance of 40.2 MPa with a better thermal resistance. On the strength of electrolyte membrane properties, this polymer electrolyte membrane was suggested had one potential used for polymer electrolyte in field of lithium battery applications.

Fauzi, Iqbal; Arcana, I. Made



Enzymatic Catalytic Beds For Oxidation Of Alcohols  

NASA Technical Reports Server (NTRS)

Modules containing beds of enzymatic material catalyzing oxidation of primary alcohols and some other organic compounds developed for use in wastewater-treatment systems of future spacecraft. Designed to be placed downstream of multifiltration modules, which contain filters and sorbent beds removing most of non-alcoholic contaminants but fail to remove significant amounts of low-molecular-weight, polar, nonionic compounds like alcohols. Catalytic modules also used on Earth to oxidize primary alcohols and other compounds in wastewater streams and industrial process streams.

Jolly, Clifford D.; Schussel, Leonard J.



Phosphorylation of Ser 402 impedes phosphatase activity of slingshot 1  

PubMed Central

By using mass spectrometry, we have identified Ser 402 as a new phosphorylation site within the catalytic domain of human slingshot 1 (SSH1). Phosphorylation at this site inhibits substrate binding and, thus, phosphatase activity in vitro, resulting in enrichment of phosphorylated cofilin in monolayer cell culture. We further demonstrate that protein kinase D (PKD) is upstream from Ser 402 phosphorylation. Accordingly, expression of active PKD in Drosophila phenotypically mimics the loss of SSH activity by inducing accumulation of phosphorylated cofilin and filamentous actin. We thus identify a universal mechanism by which PKD controls SSH1 phosphatase activity. PMID:21525957

Barisic, Sandra; Nagel, Anja C; Franz-Wachtel, Mirita; Macek, Boris; Preiss, Anette; Link, Gisela; Maier, Dieter; Hausser, Angelika



Phosphorylated tyrosine in the flagellum filament protein of Pseudomonas aeruginosa.  

PubMed Central

Purified flagella from two strains of 32P-labeled Pseudomonas aeruginosa were shown to be phosphorylated. This was confirmed by autoradiography of flagellin protein in polyacrylamide gels. Thin-layer electrophoresis and autoradiography of flagellin partial hydrolysates indicated that phosphotyrosine was the major phosphorylated amino acid. High-pressure liquid chromatographic analysis confirmed the presence of phosphotyrosine in flagellum filament protein. Preliminary data indicated that less than one tyrosine per subunit was phosphorylated. No evidence was found for phosphorylation of serine or threonine. A function related to tyrosine phosphorylation has not been determined. Images PMID:1697577

Kelly-Wintenberg, K; Anderson, T; Montie, T C



Thylakoid protein phosphorylation in evolutionally divergent species with oxygenic photosynthesis.  


Phosphothreonine antibody was used to explore reversible thylakoid protein phosphorylation in vivo in evolutionally divergent organisms with oxygenic photosynthesis. Three distinct groups of organisms were found. Cyanobacteria and red algae, both with phycobilisome antenna system, did not show phosphorylation of any of the photosystem II (PSII) proteins and belong to group 1. Group 2 species, consisting of a moss, a liverwort and a fern, phosphorylated both the light-harvesting chlorophyll alb proteins (LHCII) and the PSII core proteins D2 and CP43, but not the D1 protein. Reversible phosphorylation of the D1 protein seems to be the latest event in the evolution of PSII protein phosphorylation and was found only in seed plants, in group 3 species. Light-intensity-dependent regulation of LHCII protein phosphorylation was similar in group 2 and 3 species, with maximal phosphorylation of LHCII at low light and nearly complete dephosphorylation at high light. PMID:9512353

Pursiheimo, S; Rintamäki, E; Baena-Gonzalez, E; Aro, E M



Multistep phosphorylation systems: tunable components of biological signaling circuits.  


Multisite phosphorylation of proteins is a powerful signal processing mechanism that plays crucial roles in cell division and differentiation as well as in disease. We recently demonstrated a novel phenomenon in cell cycle regulation by showing that cyclin-dependent kinase-dependent multisite phosphorylation of a crucial substrate is performed sequentially in the N-to-C terminal direction along the disordered protein. The process is controlled by key parameters, including the distance between phosphorylation sites, the distribution of serines and threonines in sites, and the position of docking motifs. According to our model, linear patterns of phosphorylation along disordered protein segments determine the signal-response function of a multisite phosphorylation switch. Here we discuss the general advantages and engineering principles of multisite phosphorylation networks as processors of kinase signals. We also address the idea of using the mechanistic logic of linear multisite phosphorylation networks to design circuits for synthetic biology applications. PMID:25368420

Valk, Evin; Venta, Rainis; Ord, Mihkel; Faustova, Ilona; Kõivomägi, Mardo; Loog, Mart



Enzymatic and bacterial conversions during sourdough fermentation.  


Enzymatic and microbial conversion of flour components during bread making determines bread quality. Metabolism of sourdough microbiota and the activity of cereal enzymes are interdependent. Acidification, oxygen consumption, and thiols accumulation by microbial metabolism modulate the activity of cereal enzymes. In turn, cereal enzymes provide substrates for bacterial growth. This review highlights the role of cereal enzymes and the metabolism of lactic acid bacteria in conversion of carbohydrates, proteins, phenolic compounds and lipids. Heterofermentative lactic acid bacteria prevailing in wheat and rye sourdoughs preferentially metabolise sucrose and maltose; the latter is released by cereal enzymes during fermentation. Sucrose supports formation of acetate by heterofermentative lactobacilli, and the formation of exopolysaccharides. The release of maltose and glucose by cereal enzymes during fermentation determines the exopolysaccharide yield in sourdough fermentations. Proteolysis is dependent on cereal proteases. Peptidase activities of sourdough lactic acid bacteria determine the accumulation of (bioactive) peptides, amino acids, and amino acid metabolites in dough and bread. Enzymatic conversion and microbial metabolism of phenolic compounds is relevant in sorghum and millet containing high levels of phenolic compounds. The presence of phenolic compounds with antimicrobial activity in sorghum selects for fermentation microbiota that are resistant to the phenolic compounds. PMID:24230468

Gänzle, Michael G



Enzymatic deconstruction of xylan for biofuel production  

PubMed Central

The combustion of fossil-derived fuels has a significant impact on atmospheric carbon dioxide (CO2) levels and correspondingly is an important contributor to anthropogenic global climate change. Plants have evolved photosynthetic mechanisms in which solar energy is used to fix CO2 into carbohydrates. Thus, combustion of biofuels, derived from plant biomass, can be considered a potentially carbon neutral process. One of the major limitations for efficient conversion of plant biomass to biofuels is the recalcitrant nature of the plant cell wall, which is composed mostly of lignocellulosic materials (lignin, cellulose, and hemicellulose). The heteropolymer xylan represents the most abundant hemicellulosic polysaccharide and is composed primarily of xylose, arabinose, and glucuronic acid. Microbes have evolved a plethora of enzymatic strategies for hydrolyzing xylan into its constituent sugars for subsequent fermentation to biofuels. Therefore, microorganisms are considered an important source of biocatalysts in the emerging biofuel industry. To produce an optimized enzymatic cocktail for xylan deconstruction, it will be valuable to gain insight at the molecular level of the chemical linkages and the mechanisms by which these enzymes recognize their substrates and catalyze their reactions. Recent advances in genomics, proteomics, and structural biology have revolutionized our understanding of the microbial xylanolytic enzymes. This review focuses on current understanding of the molecular basis for substrate specificity and catalysis by enzymes involved in xylan deconstruction. PMID:20431716




Probing enzymatic activity inside single cells.  


We report a novel approach for determining the enzymatic activity within a single suspended cell. Using a steady-state microfluidic delivery device and timed exposure to the pore-forming agent digitonin, we controlled the plasma membrane permeation of individual NG108-15 cells. Mildly permeabilized cells (~100 pores) were exposed to a series of concentrations of fluorescein diphosphate (FDP), a fluorogenic alkaline phosphatase substrate, with and without levamisole, an alkaline phosphatase inhibitor. We generated quantitative estimates for intracellular enzyme activity and were able to construct both dose-response and dose-inhibition curves at the single-cell level, resulting in an apparent Michaelis contant Km of 15.3 ?M ± 1.02 (mean ± standard error of the mean (SEM), n = 16) and an inhibition constant Ki of 0.59 mM ± 0.07 (mean ± SEM, n = 14). Enzymatic activity could be monitored just 40 s after permeabilization, and five point dose-inhibition curves could be obtained within 150 s. This rapid approach offers a new methodology for characterizing enzyme activity within single cells. PMID:24003961

Olofsson, Jessica; Xu, Shijun; Jeffries, Gavin D M; Jesorka, Aldo; Bridle, Helen; Isaksson, Ida; Weber, Stephen G; Orwar, Owe



Enzymatic processing of municipal solid waste.  


The focus of this work was to investigate an enzymatic liquefaction of MSW organics, paper and cardboard. Liquefaction trials were conducted in different trial volumes: 50 g lab-scale trials and 5 0kg vessel-tests and evaluated based on particle size and viscosity. The viscosity results showed that Celluclast 1.5L had the singular significant effect on liquefaction of model MSW. No effect of ?-amylase, protease and interaction in between and with cellulases on viscosity and particle size distribution was found in this study. Degradable material with a particle size above 1mm after treatment was evaluated using SEM microscopy. These results showed that paper particles were the main obstacles needing additional treatment in order to become fully liquefied. In a pilot scale test treating authentic MSW; more than 90% of initial organic and paper dry matter (DM) was recovered as liquid slurry after sieving through a 5-mm sieve. These tests were performed at up to 35% DM, showing that this process can easily manage high DM loadings. MSW enzymatic liquefaction promotes the separation of organics and paper from solids, which facilitate the use of these degradable fractions, with minimal loss, capable to enter a biogas plant through existing pipes. PMID:20727726

Jensen, Jacob Wagner; Felby, Claus; Jørgensen, Henning; Rønsch, Georg Ørnskov; Nørholm, Nanna Dreyer



Palm Date Fibers: Analysis and Enzymatic Hydrolysis  

PubMed Central

Waste palm dates were subjected to analysis for composition and enzymatic hydrolysis of their flesh fibers. The fruit contained 32% glucose and 30% fructose, while the water-insoluble fibers of its flesh consisted of 49.9% lignin and 20.9% polysaccharides. Water-insoluble fibers were settled to 55% of its initial volume in 12 h. The presence of skin and flesh colloidal fibers results in high viscosity and clogging problems during industrial processes. The settling velocity of the fibers was improved by enzymatic hydrolysis. Hydrolysis resulted in 84.3% conversion of the cellulosic part of the fibers as well as reducing the settling time to 10 minutes and the final settled volume to 4% of the initial volume. It implies easier separation of the fibers and facilitates fermentation processes in the corresponding industries. Two kinds of high- and low-lignin fibers were identified from the water-insoluble fibers. The high-lignin fibers (75% lignin) settled easily, while the low-lignin fibers (41.4% lignin) formed a slurry suspension which settled very slowly. The hydrophilicity of these low-lignin fibers is the major challenge of the industrial processes. PMID:21151438

Shafiei, Marzieh; Karimi, Keikhosro; Taherzadeh, Mohammad J.



Steam pretreatment of lignocellulosic material for enhanced enzymatic hydrolysis  

SciTech Connect

Pretreatment methods were compared with steam explosion, and differing views on the relative importance of mechanical and chemical effects were outlined. Hydrolysis was desirable; pyrolysis was undesirable. The effects of initial moisture content on steam consumption, mechanism and rate of heat transfer, pentosan solubilization, and subsequent glucose yield were summarized. The insignificant effect, after treatment at 240 degrees C, of 90% pressure bleed-down before explosion on subsequent simultaneous saccharification and fermentation (SSF) yields was described. Treatment at 190 degrees C with complete bleed-down (no explosion), when compared with that at 240 degrees C with explosion from full pressure, showed at least as good solubilization of pentosan, enzymatic hydrolysis, and SSF but showed greater pentosan destruction for the same degree of pentosan removal. Water washing of unexploded steamed aspenwood chips was at least as efficient as that of similarly treated but exploded chips. Scanning electron micrographs of unexploded chips showed extensive rupturing of vessel pit membranes and other morphological features associated with steam-exploded wood. Neither the explosion nor the high temperatures (above 190 degrees C) are necessary. 28 references.

Brownell, H.H.; Saddler, J.N.



Tandem phosphorylation of Ser-911 and Thr-912 at the C terminus of yeast plasma membrane H+-ATPase leads to glucose-dependent activation.  


In recent years there has been growing interest in the post-translational regulation of P-type ATPases by protein kinase-mediated phosphorylation. Pma1 H(+)-ATPase, which is responsible for H(+)-dependent nutrient uptake in yeast (Saccharomyces cerevisiae), is one such example, displaying a rapid 5-10-fold increase in activity when carbon-starved cells are exposed to glucose. Activation has been linked to Ser/Thr phosphorylation in the C-terminal tail of the ATPase, but the specific phosphorylation sites have not previously been mapped. The present study has used nanoflow high pressure liquid chromatography coupled with electrospray electron transfer dissociation tandem mass spectrometry to identify Ser-911 and Thr-912 as two major phosphorylation sites that are clearly related to glucose activation. In carbon-starved cells with low Pma1 activity, peptide 896-918, which was derived from the C terminus upon Lys-C proteolysis, was found to be singly phosphorylated at Thr-912, whereas in glucose-metabolizing cells with high ATPase activity, the same peptide was doubly phosphorylated at Ser-911 and Thr-912. Reciprocal (14)N/(15)N metabolic labeling of cells was used to measure the relative phosphorylation levels at the two sites. The addition of glucose to carbon-starved cells led to a 3-fold reduction in the singly phosphorylated form and an 11-fold increase in the doubly phosphorylated form. These results point to a mechanism in which the stepwise phosphorylation of two tandemly positioned residues near the C terminus mediates glucose-dependent activation of the H(+)-ATPase. PMID:17932035

Lecchi, Silvia; Nelson, Clark J; Allen, Kenneth E; Swaney, Danielle L; Thompson, Katie L; Coon, Joshua J; Sussman, Michael R; Slayman, Carolyn W



Effects of phospholipid fatty acyl chain length on phosphorylation and dephosphorylation of the Ca(2+)-ATPase.  

PubMed Central

The kinetics of the Ca(2+)-ATPase purified from sarcoplasmic reticulum have been studied after reconstitution into bilayers of dimyristoleoylphosphatidylcholine [di(C14:1)PC], dioleoylphosphatidylcholine[di(C18:1)PC] and dinervonylphosphatidylcholine [di(C24:1)PC]. In di(C24:1)PC the rate of phosphorylation of the ATPase by ATP was comparable with that in di(C18:1)PC (about 70 s-1), but in di(C14:1)PC the rate was much lower (21 s-1). Fluorescence responses of the ATPase suggest changes in the phosphoryl-transfer step rather than in the preceding conformational change E1Ca2ATP<-->E1'Ca2ATP. The rate of dephosphorylation of the phosphorylated ATPase was found to decrease in the order di(C24:1)PC < di(C14:1)PC < di(C18:1)PC. For the ATPase in di(C24:1)PC the rate of dephosphorylation (3.3 s-1) was slow enough to be the rate-limiting step for ATP hydrolysis; in di(C14:1)PC, it is suggested that both phosphorylation and dephosphorylation contribute to rate limitation. Phosphorylation of the ATPase in di(C24:1)PC by Pi was normal, but no phosphoenzyme could be detected in di(C14:1)PC. The rate of the Ca(2+)-transport step was normal in di(C24:1)PC, suggesting that the single Ca2+ ion bound to the ATPase in di(C24:1)PC could be transported. PMID:7575421

Starling, A P; East, J M; Lee, A G



Mass spectrometric characterization of phosphorylated peptides using MALDI in-source decay via redox reactions.  


Matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) has been used for characterization of a phosphorylated peptides and proteins because labile phosphate group is not lost during the MALDI-ISD process. The conventional MALDI-ISD is initiated by the hydrogen transfer from reducing matrix molecules to peptide backbone, leading to c'- and z'-series ions. In contrast, when an oxidizing chemical 5-nitrosalicylic acid (5-NSA) is served as the MALDI-ISD matrix, a- and x-series ions are specifically generated by hydrogen abstraction from peptide backbone to matrix molecule. The 5-NSA provides useful complementary information to the conventional MALDI-ISD for the analysis of amino acid sequencing and site localization of phosphorylation in peptides. The MALDI-ISD with reducing and oxidizing matrix could be a useful method for the de novo peptide sequencing. PMID:22359327

Asakawa, Daiki; Takayama, Mitsuo



Phosphorylation of Tip60 Tyrosine 327 by Abl Kinase Inhibits HAT Activity through Association with FE65.  


The transfer of acetyl groups from acetyl coenzyme A to the ? amino group of internal lysine residues is catalyzed by Tip60, which is in the MYST family of nuclear histone acetyltransferases (HATs). The tyrosine phosphorylation of Tip60 seems to be a unique modification. We present evidence that Tip60 is modified on tyrosine 327 by Abl kinase. We show that this causes functional changes in HAT activity and the subcellular localization of TIP60, which forms a complex with Abl kinase. The Tip60 mutation Y327F abolished tyrosine phosphorylation, reduced the inhibition of Tip60 HAT activity, and caused G0-G1 arrest and association with FE65. Thus, our findings for the first time suggested a novel regulation mechanism of Tip60. Regulation was through phosphorylation of tyrosine 327 by Abl tyrosine kinase and depended on environmental conditions, suggesting that the tyrosine residue of Tip60 is important for the activation process. PMID:24044023

Shin, Sung Hwa; Kang, Sang Sun



In Plants, 3-O-Methylglucose Is Phosphorylated by Hexokinase But Not Perceived as a Sugar1  

PubMed Central

In plants, sugars are the main respiratory substrates and important signaling molecules in the regulation of carbon metabolism. Sugar signaling studies suggested that sugar sensing involves several key components, among them hexokinase (HXK). Although the sensing mechanism of HXK is unknown, several experiments support the hypothesis that hexose phosphorylation is a determining factor. Glucose (Glc) analogs transported into cells but not phosphorylated are frequently used to test this hypothesis, among them 3-O-methyl-Glc (3-OMG). The aim of the present work was to investigate the effects and fate of 3-OMG in heterotrophic plant cells. Measurements of respiration rates, protein and metabolite contents, and protease activities and amounts showed that 3-OMG is not a respiratory substrate and does not contribute to biosynthesis. Proteolysis and lipolysis are induced in 3-OMG-fed maize (Zea mays L. cv DEA) roots in the same way as in sugar-starved organs. However, contrary to the generally accepted idea, phosphorous and carbon nuclear magnetic resonance experiments and enzymatic assays prove that 3-OMG is phosphorylated to 3-OMG-6-phosphate, which accumulates in the cells. Insofar as plant HXK is involved in sugar sensing, these findings are discussed on the basis of the kinetic properties because the catalytic efficiency of HXK isolated from maize root tips is five orders of magnitude lower for 3-OMG than for Glc and Man. PMID:12586906

Cortes, Sandra; Gromova, Marina; Evrard, Adeline; Roby, Claude; Heyraud, Alain; Rolin, Dominique B.; Raymond, Philippe; Brouquisse, Renaud M.



Fractionation and Molecular Characteristics of Cellulose During Enzymatic Hydrolysis  

Microsoft Academic Search

The effect of enzymatic hydrolysis (EH) on the molecular characteristics as well as fractional composition of cellulose was studied using the direct size exclusion chromatography (SEC) analysis in sodium hydroxide. Bleached hardwood pulp was subjected to the action of the cellulase complex Celluclast™ supplemented with Novozyme 188™. The residues after the enzymatic treatment were fractionated by dissolution in 10% NaOH

T. Eremeeva; T. Bikova; M. Eisimonte; U. Viesturs; A. Treimanis



Enzymatic enantiomeric resolution of phenylethylamines structurally related to amphetamine.  


Both enantiomers of several phenylethylamines, structurally related to amphetamine, have been prepared in good yields and excellent enantiomeric purity by enzymatic kinetic resolution using CAL-B and ethyl methoxyacetate as the acyl donor. In the case of the 4-hydroxyderivative of amphetamine (compound 4i), the S enantiomer racemized possibly in a dynamic kinetic resolution (DKR) under the enzymatic conditions used. PMID:21971988

Muñoz, Lourdes; Rodriguez, Anna M; Rosell, Gloria; Bosch, M Pilar; Guerrero, Angel



Structure-Function Relationships in Enzymatically Modified Articular Cartilage  

Microsoft Academic Search

The present study is aimed at revealing structure-function relationships of bovine patellar articular cartilage. Collagenase, chondroitinase ABC and elastase were used for controlled and selective enzymatic modifications of cartilage structure, composition and functional properties. The effects of the enzymatic degradations were quantitatively evaluated using quantitative polarized light microscopy, digital densitometry of safranin O-stained sections as well as with biochemical and

Jarno Rieppo; Juha Töyräs; Miika T. Nieminen; Vuokko Kovanen; Mika M. Hyttinen; Rami K. Korhonen; Jukka S. Jurvelin; Heikki J. Helminen



Enzymatic hydrolysis of rawhide using papain and neutrase  

Microsoft Academic Search

Rawhide split was hydrolysed separately by two proteolytic enzymes, papain and neutrase. The effects of enzymatic conditions of the hydrolysis reaction were investigated. During the first 10min of the enzymatic hydrolysis, the yield of the hydrolysed protein increased sharply, then it slowly increased or became essentially constant due to the limited availability of the substrate. The optimum hydrolysis conditions of

Siriporn Damrongsakkul; Kongpob Ratanathammapan; Kittinan Komolpis; Wiwut Tanthapanichakoon



Enzymatically cross-linked hydrogels and their adhesive  

E-print Network

), and as surgical sealants and adhesives (4­6). An emerging approach to formation of hydrogels relies on enzymaticEnzymatically cross-linked hydrogels and their adhesive strength to biosurfaces B-H Hu PB-linked hydrogels were compared with commercial fibrin tissue adhesive. Outcome Measure ­ The shear strength between


Coulomb Interactions between Cytoplasmic Electric Fields and Phosphorylated Messenger Proteins Optimize Information Flow in Cells  

PubMed Central

Background Normal cell function requires timely and accurate transmission of information from receptors on the cell membrane (CM) to the nucleus. Movement of messenger proteins in the cytoplasm is thought to be dependent on random walk. However, Brownian motion will disperse messenger proteins throughout the cytosol resulting in slow and highly variable transit times. We propose that a critical component of information transfer is an intracellular electric field generated by distribution of charge on the nuclear membrane (NM). While the latter has been demonstrated experimentally for decades, the role of the consequent electric field has been assumed to be minimal due to a Debye length of about 1 nanometer that results from screening by intracellular Cl? and K+. We propose inclusion of these inorganic ions in the Debye-Huckel equation is incorrect because nuclear pores allow transit through the membrane at a rate far faster than the time to thermodynamic equilibrium. In our model, only the charged, mobile messenger proteins contribute to the Debye length. Findings Using this revised model and published data, we estimate the NM possesses a Debye-Huckel length of a few microns and find this is consistent with recent measurement using intracellular nano-voltmeters. We demonstrate the field will accelerate isolated messenger proteins toward the nucleus through Coulomb interactions with negative charges added by phosphorylation. We calculate transit times as short as 0.01 sec. When large numbers of phosphorylated messenger proteins are generated by increasing concentrations of extracellular ligands, we demonstrate they generate a self-screening environment that regionally attenuates the cytoplasmic field, slowing movement but permitting greater cross talk among pathways. Preliminary experimental results with phosphorylated RAF are consistent with model predictions. Conclusion This work demonstrates that previously unrecognized Coulomb interactions between phosphorylated messenger proteins and intracellular electric fields will optimize information transfer from the CM to the NM in cells. PMID:20711447

Gatenby, Robert A.; Frieden, B. Roy



Membrane-less and mediator-free enzymatic biofuel cell using carbon nanotube\\/porous silicon electrodes  

Microsoft Academic Search

Membrane-less and mediator-free direct electron transfer enzymatic biofuel cells (BFCs) with bioelectrodes comprised of single wall carbon nanotubes (SWNTs) deposited by two methods on porous silicon (pSi) substrates, are reported. In one method the SWNTs were grown by chemical vapor deposition (CVD) and then functionalized with carboxylic groups, and in the second method, pre-synthesized carboxylated SWNTs (c-SWNTs) were electrophoretically deposited

Shiunchin C. Wang; Fan Yang; Manuel Silva; Anna Zarow; Yubing Wang; Zafar Iqbal



Heavy atom isotope effects on enzymatic reactions  

NASA Astrophysics Data System (ADS)

The theory of isotope effects, which has proved to be extremely useful in providing geometrical details of transition states in a variety of chemical reactions, has recently found an application in studies of enzyme-catalyzed reactions. These reactions are multistep in nature with few steps being partially rate-limiting, thus interpretation of these isotope effects is more complex. The theoretical framework of heavy-atom isotope effects on enzymatic reactions is critically analyzed on the basis of recent results of: carbon kinetic isotope effects on carbonic anhydrase and catalytic antibodies; multiple carbon, deuterium isotope effects on reactions catalyzed by formate decarboxylase; oxygen isotope effects on binding processes in reactions catalyzed by pyruvate kinase; and equilibrium oxygen isotope effect on binding an inhibitor to lactate dehydrogenase. The advantages and disadvantages of reaction complexity in learning details of formal and molecular mechanisms are discussed in the examples of reactions catalyzed by phosphoenolpyruvate carboxylase, orotidine decarboxylase and glutamine synthetase.

Paneth, Piotr



Proteinuria: an enzymatic disease of the podocyte?  

PubMed Central

Proteinuria is a major health-care problem that affects several hundred million people worldwide. Proteinuria is a cardinal sign and a prognostic marker of kidney disease, and also an independent risk factor for cardiovascular morbidity and mortality. Microalbuminuria is the earliest cue of renal complications of diabetes, obesity, and the metabolic syndrome. It can often progress to overt proteinuria that in 10–50% of patients is associated with the development of chronic kidney disease, ultimately requiring dialysis or transplantation. Therefore, reduction or prevention of proteinuria is highly desirable. Here we review recent novel insights into the pathogenesis and treatment of proteinuria, with a special emphasis on the emerging concept that proteinuria can result from enzymatic cleavage of essential regulators of podocyte actin dynamics by cytosolic cathepsin L (CatL), resulting in a motile podocyte phenotype. Finally, we describe signaling pathways controlling the podocyte actin cytoskeleton and motility and how these pathways can be manipulated for therapeutic benefit. PMID:19924101

Mundel, Peter; Reiser, Jochen



Surface-initiated enzymatic polymerization of DNA.  


We describe a technique to synthesize DNA homopolymers on a surface using surface-initiated enzymatic polymerization (SIEP) with terminal deoxynucleotidyl transferase (TdTase), an enzyme that repetitively adds mononucleotides to the 3'-end of oligonucleotides. The thickness of the synthesized DNA layer was found to depend on the deoxymononucleotide monomer, in the order of dATP > dTTP > dGTP approximately dCTP. In addition, the composition and the surface density of oligonucleotide initiators were also important in controlling the extent of DNA polymerization. The extension of single-stranded DNA chains by SIEP was further verified by their binding to antibodies specific to oligonucleotides. TdTase-mediated SIEP can also be used to grow spatially defined three-dimensional DNA structures by soft lithography and is a new tool for bioinspired fabrication at the micro- and nanoscale. PMID:17929953

Chow, Dominic C; Chilkoti, Ashutosh



Analog Noise Reduction in Enzymatic Logic Gates  

E-print Network

In this work we demonstrate both experimentally and theoretically that the analog noise generation by a single enzymatic logic gate can be dramatically reduced to yield gate operation with virtually no input noise amplification. This is achieved by exploiting the enzyme's specificity when using a co-substrate that has a much lower affinity than the primary substrate. Under these conditions, we obtain a negligible increase in the noise output from the logic gate as compared to the input noise level. Experimental realizations of the AND logic gate with the enzyme horseradish peroxidase using hydrogen peroxide and two different co-substrates, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and ferrocyanide, with vastly different rate constants confirmed our general theoretical conclusions.

Dmitriy Melnikov; Guinevere Strack; Marcos Pita; Vladimir Privman; Evgeny Katz



Recent development of miniatured enzymatic biofuel cell  

NASA Astrophysics Data System (ADS)

Enzymatic biofuel cells (EBFCs) that oxidize biological fuels using enzyme-modified electrodes are considered a promising candidate for implantable power sources. However, there are still challenges to overcome before biofuel cells become competitive in any practical applications. Currently, the short lifespan of the catalytic enzymes and poor power density are the most critical issues in developing EBFCs. In this paper, we will review the recent development of biofuel cells and highlight the progress in Carbon-microelectromechanical system (C-MEMS) based micro biofuel cells by both computational modeling and experimental work. Also, our effort on utilizing a covalent immobilization technique for the attachment of enzymes onto the substrate which is expected to increase the enzyme loading efficiency and the power density of devices is discussed in this paper.

Song, Yin; Penmatsa, Varun; Wang, Chunlei



Enzymatic removal of toxic phenols and anilines from waste waters  

SciTech Connect

A new enzymatic method has been developed for the removal of phenols and anilines from industrial waste waters. This involves the treatment of aqueous solutions containing the pollutants with horseradish peroxidase and hydrogen peroxide. Such treatment results in precipitation of phenols and aromatic amines from water as a result of their enzymatic crosslinking. This approach was used to remove over 30 different phenols and aromatic amines from water. The effect of reaction conditions on the enzymatic removal of the pollutants was studied. For some pollutants, the efficiency of the enzymatic removal is very high (exceeding 99%), whereas for others it is significantly lower. The presence of easily removed phenols and aromatic amines greatly enhances the enzymatic precipitation of those that have low removal efficiencies.

Klibanov, A.M.; Alberti, B.N.; Morris, E.D.; Felshin, L.M.



?-cyclodextrin assistant flavonoid glycosides enzymatic hydrolysis  

PubMed Central

Background: The content of icaritin and genistein in herba is very low, preparation with relatively large quantities is an important issue for extensive pharmacological studies. Objective: This study focuses on preparing and enzymic hydrolysis of flavonoid glycosides /?-cyclodextrin inclusion complex to increase the hydrolysis rate. Materials and Methods: The physical property of newly prepared inclusion complex was tested by differential scanning calorimetry (DSC). The conditions of enzymatic hydrolysis were optimized for the bioconversion of flavonoid glycosides /?-cyclodextrin inclusion complex by mono-factor experimental design. The experiments are using the icariin and genistein as the model drugs. Results: The solubility of icariin and genistein were increased almost 17 times from 29.2 ?g/ml to 513.5 ?g/ml at 60°C and 28 times from 7.78 ?g/ml to 221.46 ?g/ml at 50°C, respectively, demonstrating that the inclusion complex could significantly increase the solubility of flavonoid glycosides. Under the optimal conditions, the reaction time of icariin and genistin decreased by 68% and 145%, when compared with that without ?-CD inclusion. By using this enzymatic condition, 473 mg icaritin (with the purity of 99.34%) and 567 mg genistein(with the purity of 99.46%), which was finally determined by melt point, ESI-MS, UV, IR, 1H NMR and 13C NMR, was obtained eventually by transforming the inclusion complex(contains 1.0 g substrates). Conclusion: This study can clearly indicate a new attempt to improve the speed of enzyme-hydrolysis of poorly water-soluble flavonoid glycosides and find a more superior condition which is used to prepare icaritin and genistein. PMID:24143039

Jin, Xin; Zhang, Zhen-hai; Sun, E.; Jia, Xiao-Bin



Ilex aquifolium: protection against enzymatic and non-enzymatic lipid peroxidation.  


The ethanolic extract of Ilex aquifolium L. (Aquifoliaceae) concentration-dependently inhibited leukotriene B4 biosynthesis in isolated bovine PMNL with an IC50 value of about 60 micrograms/ml, whereas the effect on epidermal 12(S)-HETE biosynthesis was much less pronounced. The extract also inhibited the non-enzymatic, peroxyl radical-stimulated lipid peroxidation in model membranes and was further a scavenger of the iron-dependent generation of hydroxyl radicals from hydrogen peroxide as determined by protection against deoxyribose degradation. While inhibition of leukotriene biosynthesis was not mediated by its known phenolic constituents such as hyperoside, rutoside, and chlorogenic acid, these compounds were responsible for the inhibitory effects of I. aquifolium against non-enzymatic lipid peroxidation and deoxyribose degradation. PMID:9741300

Müller, K; Ziereis, K; Paper, D H



PhosPhAt: a database of phosphorylation sites in Arabidopsis thaliana and a plant-specific phosphorylation site predictor.  


The PhosPhAt database provides a resource consolidating our current knowledge of mass spectrometry-based identified phosphorylation sites in Arabidopsis and combines it with phosphorylation site prediction specifically trained on experimentally identified Arabidopsis phosphorylation motifs. The database currently contains 1187 unique tryptic peptide sequences encompassing 1053 Arabidopsis proteins. Among the characterized phosphorylation sites, there are over 1000 with unambiguous site assignments, and nearly 500 for which the precise phosphorylation site could not be determined. The database is searchable by protein accession number, physical peptide characteristics, as well as by experimental conditions (tissue sampled, phosphopeptide enrichment method). For each protein, a phosphorylation site overview is presented in tabular form with detailed information on each identified phosphopeptide. We have utilized a set of 802 experimentally validated serine phosphorylation sites to develop a method for prediction of serine phosphorylation (pSer) in Arabidopsis. An analysis of the current annotated Arabidopsis proteome yielded in 27,782 predicted phosphoserine sites distributed across 17,035 proteins. These prediction results are summarized graphically in the database together with the experimental phosphorylation sites in a whole sequence context. The Arabidopsis Protein Phosphorylation Site Database (PhosPhAt) provides a valuable resource to the plant science community and can be accessed through the following link PMID:17984086

Heazlewood, Joshua L; Durek, Pawel; Hummel, Jan; Selbig, Joachim; Weckwerth, Wolfram; Walther, Dirk; Schulze, Waltraud X



Enzymatic and functional correction along with long-term enzyme secretion from transduced bone marrow hematopoietic stem\\/progenitor and stromal cells derived from patients with Fabry disease  

Microsoft Academic Search

Fabry disease is a lysosomal storage disorder that is due to a deficiency in ?-galactosidase A (?-gal A). Previously we have shown that a recombinant retrovirus synthesized for the transfer of the human ?-gal A coding sequence was able to engineer enzymatic correction of the hydrolase deficiency in fibroblasts and lymphoblasts from Fabry patients. The corrected cells secreted ?-gal A

Toshihiro Takenaka; Chad S Hendrickson; David M Tworek; Matthew Tudor; Raphael Schiffmann; Roscoe O Brady; Jeffrey A Medin



A sensitive and robust method for automated on-line monitoring of enzymatic activities in water and water resources.  


The realisation of a novel concept for automated on-line monitoring of enzymatic activities in water was successfully demonstrated by long-term field testing at two remote Austrian ground water resources. The ?-D-glucuronidase (GLUC) activity was selected as a representative enzymatic model parameter for the on-line determination. But the device can be adapted for any enzymatic reaction with diagnostic relevance for microbial water quality monitoring, as demonstrated for the ?-D-galactosidase activity. Automated filtration of volumes up to 5 litres supports sensitive quantification of enzymatic activities. Internet-based data transfer, using internal control parameters for verification and a dynamic determination of the limit of quantification, enabled robust enzymatic on-line monitoring during a 2-year period. A proportion of 5,313 out of 5,506 GLUC activity measurements (96.5%) could be positively verified. Hydrological (discharge, gauge, turbidity, temperature, pH, electric conductivity, spectral absorbance coefficient at 254 nm) as well as microbiological parameters (Escherichia coli, coliforms) were concurrently determined to characterise the investigated ground water resources. The enzymatic on-line measurements closely reflected the different hydrological conditions and contamination patterns of the test sites. Contrary to expectations, GLUC did not qualify as a proxy-parameter for the occurrence of cultivation-based E. coli contamination and warrants further detailed investigations on its indication capacity as a rapid means for microbial faecal pollution detection in such aquatic habitats. Microbial on-line monitoring is likely to become more important in the future, complementing existing surveillance strategies for water safety management. Further perspectives on the application of such analytical on-line technologies, such as their connection with event-triggered sampling and standardised diagnostics, are discussed. PMID:24647204

Ryzinska-Paier, G; Lendenfeld, T; Correa, K; Stadler, P; Blaschke, A P; Mach, R L; Stadler, H; Kirschner, A K T; Farnleitner, A H



Can You Hear Me Now? Regulating Transcriptional Activators by Phosphorylation  

NSDL National Science Digital Library

Extracellular signals often modulate the expression of specific genetic programs by triggering the phosphorylation of relevant transcription factors (TFs). Phosphorylation in turn regulates such TFs by altering their cellular localization, DNA binding affinity, or transcriptional activity. Structural approaches have revealed how phosphorylation turns some TFs on or off; but less is known about how phosphorylation regulates other transcription factors in a graded manner that depends on signal intensity. A recent paper by Graves and colleagues reveals how a group of phosphorylation sites in Ets-1 regulates its DNA binding activity. Their studies provide new insight into the importance of multisite phosphorylation for the graded regulation of transcription and highlight the involvement of allosteric mechanisms in this process.

Kevin H. Gardner (University of Texas Southwestern Medical Center;Departments of Biochemistry and Pharmacology REV); Marc Montminy (The Salk Institute for Biological Studies; REV)



Analysis of mutations that uncouple transport from phosphorylation in enzyme IIGlc of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system.  

PubMed Central

Mutations that uncouple glucose transport from phosphorylation were isolated in plasmid-encoded Escherichia coli enzyme IIGlc of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The uncoupled enzymes IIGlc were able to transport glucose in the absence of the general phosphoryl-carrying proteins of the PTS, enzyme I and HPr, although with relatively low affinity. Km values of the uncoupled enzymes IIGlc for glucose ranged from 0.5 to 2.5 mM, 2 orders of magnitude higher than the value of normal IIGlc. Most of the mutant proteins were still able to phosphorylate glucose and methyl alpha-glucoside (a non-metabolizable glucose analog specific for IIGlc), indicating that transport and phosphorylation are separable functions of the enzyme. Some of the uncoupled enzymes IIGlc transported glucose with a higher rate and lower apparent Km in a pts+ strain than in a delta ptsHI strain lacking the general proteins enzyme I and HPr. Since the properties of these uncoupled enzymes IIGlc in the presence of PTS-mediated phosphoryl transfer resembled those of wild-type IIGlc, these mutants appeared to be conditionally uncoupled. Sequencing of the mutated ptsG genes revealed that all amino acid substitutions occurred in a hydrophilic segment within the hydrophobic N-terminal part of IIGlc. These results suggest that this hydrophilic loop is involved in binding and translocation of the sugar substrate. Images PMID:1569016

Ruijter, G J; van Meurs, G; Verwey, M A; Postma, P W; van Dam, K



A Novel In Vitro Assay to Assess Phosphorylation of 3?-[18F]fluoro-3?-Deoxythymidine  

PubMed Central

Purpose 3?-[18F]fluoro-3’-deoxythymidine ([18F]FLT) is phosphorylated by thymidine kinase 1 (TK-1), a cell cycle regulated enzyme. Appropriate use of [18F]FLT tracer requires validation of the TK-1 activity. Here, we report development of a novel phosphoryl-transfer assay to assess phosphorylation of [18F]FLT both in tumor cell lysates and tumor cells. Procedures The intrinsic F-18 radioactivity was used to quantify both substrate and phosphorylated products using a rapid thin layer chromatography method. Phosphorylation kinetics of [18F]FLT in SW480 and DiFi tumor cell lysates and cellular uptake were measured. Results The apparent Michaelis–Menten kinetic parameters for [18F]FLT are Km = 4.8 ± 0.3 ?M and Vmax=7.4 pmol min?1per 1×106 cells with ~2-fold higher TK-1 activity in DiFi versus SW480 lysates. Conclusions The apparent Km of [18F]FLT was comparable to the value reported with purified recombinant TK-1. The uptake of [18F]FLT by SW480 cells is inhibited by nitrobenzylthioinosine or dipyridamole indicating that uptake is mediated predominantly by the equilibrative nucleoside transporters in these tumor cells. PMID:20532643

Guo, Ning; Xie, Jingping; Manning, H. Charles; Deane, Natasha G.; Ansari, M. Sib; Coffey, Robert J.; Gore, John; Price, Ronald R.; Baldwin, Ronald M.; McIntyre, J. Oliver



Regulation of cardiac C-protein phosphorylation  

SciTech Connect

Molecular mechanisms of cardiac sympathetic and parasympathetic responses were addressed by studying subcellular changes in protein phosphorylation, cAMP-dependent protein kinase activity and protein phosphatase activity in frog hearts. B-adrenergic agonists increased and muscarinic cholinergic agonists decreased (/sup 32/P)phosphate incorporation into C-protein, a thick filament component. Regulation of protein phosphatase activity by Iso and methacholine (MCh) was assayed using extracts of drug treated frog hearts and (/sup 32/P)phospho-C-protein as substrate. Total phosphatase activity decreased 21% in extracts from hearts perfused with 0.1 Iso and 17% in hearts exposed to Iso plus 1 methacholine. This decrease reflected decreased phosphatase-2A activity. No changes in total phosphatase activity were measurable in broken cells treated with Iso or MCh. The results suggest adrenergic stimulation changes contractile activity in frog hearts by activating cAMP-dependent protein kinase associated with particulate cellular elements and inactivating soluble protein phosphatase-2A. This is the first demonstration of coordinated regulation of these enzymes by B-adrenergic agonists favoring phosphorylation of effector proteins. Coordinated regulation by methacholine in the presence of Iso was not observed.

Titus, F.L.



Regulation of Sprouty Stability by Mnk1-Dependent Phosphorylation  

PubMed Central

Sprouty (Spry) proteins are negative feedback modulators of receptor tyrosine kinase pathways in Drosophila melanogaster and mammals. Mammalian Spry proteins have been shown to undergo tyrosine and serine phosphorylation in response to growth factor stimulation. While several studies have addressed the function of tyrosine phosphorylation of Spry, little is known about the significance of Spry serine phosphorylation. Here we identify mitogen-activated protein kinase-interacting kinase 1 (Mnk1) as the kinase that phosphorylates human Spry2 (hSpry2) on serines 112 and 121. Mutation of these serine residues to alanine or inhibition of Mnk1 activity increases the rate of ligand-induced degradation of hSpry2. Conversely, enhancement of serine phosphorylation achieved through either the inhibition of cellular phosphatases or the expression of active Mnk1 results in the stabilization of hSpry2. Previous studies have shown that growth factor stimulation induces the proteolytic degradation of hSpry2 by stimulating tyrosine phosphorylation on hSpry2, which in turn promotes c-Cbl binding and polyubiquitination. A mutant of hSpry2 that is deficient in serine phosphorylation displays enhanced tyrosine phosphorylation and c-Cbl binding, indicating that serine phosphorylation stabilizes hSpry2 by exerting an antagonistic effect on tyrosine phosphorylation. Moreover, loss of serine phosphorylation and the resulting enhanced degradation of hSpry2 impair its capacity to antagonize fibroblast growth factor-induced extracellular signal-regulated kinase activation. Our results imply that Mnk1-mediated serine phosphorylation of hSpry2 constitutes a regulatory mechanism to extend the temporal range of Spry activity. PMID:16479008

DaSilva, John; Xu, Lizhong; Kim, Hong Joo; Miller, W. Todd; Bar-Sagi, Dafna



Phosphorylation of ATXN1 at Ser776 in the cerebellum  

PubMed Central

Spinocerebellar ataxia type 1 (SCA1) is one of nine inherited neurodegenerative disorders caused by a mutant protein with an expanded polyglutamine tract. Phosphorylation of ataxin-1 (ATXN1) at serine 776 is implicated in SCA1 pathogenesis. Previous studies, utilizing transfected cell lines and a Drosophila photoreceptor model of SCA1, suggest that phosphorylating ATXN1 at S776 renders it less susceptible to degradation. This work also indicated that oncogene from AKR mouse thymoma (Akt) promotes the phosphorylation of ATXN1 at S776 and severity of neurodegeneration. Here, we examined the phosphorylation of ATXN1 at S776 in cerebellar Purkinje cells, a prominent site of pathology in SCA1. We found that while phosphorylation of S776 is associated with a stabilization of ATXN1 in Purkinje cells, inhibition of Akt either in vivo or in a cerebellar extract-based phosphorylation assay did not decrease the phosphorylation of ATXN1-S776. In contrast, immunodepletion and inhibition of cyclic AMP-dependent protein kinase decreased phosphorylation of ATXN1-S776. These results argue against Akt as the in vivo kinase that phosphorylates S776 of ATXN1 and suggest that cyclic AMP-dependent protein kinase is the active ATXN1-S776 kinase in the cerebellum. PMID:19500214

Jorgensen, Nathan D.; Andresen, J. Michael; Lagalwar, Sara; Armstrong, Ben; Stevens, Sam; Byam, Courtney E.; Duvick, Lisa A.; Lai, Shaojuan; Jafar-Nejad, Paymaan; Zoghbi, Huda Y.; Clark, H. Brent; Orr, Harry T.



Selenium mediated arsenic toxicity modifies cytotoxicity, reactive oxygen species and phosphorylated proteins.  


The effect of selenium on modulating arsenic cytotoxicity is well known in mammals, but not well understood. Cell cytotoxicity and reactive oxygen species (ROS) changes were performed in combinations of As(III) and selenomethionine (SeMet) toxic mixes on, HEK293, human kidney cells. Cell growth is readily restored from 20% to 60% when switching from 30 ?M As(III) as toxin to a mix of 30 ?M As(III) and 100 ?M SeMet. As(III) alone triggers ROS formation, primarily hydrogen peroxide, in a concentration dependent manner as observed through changes in the fluorescence from 2',7'-dichlorofluorescein diacetate. Importantly, SeMet induces lower ROS levels at the same concentrations used to modulate As(III) cytotoxicity (IC50). Elevated ROS is important to As(III) cytotoxicity and minimizing it is essential to the SeMet modulating function. Changes in cell signaling, through analysis of signaling changes via differential protein phosphorylation to uncover molecular level changes occurring in HEK293 human kidney cells as SeMet modulates the As(III) cytotoxicity. To discover changes in the phosphoproteome, cells were incubated under three conditions: 30 ?M As(III), 100 ?M SeMet, and 30 ?M As(III) + 100 ?M SeMet. After total protein isolation the three samples were separated into fractions using size exclusion chromatography by detecting (31)P(+). Each sample was analyzed for the phosphorylated peptides by enzymatic digestion, selective enrichment of phosphorylated peptides via TiO2, followed by nanoLC-ESIMS. Phosphorylated proteins unique to the As(III)-SeMet mixture were then identified. The molecular level changes to the cells show uniquely that the As(III)-SeMet mixture details proteins involved in ROS detoxification, cell cycle arrest, and protein/DNA damage. This study shows that SeMet not only lowers the total amount of ROS in a cell but also confers upon HEK293 cells the ability to detoxify. Thus, SeMet is not only a potent antioxidant in this system, but induces molecular changes that confer survival. PMID:23503360

Chitta, Karnakar R; Landero Figueroa, Julio A; Caruso, Joseph A; Merino, Edward J



Hits, Fhits and Nits: beyond enzymatic function.  


We have briefly summarized what is known about these proteins, but in closing wish to feature the outstanding questions. Hint1 was discovered mistakenly as an inhibitor of Protein Kinase C and designated Pkci, a designation that still confuses the literature. The other Hint family members were discovered by homology to Hint1. Aprataxin was discovered as a result of the hunt for a gene responsible for AOA1. Fhit was discovered through cloning of a familial chromosome translocation breakpoint on chromosome 3 that interrupts the large FHIT gene within an intron, in the FRA3B chromosome region (Ohta et al., 1996), now known to be the region of the human genome most susceptible to DNA damage due to replication stress (Durkin et al., 2008). The NitFhit fusion genewas discovered during searches for Fhit homologs in flies and worms because the fly/worm Nit polypeptide is fused to the 5'-end of the Fhit gene; the mammalian Nit gene family was discovered because of the NitFhit fusion gene, in searches for homologs to the Nit polypeptide of the NitFhit gene. Each of the Hit family member proteins is reported to have enzymatic activities toward putative substrates involving nucleosides or dinucleosides. Most surprisingly, each of the Hit family proteins discussed has been implicated in important DNA damage response pathways and/or tumor suppression pathways. And for each of them it has been difficult to assign definite substrates, to know if the substrates and catalytic products have biological functions, to know if that function is related to the DNA damage response and suppressor functions, and to precisely define the pathways through which tumor suppression occurs. When the fly Nit sequence was found at the 5'-end of the fly Fhit gene, this gene was hailed as a Rosetta stone gene/protein that would help in discovery of the function of Fhit, because the Nit protein should be in the same signal pathway (Pace et al., 2000). However, the mammalian Nit family proteins have turned out to be at least as mysterious as the Fhit proteins, with the Nit1 substrate still unknown and the surprising finding that Nit proteins also appear to behave as tumor suppressor proteins. Whether the predicted enzymatic functions of these proteins are relevant to the observed biological functions, remain among the outstanding unanswered puzzles and raise the question: have these mammalian proteins evolved beyond the putative original enzymatic purpose, such that the catalytic function is now vestigial and subservient to signal pathways that use the protein-substrate complexes in pathways that signal apoptosis or DNA damage response? Or can these proteins be fulfilling catalytic functions independently but in parallel with signal pathway functions, as perhaps observed for Aprataxin? Or is the catalytic function indeed part of the observed biological functions, such as apoptosis and tumor suppression? Perhaps the recent, post-genomic focus on metabolomics and genome-wide investigations of signal pathway networks will lead to answers to some of these outstanding questions. PMID:21035495

Huebner, Kay; Saldivar, Joshua C; Sun, Jin; Shibata, Hidetaka; Druck, Teresa



In vivo phosphorylation of adaptors regulates their interaction with clathrin  

PubMed Central

The coat proteins of clathrin-coated vesicles (CCV) spontaneously self- assemble in vitro, but, in vivo, their self-assembly must be regulated. To determine whether phosphorylation might influence coat formation in the cell, the in vivo phosphorylation state of CCV coat proteins was analyzed. Individual components of the CCV coat were isolated by immunoprecipitation from Madin-Darby bovine kidney cells, labeled with [32P]orthophosphate under normal culture conditions. The predominant phosphoproteins identified were subunits of the AP1 and AP2 adaptors. These included three of the four 100-kD adaptor subunits, alpha and beta 2 of AP2 and beta 1 of AP1, but not the gamma subunit of AP1. In addition, the mu 1 and mu 2 subunits of AP1 and AP2 were phosphorylated under these conditions. Lower levels of in vivo phosphorylation were detected for the clathrin heavy and light chains. Analysis of phosphorylation sites of the 100-kD adaptor subunits indicated they were phosphorylated on serines in their hinge regions, domains that have been implicated in clathrin binding. In vitro clathrin-binding assays revealed that, upon phosphorylation, adaptors no longer bind to clathrin. In vivo analysis further revealed that adaptors with phosphorylated 100-kD subunits predominated in the cytosol, in comparison with adaptors associated with cellular membranes, and that phosphorylated beta 2 subunits of AP2 were exclusively cytosolic. Kinase activity, which converts adaptors to a phosphorylated state in which they no longer bind clathrin, was found associated with the CCV coat. These results suggest that adaptor phosphorylation influences adaptor-clathrin interactions in vivo and could have a role in controlling coat disassembly and reassembly. PMID:8909539



Computational studies on nonenzymatic and enzymatic pyridoxal phosphate catalyzed decarboxylations of 2-aminoisobutyrate.  


A computational study of nonenzymatic and enzymatic pyridoxal phosphate-catalyzed decarboxylation of 2-aminoisobutyrate (AIB) is presented. Four prototropic isomers of a model aldimine between AIB and 5'-deoxypyridoxal, with acetate interacting with the pyridine nitrogen, were employed in calculations of both gas phase and water model (PM3 and PM3-SM3) decarboxylation reaction paths. Calculations employing the transition state structures obtained for the four isomers allow the demonstration of stereoelectronic effects in transition state stabilization as well as a separation of the contributions of the Schiff base and pyridine ring moieties to this stabilization. The unprotonated Schiff base contribution (approximately 16 kcal/mol) is larger than that of the pyridine ring even when it is protonated (approximately 10 kcal/mol), providing an explanation of the catalytic power of pyruvoyl-dependent amino acid decarboxylases. An active site model of dialkylglycine decarboxylase was constructed and validated, and enzymatic decarboxylation reaction paths were calculated. The reaction coordinate is shown to be complex, with proton transfer from Lys272 to the coenzyme C4' likely simultaneous with C alpha--CO(2)(-) bond cleavage. The proposed concerted decarboxylation/proton-transfer mechanism provides a simple explanation for the observed specificity of this enzyme toward oxidative decarboxylation. PMID:11170465

Toney, M D



Non-Enzymatic Dynamic Kinetic Resolution of Secondary Alcohols via Enantioselective Acylation: Synthetic and Mechanistic Studies  

PubMed Central

Because of the ubiquity of the secondary carbinol subunit, the development of new methods for its enantioselective synthesis remains an important ongoing challenge. In this report, we describe the first non-enzymatic method for the dynamic kinetic resolution (DKR) of secondary alcohols (specifically, aryl alkyl carbinols) through enantioselective acylation, and we substantially expand the scope of this approach, vis-à-vis enzymatic reactions. Simply combining an effective process for the kinetic resolution of alcohols with an active catalyst for the racemization of alcohols did not lead to DKR, due to the incompatibility of the ruthenium-based racemization catalyst with the acylating agent (Ac2O) used in the kinetic resolution. A mechanistic investigation revealed that the ruthenium catalyst is deactivated through the formation of a stable ruthenium-acetate complex; this deleterious pathway was circumvented through the appropriate choice of acylating agent (an acyl carbonate). Mechanistic studies of this new process point to reversible N-acylation of the nucleophilic catalyst, acyl transfer from the catalyst to the alcohol as the rate-determining step, and carbonate anion serving as the Brønsted base in that acyl-transfer step. PMID:22934603

Lee, Sarah Yunmi; Murphy, Jaclyn M.; Ukai, Atsushi; Fu, Gregory C.



Dynamic transfer 1 DYNAMIC TRANSFER  

E-print Network

Dynamic transfer 1 DYNAMIC TRANSFER: A PERSPECTIVE FROM PHYSICS EDUCATION RESEARCH Running head: Dynamic transfer N. Sanjay Rebello Physics Department, 116 Cardwell Hall, Kansas State University. Phone: (760) 768 5606 Fax: (785) 768 5631 Email: #12;Dynamic transfer 2 ABSTRACT We

Zollman, Dean


Effects of calcium, strontium, and barium ions on phosphorylation of hippocampal proteins in vitro.  


Calcium ion alone or in the presence of added calmodulin stimulated in vitro transfer of 32P from [gamma 32P]ATP into several proteins of mitochondrial and synaptosomal particulate fractions from rat brain. Strontium ion was capable of substituting for calcium ion in this stimulation, but barium ion lacked this capacity. These results bring into question the hypothesis that calcium-dependent protein phosphorylation of synaptic proteins is intrinsic to neurotransmitter release during neurotransmission, but they do not rule out that possibility. PMID:6689699

Hoch, D B; Wilson, J E



Use of microbial transglutaminase for the enzymatic biotinylation of antibodies.  


Nowadays many reagents are available for the biotinylation of proteins. As most of them bind to amino groups of the protein the degree of labelling differs from batch to batch and the possibility exists that the biological activity of the target protein may be affected by the labelling procedure. In the present study we have investigated an enzymatic approach to biotinylation using microbial transglutaminase (MTGase) from Streptoverticillium mobaraense. The proposed method is particularly suitable when only a few biotin molecules need to be attached to the target proteins. The enzyme catalyses the acyl transfer reaction between gamma-carboxyamide groups and various primary amines. This was exploited for biotinylation using two amino-modified biotin derivatives, biotinamido-5-pentylamin (BIAPA) and biotinoyl-1,8-diamino-3, 6-dioxaoctane (BIDADOO) as acyl acceptors and a monoclonal IgG against the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as the acyl donor. Kinetic studies revealed that the MTGase-mediated reaction proceeds with low velocity and is almost complete after 34 h. Conjugation ratios ranging from 1.1 to 1.9 biotins per IgG were found by mass spectrometry. To investigate the influence of antibody conjugation on antigen binding a competitive ELISA for the determination of 2,4-D employing MTGase-biotinoylated IgGs was developed. In this assay lower limits of detection of 0.3 and 1.0 microg/l of 2,4-D were achieved with BIDADOO- and BIAPA-modified antibodies, respectively. PMID:10854600

Josten, A; Haalck, L; Spener, F; Meusel, M



Examination of Enzymatic H-Tunneling through Kinetics and Dynamics  

PubMed Central

In recent years, kinetic measurements of isotope effects of enzyme catalyzed reactions and their temperature dependence led to the development of theoretical models that were used to rationalize the findings. These models suggested that motions at the femto- to pico-second (fs to ps) time scale modulate the environment of the catalyzed reaction. Due to the fast nature of motions that directly affect the cleavage of a covalent bond, it is challenging to correlate the enzyme kinetics and dynamics related to that step. We report a study of formate dehydrogenase (FDH) that compares the temperature dependence of intrinsic kinetic isotope effects (KIEs) to measurements of the environmental dynamics at the fs-ps time scale (Bandaria et al., J. Am. Chem. Soc. 2008, 130, 22–23). The findings from this comparison of experimental kinetics and dynamics are consistent with models of environmentally coupled H-tunneling models, also known as Marcus-like models. Apparently, at tunneling ready conformations, the donor-acceptor distance, orientation, and fluctuations, seems to be well tuned for H- transfer and are not affected by thermal fluctuations slower than 20 ps. This phenomenon has been suggested in the past to be quite general in enzymatic reactions. Here, the kinetics and the dynamics measurements on a single chemical step and on fs-ps time scale, respectively, provide new insight and support for the relevant theoretical models. Furthermore, this methodology could be applied to other systems and be used to examine mutants for which the organization of the donor and acceptor is not ideal, or enzymes with different rigidity and different temperature optimum. PMID:19621965

Bandaria, Jigar N.; Cheatum, Christopher M.; Kohen, Amnon



Engineering Kinases to Phosphorylate Nucleoside Analogs for Antiviral and Cancer Therapy  

PubMed Central

Enzyme engineering by directed evolution presents a powerful strategy for tailoring the function and physicochemical properties of biocatalysts to therapeutic and industrial applications. Our laboratory’s research focuses on developing novel molecular tools for protein engineering, as well as on utilizing these methods to customize enzymes and to study fundamental aspects of their structure and function. Specifically, we are interested in nucleoside and nucleotide kinases which are responsible for the intracellular phosphorylation of nucleoside analog (NA) prodrugs to their biologically active triphosphates. The high substrate specificity of the cellular kinases often interferes with prodrug activation and consequently lowers the potency of NAs as antiviral and cancer therapeutics. A working solution to the problem is the co-adminstration of a promiscuous kinase from viruses, bacteria, and other mammals. However, further therapeutic enhancements of NAs depend on the selective and efficient prodrug phosphorylation. In the absence of true NA kinases in nature, we are pursuing laboratory evolution strategies to generate efficient phosphoryl-transfer catalysts. This review summarizes some of our recent work in the field and outlines future challenges. PMID:20305804

Lutz, Stefan; Liu, Lingfeng; Liu, Yichen



Enzymatic detection of D-amino acids.  


D: -Amino acids play several key roles and are widely diffused in living organisms, from bacteria (in which D-alanine is a component of the cell wall) to mammals (where D-serine is involved in glutamatergic neurotransmission in the central nervous system). The study of the biological processes involving D-amino acids and their use as clinical or biotechnological biomarkers requires reliable methods of quantifying them. Although "traditional" analytical techniques have been (and still are) employed for such tasks, enzymatic assays based on enzymes which possess a strict stereospecificity (i.e., that are only active on the D-enantiomers of amino acids) allowed the set-up of low-cost protocols with a high sensitivity and selectivity and suitable for determining the D-amino acid content of complex biological samples. The most exploited enzyme in these assays is D-amino acid oxidase, a flavoenzyme that exclusively oxidizes D-amino acids and possesses with a broad substrate specificity and a high kinetic efficiency. PMID:21956570

Molla, Gianluca; Piubelli, Luciano; Volontè, Federica; Pilone, Mirella S



Enzymatic hydrolysis of spent coffee ground.  


Spent coffee ground (SCG) is the main residue generated during the production of instant coffee by thermal water extraction from roasted coffee beans. This waste is composed mainly of polysaccharides such as cellulose and galactomannans that are not solubilised during the extraction process, thus remaining as unextractable, insoluble solids. In this context, the application of an enzyme cocktail (mannanase, endoglucanase, exoglucanase, xylanase and pectinase) with more than one component that acts synergistically with each other is regarded as a promising strategy to solubilise/hydrolyse remaining solids, either to increase the soluble solids yield of instant coffee or for use as raw material in the production of bioethanol and food additives (mannitol). Wild fungi were isolated from both SCG and coffee beans and screened for enzyme production. The enzymes produced from the selected wild fungi and recombinant fungi were then evaluated for enzymatic hydrolysis of SCG, in comparison to commercial enzyme preparations. Out of the enzymes evaluated on SCG, the application of mannanase enzymes gave better yields than when only cellulase or xylanase was utilised for hydrolysis. The recombinant mannanase (Man1) provided the highest increments in soluble solids yield (17 %), even when compared with commercial preparations at the same protein concentration (0.5 mg/g SCG). The combination of Man1 with other enzyme activities revealed an additive effect on the hydrolysis yield, but not synergistic interaction, suggesting that the highest soluble solid yields was mainly due to the hydrolysis action of mannanase. PMID:23436225

Jooste, T; García-Aparicio, M P; Brienzo, M; van Zyl, W H; Görgens, J F



Enzymatic production of hydrogen from glucose  

SciTech Connect

The objective of this research is to optimize conditions for the enzymatic production of hydrogen gas from biomass-derived glucose. This new project is funded at 0.5 PY level of effort for FY 1995. The rationale for the work is that cellulose is, potentially, a vast source of hydrogen and that enzymes offer a specific and efficient method for its extraction with minimal environmental impact. This work is related to the overall hydrogen program goal of technology development and validation. The approach is based on knowledge that glucose is oxidized by the NADP{sup +} requiring enzyme glucose dehydrogenase (GDH) and that the resulting NADPH can donate its electrons to hydrogenase (H{sub 2}ase) which catalyzes the evolution of H{sub 2}. Thus hydrogen production from glucose was achieved using calf liver GDH and Pyrococcus furiosus H{sub 2}ase yielding 17% of theoretical maximum expected. The cofactor NADP{sup +} for this reaction was regenerated and recycled. Current and future work includes understanding the rate limiting steps of this process and the stabilization/immobilization of the enzymes for long term hydrogen production. Cooperative interactions with the Universities of Georgia and Bath for obtaining thermally stable enzymes are underway.

Woodward, J. [Oak Ridge National Lab., TN (United States); Mattingly, S.M.



Natural Product Sugar Biosynthesis and Enzymatic Glycodiversification**  

PubMed Central

Many biologically active small molecule natural products produced by microorganisms derive their activities from sugar substituents. Changing the structures of these sugars can have a profound impact on the biological properties of the parent compounds. This realization has inspired attempts to derivatize the sugar moieties of these natural products through exploitation of the sugar biosynthetic machinery. This approach requires an understanding of the biosynthetic pathway of each target sugar and detailed mechanistic knowledge of the key enzymes. Scientists have begun to unravel the biosynthetic logic behind the assembly of many glycosylated natural products, and have found that a core set of enzyme activities is mixed and matched to synthesize the diverse sugar structures observed in nature. Remarkably, many of these sugar biosynthetic enzymes and glycosyltransferases also exhibit relaxed substrate specificity. The promiscuity of these enzymes has prompted efforts to modify the sugar structures and/or alter the glycosylation patterns of natural products via metabolic pathway engineering and/or enzymatic glycodiversification. In applied biomedical research, these studies will enable the development of new glycosylation tools and generate novel glycoforms of secondary metabolites with useful biological activity. PMID:19058170

Thibodeaux, Christopher J.; Melancon, Charles E.; Liu, Hung-wen



Nanostructured Organometallic Polymers for Enzymatic Bioenergy  

NASA Astrophysics Data System (ADS)

The development of efficient enzymatic biofuel cell is a subject of considerable studies in past decades for potential applications such as biomedical devices and microchip systems. One of the key challenges in advancing the technology lies in the power densities of the system. Limitations have been arisen from the buried redox active sites within enzyme structure and poor interplay between redox reactions. In present study, a glucose oxidase is employed as a model enzyme and ferrocene-containing organometallic block copolymers are chosen for the electron mediators. Wiring of glucose oxidase into electrode surface was successfully achieved by cross-linked networks of organometallic polymers and remarkably, catalytic current densities of the fabricated electrodes have proven be a sensitive function of the morphologies of electron mediators. Different nanoscale morphologies, i.e., bicontinous structure, nanowires, and nanoparticles, have been derived and the use of bicontinous morphology confirms 2-50 times improved catalytic current response than the values obtained from other morphologies. The bio-sensing ability of the fabricated electrode with structural optimization was also exploited and good sensitivity is obtained at the physiological concentration of glucose in blood.

Park, Moon Jeong; Lee, Jungphil




PubMed Central

Meloche, H. P., Jr. (Northern Regional Research Laboratory, Peoria, Ill.). Enzymatic utilization of glucose by a basidiomycete. J. Bacteriol. 83:766–774. 1962.—Cell-free extracts of acetone-dried Lactarius torminosus NRRL 2900 were prepared. These extracts contained hexokinase. They also contained triphosphopyridine nucleotide (TPN)-specific glucose-6-phosphate dehydrogenase and catalyzed the reduction of TPN in the presence of d-fructose-6-phosphate, 6-phospho-d-gluconic acid (6PG), and d-ribose-5-phosphate (R5P). Aged preparations oxidized d-glucose-6-phosphate (G6P) to 6PG, whereas fresh preparations oxidized G6P to a pentose with the uptake of 1 ?mole of O2 and the evolution of 1 ?mole of CO2 per ?mole of G6P. Evidence for the action of transketolase in the metabolism of R5P by cell-free extracts was obtained. Cell-free preparations lacked hexosediphosphate enzymes. Triosephosphate isomerase and F6P kinase could not be demonstrated; however, aldolase activity was present. Evidence is presented for the conversion of d-glyceraldehyde-3-phosphate to pyruvate. In addition, phosphohexoisomerase was demonstrated. It appears that a hexosemonophosphate pathway operates in L. torminosus extracts and may be the major mechanism of glucose dissimilation in this organism. PMID:14472442

Meloche, H. P.



The enzymatic synthesis of rubber polymer  

SciTech Connect

Washed rubber particles (WRP) isolated from stem homogenates of Parthenium argentatum by ultracentrifugation and gel filtration on columns of LKB Ultrogel AcA34 contain a tightly bound rubber transferase which catalyzes the polymerization of IPP into rubber polymer. The polymerization reaction requires Mg{sup 2+}, IPP and an allylic-PP. The Km values for Mg{sup 2+}, IPP and DMAPP are 5.2{times}10{sup {minus}4}M, 8.3{times}10{sup {minus}5} M and 9.6{times}10{sup {minus}5}M respectively. Gel permeation chromatography of the enzymatic polymer product on 3 linear columns of 1{times}10{sup 6} to 500 {angstrom} Ultrastyragel shows that the in vitro formed polymer has a similar mol wt to natural rubber. Over 90% of the in vitro formation of the rubber polymer was a de novo polymerization reaction from DMAPP initiator and IPP monomers. The bound rubber polymerase substantially differs from cytosolic rubber transferase which catalyzes only chain lengthening reactions. Treatment of the WRP with Chaps solubilized the bound rubber transferase which was further purified by DEAE-cellulose chromatography. The purified preparation primarily consists of a 52 kD polypeptide which binds to a photolabile substrate analog. The soluble rubber transferase catalyzes the synthesis of a 1{times}10{sup 5} mol wt rubber polymer from Mg{sup 2+}, DMAPP, IPP and detergent.

Venkatachalam, K.V.; Wooten, L.; Benedict, C.R. (Texas A M Univ., College Station (USA))



Enzymatic ligation of large biomolecules to DNA.  


The ability to synthesize, characterize, and manipulate DNA forms the foundation of a range of advanced disciplines including genomics, molecular biology, and biomolecular engineering. In particular for the latter field, DNA has proven useful as a structural or functional component in nanoscale self-assembled structures, antisense therapeutics, microarray diagnostics, and biosensors. Such applications frequently require DNA to be modified and conjugated to other macromolecules, including proteins, polymers, or fatty acids, in order to equip the system with properties required for a particular application. However, conjugation of DNA to large molecular components using classical chemistries often suffers from suboptimal yields. Here, we report the use of terminal deoxynucleotidyl transferase (TdT) for direct enzymatic ligation of native DNA to nucleotide triphosphates coupled to proteins and other large macromolecules. We demonstrate facile synthesis routes for a range of NTP-activated macromolecules and subsequent ligation to the 3' hydroxyl group of oligodeoxynucleotides using TdT. The reaction is highly specific and proceeds rapidly and essentially to completion at micromolar concentrations. As a proof of principle, parallelly labeled oligonucleotides were used to produce nanopatterned DNA origami structures, demonstrating rapid and versatile incorporation of non-DNA components into DNA nanoarchitectures. PMID:23927463

Sørensen, Rasmus Schøler; Okholm, Anders Hauge; Schaffert, David; Kodal, Anne Louise Bank; Gothelf, Kurt V; Kjems, Jørgen



Mitochondrial Phosphorylation in Apoptosis: Flipping the Death Switch  

PubMed Central

Abstract Significance: Apoptosis is a complex cellular process subject to multiple layers of regulation. One such layer of regulation includes post-translational modifications, including acetylation and phosphorylation. In particular, phosphorylation of proteins directly implicated in the apoptotic process has been extensively documented. Importantly, these phosphorylation events often have functional consequences, affecting the onset of apoptotic cell death. Recent Advances: Large-scale proteomics studies have identified multiple novel phosphorylation sites on proteins involved in the apoptotic process. The delineation of the regulation and functional consequences of these phosphorylation events will be important in understanding the regulatory complexity of apoptosis. Critical Issues: Multiple mitochondrial-localized proteins involved in apoptosis are functionally affected by phosphorylation, which can ultimately dictate whether a cell lives or dies. The dynamic interplay between these phosphorylated proteins and their regulatory enzymes is critical for understanding the complex cellular decision to undergo apoptosis. Future Directions: Detailed analysis of the kinetic and spatial regulation of phosphorylation events on apoptotic proteins, as well as how these dynamics influence the cell death process, will illuminate the complex interplay between the network of proteins that control the decision to undergo cell death. Antioxid. Redox Signal. 19, 572–582. PMID:23088365

Niemi, Natalie M.



Myosin light chain kinase phosphorylation in tracheal smooth muscle  

SciTech Connect

Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in {sup 32}P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+.

Stull, J.T.; Hsu, L.C.; Tansey, M.G.; Kamm, K.E. (Univ. of Texas Southwestern Medical Center, Dallas (USA))



Turnover of protein phosphorylation evolving under stabilizing selection  

PubMed Central

Most proteins are regulated by posttranslational modifications and changes in these modifications contribute to evolutionary changes as well as to human diseases. Phosphorylation of serines, threonines, and tyrosines are the most common modifications identified to date in eukaryotic proteomes. While the mode of action and the function of most phosphorylation sites remain unknown, functional studies have shown that phosphorylation affects protein stability, localization and ability to interact. Two broad modes of action have been described for protein phosphorylation. The first mode corresponds to the canonical and qualitative view whereby single phosphorylation sites act as molecular switches that either turn on or off specific protein functions through direct or allosteric effects. The second mode is more akin to a rheostat than a switch. In this case, a group of phosphorylation sites in a given protein region contributes collectively to the modification of the protein, irrespective of the precise position of individual sites, through an aggregate property. Here we discuss these two types of regulation and examine how they affect the rate and patterns of protein phosphorylation evolution. We describe how the evolution of clusters of phosphorylation sites can be studied under the framework of complex traits evolution and stabilizing selection. PMID:25101120

Landry, Christian R.; Freschi, Luca; Zarin, Taraneh; Moses, Alan M.



Enzymatic treatment of duck hepatitis B virus: Topology of the surface proteins for virions and noninfectious subviral particles  

SciTech Connect

The large surface antigen L of duck hepatitis B virus exhibits a mixed topology with the preS domains of the protein alternatively exposed to the particles' interior or exterior. After separating virions from subviral particles (SVPs), we compared their L topologies and showed that both particle types exhibit the same amount of L with the following differences: 1-preS of intact virions was enzymatically digested with chymotrypsin, whereas in SVPs only half of preS was accessible, 2-phosphorylation of L at S118 was completely removed by phosphatase treatment only in virions, 3-iodine-125 labeling disclosed a higher ratio of exposed preS to S domains in virions compared to SVPs. These data point towards different surface architectures of virions and SVPs. Because the preS domain acts in binding to a cellular receptor of hepatocytes, our findings implicate the exclusion of SVPs as competitors for the receptor binding and entry of virions.

Franke, Claudia [Heinrich-Pette-Institut fuer Experimentelle Virologie und Immunologie an der Universitaet Hamburg, Martinistrasse 52, D-20251 Hamburg (Germany); Matschl, Urte [Heinrich-Pette-Institut fuer Experimentelle Virologie und Immunologie an der Universitaet Hamburg, Martinistrasse 52, D-20251 Hamburg (Germany); Bruns, Michael [Heinrich-Pette-Institut fuer Experimentelle Virologie und Immunologie an der Universitaet Hamburg, Martinistrasse 52, D-20251 Hamburg (Germany)]. E-mail:



Mapping of phosphorylation sites by LC-MS/MS.  


Reversible protein phosphorylation ranks among the most important post-translational modifications that occurs in the cell. It is therefore highly relevant to elucidate the phosphorylation states of a given biological system,?albeit challenging. Most notably the often low stoichiometry of phosphorylation is inherently incompatible with standard LC-MS analysis of a complex protein digest mixture, primarily due to the relative low dynamic range of current mass analyzers. Therefore a need for specific enrichment of phosphorylated peptides or proteins exists. Significant progress surrounding the biochemical analysis of reversible protein phosphorylation in the past years has led to the development of several new techniques to isolate or enrich phosphopeptides, particularly in large-scale analyses. This chapter deals with three such examples. PMID:20839101

Gerrits, Bertran; Bodenmiller, Bernd



Exploiting holistic approaches to model specificity in protein phosphorylation  

PubMed Central

Phosphate plays a chemically unique role in shaping cellular signaling of all current living systems, especially eukaryotes. Protein phosphorylation has been studied at several levels, from the near-site context, both in sequence and structure, to the crowded cellular environment, and ultimately to the systems-level perspective. Despite the tremendous advances in mass spectrometry and efforts dedicated to the development of ad hoc highly sophisticated methods, phosphorylation site inference and associated kinase identification are still unresolved problems in kinome biology. The sequence and structure of the substrate near-site context are not sufficient alone to model the in vivo phosphorylation rules, and they should be integrated with orthogonal information in all possible applications. Here we provide an overview of the different contexts that contribute to protein phosphorylation, discussing their potential impact in phosphorylation site annotation and in predicting kinase-substrate specificity. PMID:25324856

Palmeri, Antonio; Ferrè, Fabrizio; Helmer-Citterich, Manuela




E-print Network

PROTEIN FLEXIBILITY AND ENZYMATIC CATALYSIS By M. KOKKINIDIS,*, N.M. GLYKOS, AND V.E. FADOULOGLOU ....................................................................... 182 II. Selected Methods Used for Studying Protein Flexibility. ......................... 185 A Flexibility ............................................ 191 A. Flexible Active Site Loops

Glykos, Nikolaos



EPA Science Inventory

An on-chip enzymatic assay for screening organophosphate (OP) nerve agents, based on a pre-column reaction of organophosphorus hydrolase (OPH), electrophoretic separation of the phosphonic acid products, and their contactless-conductivity detection, is described. Factors affec...


Ionizing radiation causes increased tau phosphorylation in primary neurons.  


Radiotherapy is the major treatment modality for primary and metastatic brain tumors which involves the exposure of brain to ionizing radiation. Ionizing radiation can induce various detrimental pathophysiological effects in the adult brain, and Alzheimer's disease and related neurodegenerative disorders are considered to be late effects of radiation. In this study, we investigated whether ionizing radiation causes changes in tau phosphorylation in cultured primary neurons similar to that in Alzheimer's disease. We demonstrated that exposure to 0.5 or 2 Gy ? rays causes increased phosphorylation of tau protein at several phosphorylation sites in a time- and dose-dependent manner. Consistently, we also found ionizing radiation causes increased activation of GSK3?, c-Jun N-terminal kinase and extracellular signal-regulated kinase before radiation-induced increase in tau phosphorylation. Specific inhibitors of these kinases almost fully blocked radiation-induced tau phosphorylation. Our studies further revealed that oxidative stress plays an important role in ionizing radiation-induced tau phosphorylation, likely through the activation of c-Jun N-terminal kinase and extracellular signal-regulated kinase, but not GSK3?. Overall, our studies suggest that ionizing radiation may cause increased risk for development of Alzheimer's disease by promoting abnormal tau phosphorylation. Alzheimer disease is considered as a significant radiation late effect. In this study, we investigated whether ionizing radiation causes changes in tau phosphorylation and found that ? rays cause increased tau phosphorylation at multiple sites in a dose and time-dependent manner in primary neurons which are mediated by oxidative stress-dependent activation of JNK and ERK. Oxidative stress-independent activation of GSK3? is also involved. Overall, our studies suggest that ionizing radiation may cause increased risk for Alzheimer disease development by promoting abnormal tau phosphorylation. PMID:24861936

Li, Li; Wang, Wenzhang; Welford, Scott; Zhang, Teng; Wang, Xinglong; Zhu, Xiongwei



Nanocrystal Bioassembly: Asymmetry, Proximity, and Enzymatic Manipulation  

SciTech Connect

Research at the interface between biomolecules and inorganic nanocrystals has resulted in a great number of new discoveries. In part this arises from the synergistic duality of the system: biomolecules may act as self-assembly agents for organizing inorganic nanocrystals into functional materials; alternatively, nanocrystals may act as microscopic or spectroscopic labels for elucidating the behavior of complex biomolecular systems. However, success in either of these functions relies heavily uponthe ability to control the conjugation and assembly processes.In the work presented here, we first design a branched DNA scaffold which allows hybridization of DNA-nanocrystal monoconjugates to form discrete assemblies. Importantly, the asymmetry of the branched scaffold allows the formation of asymmetric2assemblies of nanocrystals. In the context of a self-assembled device, this can be considered a step toward the ability to engineer functionally distinct inputs and outputs.Next we develop an anion-exchange high performance liquid chromatography purification method which allows large gold nanocrystals attached to single strands of very short DNA to be purified. When two such complementary conjugates are hybridized, the large nanocrystals are brought into close proximity, allowing their plasmon resonances to couple. Such plasmon-coupled constructs are of interest both as optical interconnects for nanoscale devices and as `plasmon ruler? biomolecular probes.We then present an enzymatic ligation strategy for creating multi-nanoparticle building blocks for self-assembly. In constructing a nanoscale device, such a strategy would allow pre-assembly and purification of components; these constructs can also act as multi-label probes of single-stranded DNA conformational dynamics. Finally we demonstrate a simple proof-of-concept of a nanoparticle analog of the polymerase chain reaction.

Claridge, Shelley A



[Enzymatic hydrolysis of cellulose in reverse micelles].  


Several types of surfactants were adopted to construct reverse micelles, in order to investigate the characteristics of cellulose hydrolysis, we used the carboxymethyl cellulose as substrate. The electrical conductivity was measured to determine the maximum water solubilization W0( W0 = [H2O]/[SA] ) of CTAB, SDS, Tween-80 and rhamnolipid reverse micellar systems were 15.2, 20.1, 2.3 and 40.3. In this condition we studied the effects of surfactants concentrations and cellulose dosage on the enzymatic hydrolysis of reverse micelle,and compared with aqueous systems. It was shown by the results that when the cellulose dosage was 0.15 FPU/g substrate, the maximum yield of reducing sugar in reverse micelles was obtained at 1 cmc of CTAB, SDS, Tween-80 and rhamnolipid, in which the rhamnolipid yield was the highest of 198.03 mg substrate. When the concentrations of CTAB, SDS, Tween-80 and rhamnolipid were 1 cmc, the productions of reverse micelles systems were higher than that of aqueous systems of 34.36%, 21.24%, 11.44% and 34.62%. In the optimum conditions of the surfactant concentration, taking the saving cost and sugar yield into consideration, the cellulose dosage of 5 FPU substrate was the most suitable. The reducing sugar's yield of biosurfactant rhamnolipid reverse micellar system was higher than those of three chemical surfactant systems, it was shown that the adoption of biosurfactant has technologically promising prospect in constructing reverse micelles and enhancing the stability of reverse micelles. PMID:21072947

Wang, Wei-Wei; Yuan, Xing-Zhong; Zeng, Guang-Ming; Liang, Yun-Shan; Chao, Yang



Enzyme adsorption, precipitation and crosslinking of glucose oxidase and laccase on polyaniline nanofibers for highly stable enzymatic biofuel cells.  


Enzymatic biofuel cells have many great features as a small power source for medical, environmental and military applications. Both glucose oxidase (GOx) and laccase (LAC) are widely used anode and cathode enzymes for enzymatic biofuel cells, respectively. In this paper, we employed three different approaches to immobilize GOx and LAC on polyaniline nanofibers (PANFs): enzyme adsorption (EA), enzyme adsorption and crosslinking (EAC) and enzyme adsorption, precipitation and crosslinking (EAPC) approaches. The activity of EAPC-LAC was 32 and 25 times higher than that of EA-LAC and EAC-LAC, respectively. The half-life of EAPC-LAC was 53 days, while those of EA-LAC and EAC-LAC were 6 and 21 days, respectively. Similar to LAC, EAPC-GOx also showed higher activity and stability than EA-GOx and EAC-GOx. For the biofuel cell application, EAPC-GOx and EAPC-LAC were applied over the carbon papers to form enzyme anode and cathode, respectively. In order to improve the power density output of enzymatic biofuel cell, 1,4-benzoquinone (BQ) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were introduced as the electron transfer mediators on the enzyme anode and enzyme cathode, respectively. BQ- and ABTS-mediated enzymatic biofuel cells fabricated by EAPC-GOx and EAPC-LAC showed the maximum power density output of 37.4 ?W/cm(2), while the power density output of 3.1 ?W/cm(2) was shown without mediators. Under room temperature and 4°C for 28 days, enzymatic biofuel cells maintained 54 and 70% of its initial power density, respectively. PMID:25248697

Kim, Ryang Eun; Hong, Sung-Gil; Ha, Su; Kim, Jungbae



Technology transfer  

NASA Technical Reports Server (NTRS)

The requirements for a successful technology transfer program and what such a program would look like are discussed. In particular, the issues associated with technology transfer in general, and within the Jet Propulsion Laboratory (JPL) environment specifically are addressed. The section on background sets the stage, identifies the barriers to successful technology transfer, and suggests actions to address the barriers either generally or specifically. The section on technology transfer presents a process with its supporting management plan that is required to ensure a smooth transfer process. Viewgraphs are also included.

Handley, Thomas



Mucin biosynthesis revisited. The enzymatic transfer of Gal in beta 1,3 linkage to the GalNAc moiety of the core structure R1-GlcNAc beta 1,6GalNAc alpha-O-R2.  


Synthetic glycosides containing the core, -Glc-NAc beta 1,6GalNAc alpha-, acted as acceptors for beta-galactosyltransferase of human ovarian tumor. A significant amount of Gal was transferred from UDP-Gal (100 nmol) to the alpha-benzylglycoside of LacNAc beta 1,6GalNAc (LGBn) (25.1 nmol of Gal) and the alpha-ortho-nitrophenylglycosides of LacNAc beta 1,6GalNAc (22.0 nmol of Gal), GlcNAc beta 1,6GalNAc (15.5 nmol of Gal), and Fuc alpha 1,3GlcNAc beta 1,6GalNAc (25.9 nmol of Gal); LacNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn (where Bn is benzyl) was almost inactive (only 1.2 nmol of Gal), indicating the Gal transfer to the alpha-GalNAc moiety. The product from LGBn was isolated in microgram quantities and identified by fast atom bombardment mass spectrometry as LacNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn. The alpha GalNAc:beta 1,3Gal transferase was present in high concentration in ovarian tumor tissue (ovarian cancer serum----1.4; ascitic fluid----0.9; tumor----17.4). Asialo Cowper's gland mucin (ACGM) at 5 mg/ml reaction mixture inhibited the transfer of Gal to LGBn (25.2 and 53.4% respectively for 2 and 18 h incubation at 37 degrees C); inhibition by LGBn was 13.4 and 24.5%, respectively. In contrast to the inhibition by ACGM (25.2-31.6%), there was substantial increase (13.4-35.7%) in the inhibition by LGBn, when the incubation for 2 h at 37 degrees C was continued for 40 h at 4 degrees C, indicating the high affinity of LGBn for the enzyme at lower temp. Km for LGBn in presence of ACGM was 7.6 mM and in absence, 2.7 mM; Km for ACGM (M(r) 200,000) in presence of LGBn was 16.1 microM and Ki for ACGM (as the inhibitor) was 41.7 microM. In comparison with two normal ovarian tissues, the enzyme was found to be low (55-67%) in three ovarian tumors and high (146-260%) in two ovarian and one uterus tumors, as measured with ACGM; the synthetic acceptors showed similar activities. The enzyme had nearly the same extent of activity in the pH range 6-8. Fuc alpha 1,3GlcNAc beta 1,6GalNAc alpha-O-ONP had the highest affinity for the enzyme. The present study demonstrates the feasibility of beta 1,3Gal attachment on alpha GalNAc, which has already been substituted by beta 1,6GlcNAc, then elongated by beta 1,4Gal and also terminated by alpha 1,3Fuc. PMID:1400309

Chandrasekaran, E V; Jain, R K; Matta, K L



A systematic approach to the analysis of protein phosphorylation.  


Reversible protein phosphorylation has been known for some time to control a wide range of biological functions and activities. Thus determination of the site(s) of protein phosphorylation has been an essential step in the analysis of the control of many biological systems. However, direct determination of individual phosphorylation sites occurring on phosphoproteins in vivo has been difficult to date, typically requiring the purification to homogeneity of the phosphoprotein of interest before analysis. Thus, there has been a substantial need for a more rapid and general method for the analysis of protein phosphorylation in complex protein mixtures. Here we describe such an approach to protein phosphorylation analysis. It consists of three steps: (1) selective phosphopeptide isolation from a peptide mixture via a sequence of chemical reactions, (2) phosphopeptide analysis by automated liquid chromatography-tandem mass spectrometry (LC-MS/MS), and (3) identification of the phosphoprotein and the phosphorylated residue(s) by correlation of tandem mass spectrometric data with sequence databases. By utilizing various phosphoprotein standards and a whole yeast cell lysate, we demonstrate that the method is equally applicable to serine-, threonine- and tyrosine-phosphorylated proteins, and is capable of selectively isolating and identifying phosphopeptides present in a highly complex peptide mixture. PMID:11283598

Zhou, H; Watts, J D; Aebersold, R



Signal processing by protein tyrosine phosphorylation in plants  

PubMed Central

Protein phosphorylation is a reversible post-translational modification controlling many biological processes. Most phosphorylation occurs on serine and threonine, and to a less extend on tyrosine (Tyr). In animals, Tyr phosphorylation is crucial for the regulation of many responses such as growth or differentiation. Only recently with the development of mass spectrometry, it has been reported that Tyr phosphorylation is as important in plants as in animals. The genes encoding protein Tyr kinases and protein Tyr phosphatases have been identified in the Arabidopsis thaliana genome. Putative substrates of these enzymes, and thus Tyr-phosphorylated proteins have been reported by proteomic studies based on accurate mass spectrometry analysis of the phosphopeptides and phosphoproteins. Biochemical approaches, pharmacology and genetic manipulations have indicated that responses to stress and developmental processes involve changes in protein Tyr phosphorylation. The aim of this review is to present an update on Tyr phosphorylation in plants in order to better assess the role of this post-translational modification in plant physiology. PMID:21628997



Systematic Phosphorylation Analysis of Human Mitotic Protein Complexes  

PubMed Central

Progression through mitosis depends on a large number of protein complexes that regulate the major structural and physiological changes necessary for faithful chromosome segregation. Most, if not all, of the mitotic processes are regulated by a set of mitotic protein kinases that control protein activity by phosphorylation. Although many mitotic phosphorylation events have been identified in proteome-scale mass spectrometry studies, information on how these phosphorylation sites are distributed within mitotic protein complexes and which kinases generate these phosphorylation sites is largely lacking. We used systematic protein-affinity purification combined with mass spectrometry to identify 1818 phosphorylation sites in more than 100 mitotic protein complexes. In many complexes the phosphorylation sites were concentrated on a few subunits, suggesting that these subunits serve as “switchboards” to relay the kinase-regulatory signals within the complexes. Consequent bioinformatic analyses identified potential kinase – substrate relationships for most of these sites. In a subsequent in-depth analysis of key mitotic regulatory complexes using the Aurora kinase B (AURKB) inhibitor Hesperadin and a new Pololike kinase (PLK1) inhibitor, BI 4834, we determined the kinase-dependency for 172 phosphorylation sites on 41 proteins. Combination of the results of the cellular studies with Scansite motif prediction enabled us to identify 14 sites on 6 proteins as direct candidate substrates of AURKB or PLK1. PMID:22067460

Hegemann, Bjorn; Hutchins, James R.A.; Hudecz, Otto; Novatchkova, Maria; Rameseder, Jonathan; Sykora, Martina M.; Liu, Sihan; Mazanek, Michael; Lenart, Peter; Heriche, Jean-Karim; Poser, Ina; Kraut, Norbert; Hyman, Anthony A.; Yaffe, Michael B.; Mechtler, Karl; Peters, Jan-Michael



Systematic phosphorylation analysis of human mitotic protein complexes.  


Progression through mitosis depends on a large number of protein complexes that regulate the major structural and physiological changes necessary for faithful chromosome segregation. Most, if not all, of the mitotic processes are regulated by a set of mitotic protein kinases that control protein activity by phosphorylation. Although many mitotic phosphorylation events have been identified in proteome-scale mass spectrometry studies, information on how these phosphorylation sites are distributed within mitotic protein complexes and which kinases generate these phosphorylation sites is largely lacking. We used systematic protein-affinity purification combined with mass spectrometry to identify 1818 phosphorylation sites in more than 100 mitotic protein complexes. In many complexes, the phosphorylation sites were concentrated on a few subunits, suggesting that these subunits serve as "switchboards" to relay the kinase-regulatory signals within the complexes. Consequent bioinformatic analyses identified potential kinase-substrate relationships for most of these sites. In a subsequent in-depth analysis of key mitotic regulatory complexes with the Aurora kinase B (AURKB) inhibitor Hesperadin and a new Polo-like kinase (PLK1) inhibitor, BI 4834, we determined the kinase dependency for 172 phosphorylation sites on 41 proteins. Combination of the results of the cellular studies with Scansite motif prediction enabled us to identify 14 sites on six proteins as direct candidate substrates of AURKB or PLK1. PMID:22067460

Hegemann, Björn; Hutchins, James R A; Hudecz, Otto; Novatchkova, Maria; Rameseder, Jonathan; Sykora, Martina M; Liu, Sihan; Mazanek, Michael; Lénárt, Péter; Hériché, Jean-Karim; Poser, Ina; Kraut, Norbert; Hyman, Anthony A; Yaffe, Michael B; Mechtler, Karl; Peters, Jan-Michael



A Systems Model of Phosphorylation for Inflammatory Signaling Events  

PubMed Central

Phosphorylation is a fundamental biochemical reaction that modulates protein activity in cells. While a single phosphorylation event is relatively easy to understand, multisite phosphorylation requires systems approaches for deeper elucidation of the underlying molecular mechanisms. In this paper we develop a mechanistic model for single- and multi-site phosphorylation. The proposed model is compared with previously reported studies. We compare the predictions of our model with experiments published in the literature in the context of inflammatory signaling events in order to provide a mechanistic description of the multisite phosphorylation-mediated regulation of Signal Transducer and Activator of Transcription 3 (STAT3) and Interferon Regulatory Factor 5 (IRF-5) proteins. The presented model makes crucial predictions for transcription factor phosphorylation events in the immune system. The model proposes potential mechanisms for T cell phenotype switching and production of cytokines. This study also provides a generic framework for the better understanding of a large number of multisite phosphorylation-regulated biochemical circuits. PMID:25333362

Sadreev, Ildar I.; Chen, Michael Z. Q.; Welsh, Gavin I.; Umezawa, Yoshinori; Kotov, Nikolay V.; Valeyev, Najl V.



A systems model of phosphorylation for inflammatory signaling events.  


Phosphorylation is a fundamental biochemical reaction that modulates protein activity in cells. While a single phosphorylation event is relatively easy to understand, multisite phosphorylation requires systems approaches for deeper elucidation of the underlying molecular mechanisms. In this paper we develop a mechanistic model for single- and multi-site phosphorylation. The proposed model is compared with previously reported studies. We compare the predictions of our model with experiments published in the literature in the context of inflammatory signaling events in order to provide a mechanistic description of the multisite phosphorylation-mediated regulation of Signal Transducer and Activator of Transcription 3 (STAT3) and Interferon Regulatory Factor 5 (IRF-5) proteins. The presented model makes crucial predictions for transcription factor phosphorylation events in the immune system. The model proposes potential mechanisms for T cell phenotype switching and production of cytokines. This study also provides a generic framework for the better understanding of a large number of multisite phosphorylation-regulated biochemical circuits. PMID:25333362

Sadreev, Ildar I; Chen, Michael Z Q; Welsh, Gavin I; Umezawa, Yoshinori; Kotov, Nikolay V; Valeyev, Najl V



Biofabricated film with enzymatic and redox-capacitor functionalities to harvest and store electrons.  


Exciting opportunities in bioelectronics will be facilitated by materials that can bridge the chemical logic of biology and the digital logic of electronics. Here we report the fabrication of a dual functional hydrogel film that can harvest electrons from its chemical environment and store these electrons by switching the film's redox-state. The hydrogel scaffold was formed by the anodic deposition of the aminopolysaccharide chitosan. Electron-harvesting function was conferred by co-depositing the enzyme glucose dehydrogenase (GDH) with chitosan. GDH catalyzes the transfer of electrons from glucose to the soluble redox-shuttle NADP(+). Electron-storage function was conferred by the redox-active food phenolic chlorogenic acid (CA) that was enzymatically grafted to the chitosan scaffold using tyrosinase. The grafted CA undergoes redox-cycling reactions with NADPH resulting in the net transfer of electrons to the film where they are stored in the reduced state of CA. The individual and dual functionalities of these films were demonstrated experimentally. There are three general conclusions from this proof-of-concept study. First, enzymatically-grafted catecholic moieties confer redox-capacitor function to the chitosan scaffold. Second, biological materials (i.e. chitosan and CA) and mechanisms (i.e. tyrosinase-mediated grafting) allow the reagentless fabrication of functional films that should be environmentally-friendly, safe and potentially even edible. Finally, the film's ability to mediate the transfer of electrons from a biological metabolite to an electrode suggests an approach to bridge the chemical logic of biology with the digital logic of electronics. PMID:23303212

Liba, Benjamin D; Kim, Eunkyoung; Martin, Alexandra N; Liu, Yi; Bentley, William E; Payne, Gregory F



Measuring brain glucose phosphorylation with labeled glucose  

SciTech Connect

This study tested whether glucose labeled at the C-6 position generates metabolites that leave brain so rapidly that C-6-labeled glucose cannot be used to measure brain glucose phosphorylation (CMRGlc). In pentobarbital-anesthetized rats, the parietal cortex uptake of (/sup 14/C)glucose labeled in the C-6 position was followed for times ranging from 10 s to 60 min. We subtracted the observed radioactivity from the radioactivity expected with no loss of labeled metabolites from brain by extrapolation of glucose uptake in an initial period when loss was negligible. The observed radioactivity was a monoexponentially declining function of the total radioactivity expected in the absence of metabolite loss. The constant of decline was 0.0077.min-1 for parietal cortex. Metabolites were lost from the beginning of the experiment. However, with correction for the loss of labeled metabolites, it was possible to determine an average CMRGlc between 4 and 60 min of circulation of 64 +/- 4 (SE; n = 49) mumol.hg-1.min-1.

Brondsted, H.E.; Gjedde, A.



Modelling auxin efflux carrier phosphorylation and localization.  


Regulation of the activity and localization of PIN-FORMED (PIN) membrane proteins, which facilitate efflux of the plant hormone auxin from cells, is important for plants to respond to environmental stimuli and to develop new organs. The protein kinase PINOID (PID) is involved in regulating PIN phosphorylation, and this is thought to affect PIN localization by biasing recycling towards shootwards (apical) (rather than rootwards (basal)) membrane domains. PID has been observed to undergo transient internalization following auxin treatment, and it has been suggested that this may be a result of calcium-dependent sequestration of PID by the calcium-binding protein TOUCH3 (TCH3). We present a mathematical formulation of these processes and examine the resulting steady-state and time-dependent behaviours in response to transient increases in cytosolic calcium. We further combine this model with one for the recycling of PINs in polarized cells and also examine its behaviour. The results provide insight into the behaviour observed experimentally and provide the basis for subsequent studies of the tissue-level implications of these subcellular processes for phenomena such as gravitropism. PMID:23160141

Fozard, J A; King, J R; Bennett, M J



Rosamines Targeting the Cancer Oxidative Phosphorylation Pathway  

PubMed Central

Reprogramming of energy metabolism is pivotal to cancer, so mitochondria are potential targets for anticancer therapy. A prior study has demonstrated the anti-proliferative activity of a new class of mitochondria-targeting rosamines. This present study describes in vitro cytotoxicity of second-generation rosamine analogs, their mode of action, and their in vivo efficacies in a tumor allografted mouse model. Here, we showed that these compounds exhibited potent cytotoxicity (average IC50<0.5 µM), inhibited Complex II and ATP synthase activities of the mitochondrial oxidative phosphorylation pathway and induced loss of mitochondrial transmembrane potential. A NCI-60 cell lines screen further indicated that rosamine analogs 4 and 5 exhibited potent antiproliferative effects with Log10GI50?=??7 (GI50?=?0.1 µM) and were more effective against a colorectal cancer sub-panel than other cell lines. Preliminary in vivo studies on 4T1 murine breast cancer-bearing female BALB/c mice indicated that treatment with analog 5 in a single dosing of 5 mg/kg or a schedule dosing of 3 mg/kg once every 2 days for 6 times (q2d×6) exhibited only minimal induction of tumor growth delay. Our results suggest that rosamine analogs may be further developed as mitochondrial targeting agents. Without a doubt proper strategies need to be devised to enhance tumor uptake of rosamines, i.e. by integration to carrier molecules for better therapeutic outcome. PMID:24622277

Lim, Siang Hui; Wu, Liangxing; Kiew, Lik Voon; Chung, Lip Yong; Burgess, Kevin; Lee, Hong Boon



Sirt3 Regulates Metabolic Flexibility of Skeletal Muscle Through Reversible Enzymatic Deacetylation  

PubMed Central

Sirt3 is an NAD+-dependent deacetylase that regulates mitochondrial function by targeting metabolic enzymes and proteins. In fasting mice, Sirt3 expression is decreased in skeletal muscle resulting in increased mitochondrial protein acetylation. Deletion of Sirt3 led to impaired glucose oxidation in muscle, which was associated with decreased pyruvate dehydrogenase (PDH) activity, accumulation of pyruvate and lactate metabolites, and an inability of insulin to suppress fatty acid oxidation. Antibody-based acetyl-peptide enrichment and mass spectrometry of mitochondrial lysates from WT and Sirt3 KO skeletal muscle revealed that a major target of Sirt3 deacetylation is the E1? subunit of PDH (PDH E1?). Sirt3 knockout in vivo and Sirt3 knockdown in myoblasts in vitro induced hyperacetylation of the PDH E1? subunit, altering its phosphorylation leading to suppressed PDH enzymatic activity. The inhibition of PDH activity resulting from reduced levels of Sirt3 induces a switch of skeletal muscle substrate utilization from carbohydrate oxidation toward lactate production and fatty acid utilization even in the fed state, contributing to a loss of metabolic flexibility. Thus, Sirt3 plays an important role in skeletal muscle mitochondrial substrate choice and metabolic flexibility in part by regulating PDH function through deacetylation. PMID:23835326

Jing, Enxuan; O'Neill, Brian T.; Rardin, Matthew J.; Kleinridders, Andre; Ilkeyeva, Olga R.; Ussar, Siegfried; Bain, James R.; Lee, Kevin Y.; Verdin, Eric M.; Newgard, Christopher B.; Gibson, Bradford W.; Kahn, C. Ronald



Silicon nanowires as a rechargeable template for hydride transfer in redox biocatalysis  

NASA Astrophysics Data System (ADS)

We report a new possible application of hydrogen-terminated silicon nanowires (H-SiNWs) as a rechargeable template for hydride transfer in redox biocatalysis. H-SiNWs transfer hydride efficiently to regenerate NADH by oxidizing Si-Hx bonds. The oxidized H-SiNWs were readily recharged for the continuous regeneration of NADH and enzymatic reactions.

Lee, Hwa Young; Kim, Jae Hong; Son, Eun Jin; Park, Chan Beum



Cyanogen induced phosphorylation of D-fructose. [prebiotic modeling  

NASA Technical Reports Server (NTRS)

It has been demonstrated that a phosphorylated sugar, identified as alpha-D-fructopyranose, can be formed as the result of cyanogen-induced phosphorylation of D-fructose at pH 8.8. The product was isolated from barium and cyclohexylammonium salts and identified on the basis of its chromatographic and electrophoretic properties, its lability to hydrolysis by alkaline phosphatase, the rate of its acid-catalyzed hydrolysis, and the results of periodate oxidation and optical rotatory measurements. These results support the suggestion that the cyanogen-induced phosphorylation of free sugars could be a possible process for formation of sugar phosphates under prebiotic conditions (Halman et al., 1969).

Degani, CH.; Kawatsuji, M.; Halmann, M.



Correlation between changes in light energy distribution and changes in thylakoid membrane polypeptide phosphorylation in Chlamydomonas reinhardtii  

SciTech Connect

We have used a new method to extensively modify the redox state of the plastoquinone pool in Chlamydomonas reinhardtii intact cells. This was achieved by an anaerobic treatment that inhibits the chlororespiratory pathway recently described by P. Bennoun. A state I (plus 3,4-dichlorophenyl-1,1-dimethylurea) ..-->.. anaerobic state transition induced a decrease in the maximal fluorescence yield at room temperature and in the F/sub PSII//F/sub PSI/ ratio at 77/sup 0/K, which was three times larger than in a classical state I ..-->.. state II transition. The fluorescence changes observed in vivo were similar in amplitude to those observed in vitro upon transfer to the light of dark-adapted, broken chloroplasts incubated in the presence of ATP. We then compared the phosphorylation pattern of thylakoid polypeptides in C. reinhardtii in vitro and in vivo using ..gamma..-(/sup 32/P)ATP and (/sup 32/P)orthophosphate labeling, respectively. The same set of polypeptides, mainly light-harvesting complex polypeptides, was phosphorylated in both cases. We observed that this phosphorylation process is reversible and is mediated by the redox state of the plastoquinone pool in vivo as well as in vitro. Similar changes of even larger amplitude were observed with the F34 mutant intact cells lacking in photosystem II centers. The presence of the photosystem II centers is then not required for the occurrence of the plastoquinone-mediated phosphorylation of light-harvesting complex polypeptides.

Wollman, F.A.; Delepelaire, P.



A comparison of glucose oxidase and aldose dehydrogenase as mediated anodes in printed glucose/oxygen enzymatic fuel cells using ABTS/laccase cathodes.  


Current generation by mediated enzyme electron transfer at electrode surfaces can be harnessed to provide biosensors and redox reactions in enzymatic fuel cells. A glucose/oxygen enzymatic fuel cell can provide power for portable and implantable electronic devices. High volume production of enzymatic fuel cell prototypes will likely require printing of electrode and catalytic materials. Here we report on preparation and performance of, completely enzymatic, printed glucose/oxygen biofuel cells. The cells are based on filter paper coated with conducting carbon inks, enzyme and mediator. A comparison of cell performance using a range of mediators for either glucose oxidase (GOx) or aldose dehydrogenase (ALDH) oxidation of glucose at the anode and ABTS and a fungal laccase, for reduction of oxygen at the cathode, is reported. Highest power output, although of limited stability, is observed for ALDH anodes mediated by an osmium complex, providing a maximum power density of 3.5 ?W cm(-2) at 0.34 V, when coupled to a laccase/ABTS cathode. The stability of cell voltage in a biobattery format, above a threshold of 200 mV under a moderate 75 k? load, is used to benchmark printed fuel cell performance. Highest stability is obtained for printed fuel cells using ALDH, providing cell voltages over the threshold for up to 74 h, compared to only 2 h for cells with anodes using GOx. These results provide promising directions for further development of mass-producible, completely enzymatic, printed biofuel cells. PMID:22200380

Jenkins, Peter; Tuurala, Saara; Vaari, Anu; Valkiainen, Matti; Smolander, Maria; Leech, Dónal



Redox polymer mediation for enzymatic biofuel cells  

NASA Astrophysics Data System (ADS)

Mediated biocatalytic cathodes prepared from the oxygen-reducing enzyme laccase and redox-conducting osmium hydrogels were characterized for use as cathodes in enzymatic biofuel cells. A series of osmium-based redox polymers was synthesized with redox potentials spanning the range from 0.11 V to 0.85 V (SHE), and the resulting biocatalytic electrodes were modeled to determine reaction kinetic constants using the current response, measured osmium concentration, and measured apparent electron diffusion. As in solution-phase systems, the bimolecular rate constant for mediation was found to vary greatly with mediator potential---from 250 s-1M-1 when mediator and enzyme were close in potential to 9.4 x 10 4 s-1M-1 when this overpotential was large. Optimum mediator potential for a cell operating with a non-limiting platinum anode and having no mass transport limitation from bulk solution was found to be 0.66 V (SHE). Redox polymers were synthesized under different concentrations, producing osmium variation. An increase from 6.6% to 7.2% osmium increased current response from 1.2 to 2.1 mA/cm2 for a planar film in 40°C oxygen-saturated pH 4 buffer, rotating at 900 rpm. These results translated to high surface area electrodes, nearly doubling current density to 13 mA/cm2, the highest to date for such an electrode. The typical fungal laccase from Trametes versicolor was replaced by a bacterially-expressed small laccase from Streptomyces coelicolor, resulting in biocatalytic films that reduced oxygen at increased pH, with full functionality at pH 7, producing 1.5 mA/cm 2 in planar configuration. Current response was biphasic with pH, matching the activity profile of the free enzyme in solution. The mediated enzyme electrode system was modeled with respect to apparent electron diffusion, mediator concentration, and transport of oxygen from bulk solution, all of which are to some extent controlled by design. Each factor was found to limit performance in certain circumstances. In systems relying on stagnant solution, oxygen transport was found to dominate. However, if mass transport was efficient, differences in mediator design greatly affected performance.

Gallaway, Joshua


Effects of permeant buffers on the initiation of photosynchronous phosphorylation and postillumination phosphorylation in chloroplasts.  


Under canonical chemiosmotic formulations, the development of a delocalized transmembrane proton gradient should precede and, in the absence of a membrane potential, should account for all the capacity of an energy transducing system to synthesize ATP. Furthermore, any agents, such as permeant proton-absorbing buffers, that slow down the kinetics of the development of this gradient should, consequently, delay ATP synthesis. We have studied the very early (0 through 1000 ms) steps of photosynthetic ATP synthesis utilizing real-time, rapid flow-quench techniques. We have investigated the effect(s) that permeant buffers exert on this process where these buffers show no uncoupling effects, and the transmembrane potential has been collapsed by valinomycin and K+. Experimentally this system was dissected into two ATP synthesizing components, as follows: synthesis of ATP strictly concomitant with light influx and unaffected by the addition of permeant buffers. We refer to this as photosynchronous phosphorylation and synthesis of ATP monitored after the light was extinguished and which was greatly diminished by the addition of proton-absorbing permeant buffers, thus exhibiting the characteristics of conventional postillumination phosphorylation, and we suggest that it represents part of capacitance phosphorylation. The potential for capacitance phosphorylation initiates very rapidly under light and gradually builds up to steady-state level, and it is governed by canonical chemiosmotic principles. We estimate that its contribution to overall ATP yield is minimal during the first few cycles of the system and that it increases gradually towards steady state when it contributes to the majority of ATP synthesized. Neither a delocalized transmembrane proton gradient nor a strictly localized intramembrane proton pathway can account for these observations so we have proposed that a gating mechanism exists which delivers intramembrane protons initially directly to the ATP synthetase complex but subsequently to the lumen as well, and thus, allows the lumen to act as a capacitor during the steady state. This study can reconcile the findings of Ort et al. (Ort, D. R., Dilley, R. A., and Good, N. E. (1976) Biochim. Biophys. Acta 449, 108-124) with the contrasting findings of Vinkler et al. (Vinkler, C., Avron, M., and Boyer, P. D. (1980) J. Biol. Chem. 255, 2263-2266) through the opposite effects which osmotic strength and KCl concentration exert on the two ATP synthetic phases (during and after illumination) of the rapid flash technique used in those studies.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2875995

Horner, R D; Moudrianakis, E N



Ion-radical mechanism of enzymatic ATP synthesis: DFT calculations and experimental control.  


A new, ion-radical mechanism of enzymatic ATP synthesis was recently discovered by using magnesium isotopes. It functions at a high concentration of MgCl(2) and includes electron transfer from the Mg(H(2)O)(m)(2+)(ADP(3-)) complex (m = 0-4) to the Mg(H(2)O)(n)(2+) complex as a primary reaction of ATP synthesis in catalytic sites of ATP synthase and kinases. Here, the structures and electron transfer reaction energies of magnesium complexes related to ATP synthesis are calculated in terms of DFT. ADP is modeled by pyrophosphate anions, protonated (HP(2)O(7)H(2-), HP(2)O(7)CH(3)(2-)) and deprotonated (HP(2)O(7)(3-), CH(3)P(2)O(7)(3-)). The reaction generates an ion-radical pair, composed of Mg(H(2)O)(n)(+) ion and pyrophosphate anion-radical coordinated to Mg(2+) ion. The addition of the latter to the substrate P=O bond results in ATP formation. Populations of the singlet and triplet states and singlet-triplet spin conversion in the pair are controlled by hyperfine coupling of unpaired electrons with magnetic (25)Mg and (31)P nuclei and by Zeeman interaction. Due to these two interactions, the yield of ATP is a function of nuclear magnetic moment and magnetic field; both of these effects were experimentally detected. Electron transfer reaction does not depend on m but strongly depends on n. It is exoergic and energy allowed at 0 < or = n < infinity for the deprotonated pyrophosphate anions and at 0 < or = n < 4 for the protonated ones; for other values of n, the reaction is energy deficient and forbidden. The boundary between exoergic and endoergic regimes corresponds to the trigger magnitude n* (n* = 4 for protonated anions and 6 < n* < infinity for deprotonated ones). These results explain why ATP synthesis occurs only in special devices, molecular enzymatic machines, but not in water (n = infinity). Biomedical consequences of the ion-radical enzymatic ATP synthesis are also discussed. PMID:20095588

Buchachenko, Anatoly L; Kuznetsov, Dmitry A; Breslavskaya, Natalia N



Stereochemistry and Mechanism of Enzymatic and Non-Enzymatic Hydrolysis of Benzylic sec-Sulfate Esters  

PubMed Central

The substrate scope of inverting alkylsulfatase Pisa1 was extended towards benzylic sec-sulfate esters by suppression of competing non-enzymatic autohydrolysis by addition of dimethyl sulfoxide as co-solvent. Detailed investigation of the mechanism of autohydrolysis in 18O-labeled buffer by using an enantiopure sec-benzylic sulfate ester as substrate revealed that from the three possible pathways (i) inverting SN2-type nucleophilic attack of [OH?] at the benzylic carbon represents the major pathway, whereas (ii) SN1-type formation of a planar benzylic carbenium ion leading to racemization was a minor event, and (iii) Retaining SN2-type nucleophilic attack at sulfur took place at the limits of detection. The data obtained are interpreted by analysis of Hammett constants of meta substituents. PMID:25232289

Toesch, Michael; Schober, Markus; Breinbauer, Rolf; Faber, Kurt



Computational enzymatic catalysis--clarifying enzymatic mechanisms with the help of computers.  


Enzymes play a biologically essential role in performing and controlling an important share of the chemical processes occurring in life. However, despite their critical role in nature, attaining a clear understanding of the way an enzyme acts, i.e. its catalytic mechanism, is a cumbersome task that requires the cooperative efforts of a large number of different scientific techniques. Computational methods offer a particularly insightful way to study such mechanisms, always beautifully complementing the information arising from experimental techniques and working as an excellent alternative for assessing the viability of different mechanistic proposals. This review highlights two important computational strategies to study enzymatic catalysis - the cluster modeling approach and the hybrid quantum mechanical/molecular mechanical (QM/MM) method - complemented with a selection of hand-picked examples of our own work. PMID:22870506

Sousa, Sérgio Filipe; Fernandes, Pedro Alexandrino; Ramos, Maria João



Methods for generating phosphorylation site-specific immunological reagents  


The present invention provides methods for generating phosphorylation site-specific immunological reagents. More specifically, a phosphopeptide mimetic is incorporated into a polypeptide in place of a phosphorylated amino acid. The polypeptide is used as antigen by standard methods to generate either monoclonal or polyclonal antibodies which cross-react with the naturally phosphorylated polypeptide. The phosphopeptide mimetic preferably contains a non-hydrolyzable linkage from the appropriate carbon atom of the amino acid residue to a phosphate group. A preferred linkage is a CF.sub.2 group. Such a linkage is used to generate the phosphoserine mimetic F.sub.2 Pab, which is incorporated into a polypeptide sequence derived from p53 to produce antibodies which recognize a specific phosphorylation state of p53. A CF.sub.2 group linkage is also used to produce the phosphothreonine mimetic F.sub.2 Pmb, and to produce the phosphotyrosine mimetic, F.sub.2 Pmp.

Anderson, Carl W. (Stony Brook, NY); Appella, Ettore (Montgomery, MD); Sakaguchi, Kazuyasu (Montgomery, MD)



The importance of intrinsic disorder for protein phosphorylation  

E-print Network

important protein domains, i.e. the majority of phosphorylation sites of Mdm2 are located in its p53- and p the binding af®nity of transcription factors to their coactivators and DNA thereby altering gene expression

Radivojac, Predrag


Neurofilament protein is phosphorylated in the squid giant axon  

PubMed Central

We have observed the phosphorylation of neurofilament protein from squid axoplasm. Phosphorylation is demonstrated by 32P labeling of protein during incubation of axoplasm with [gamma-32P]ATP. When the labeled proteins are separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two bands, at 2.0 x 10(5) daltons and greater than 4 x 10(5) daltons, contain the bulk of the 32P. The 2.0 x 10(5)-dalton phosphorylated polypeptide comigrates on SDS-PAGE with one of the subunits of squid neurofilament protein. Both major phosphorylated polypeptides co-fractionate with neurofilaments in discontinuous sucrose gradient centrifugation and on gel filtration chromatography on Sepharose 4B. The protein-phosphate bond behaves like a phospho-ester, and labeled phospho-serine is identified in an acid hydrolysate of the protein. The generality of this phenomenon in various species and its possible physiological significance are discussed. PMID:690167

Pant, H. C.; Shecket, G.; Gainer, H.; Lasek, R. J.



Phosphorylation of dGMP analogs by vaccinia virus TMP kinase and human GMP kinase.  


Vaccinia virus thymidylate kinase, although similar in sequence to human TMP kinase, has broader substrate specificity and phosphorylates (E)-5-(2-bromovinyl)-dUMP and dGMP. Modified guanines such as glyoxal-dG, 8-oxo-dG, O(6)-methyl-dG, N(2)-ethyl-dG and N(7)-methyl-dG were found present in cancer cell DNA. Alkylated and oxidized dGMP analogs were examined as potential substrates for vaccinia TMP kinase and also for human TMP and GMP kinases. Molecular models obtained from structure-based docking rationalized the enzymatic data. All tested nucleotides are found surprisingly substrates of vaccinia TMP kinase and also of human GMP kinase. Interestingly, O(6)-methyl-dGMP is the only analog specific for the vaccinia enzyme. Thus, O(6)-Me-dGMP could be useful for designing new compounds of medical interest either in antipoxvirus therapy or in experimental combined gene/chemotherapy of cancer. These results also provide new insights regarding dGMP analog reaction with human GMP kinase and their slow recycling by salvage pathway nucleotide kinases. PMID:19631609

Auvynet, Constance; Topalis, Dimitri; Caillat, Christophe; Munier-Lehmann, Hélène; Seclaman, Edward; Balzarini, Jan; Agrofoglio, Luigi André; Kaminski, Pierre Alexandre; Meyer, Philippe; Deville-Bonne, Dominique; El Amri, Chahrazade



Mengovirus-induced rearrangement of the nuclear pore complex: hijacking cellular phosphorylation machinery.  


Representatives of several picornavirus genera have been shown previously to significantly enhance non-controllable bidirectional exchange of proteins between nuclei and cytoplasm. In enteroviruses and rhinoviruses, enhanced permeabilization of the nuclear pores appears to be primarily due to proteolytic degradation of some nucleoporins (protein components of the pore), whereas this effect in cardiovirus-infected cells is triggered by the leader (L) protein, devoid of any enzymatic activities. Here, we present evidence that expression of L alone was sufficient to cause permeabilization of the nuclear envelope in HeLa cells. In contrast to poliovirus, mengovirus infection of these cells did not elicit loss of nucleoporins Nup62 and Nup153 from the nuclear pore complex. Instead, nuclear envelope permeabilization was accompanied by hyperphosphorylation of Nup62 in cells infected with wild-type mengovirus, whereas both of these alterations were suppressed in L-deficient virus mutants. Since phosphorylation of Nup62 (although less prominent) did accompany permeabilization of the nuclear envelope prior to its mitotic disassembly in uninfected cells, we hypothesize that cardiovirus L alters the nucleocytoplasmic traffic by hijacking some components of the normal cell division machinery. The variability and biological significance of picornaviral interactions with the nucleocytoplasmic transport in the infected cells are discussed. PMID:19144712

Bardina, Maryana V; Lidsky, Peter V; Sheval, Eugene V; Fominykh, Ksenia V; van Kuppeveld, Frank J M; Polyakov, Vladimir Y; Agol, Vadim I



Mengovirus-Induced Rearrangement of the Nuclear Pore Complex: Hijacking Cellular Phosphorylation Machinery?  

PubMed Central

Representatives of several picornavirus genera have been shown previously to significantly enhance noncontrollable bidirectional exchange of proteins between nuclei and cytoplasm. In enteroviruses and rhinoviruses, enhanced permeabilization of the nuclear pores appears to be primarily due to proteolytic degradation of some nucleoporins (protein components of the pore), whereas this effect in cardiovirus-infected cells is triggered by the leader (L) protein, devoid of any enzymatic activities. Here, we present evidence that expression of L alone was sufficient to cause permeabilization of the nuclear envelope in HeLa cells. In contrast to poliovirus, mengovirus infection of these cells did not elicit loss of nucleoporins Nup62 and Nup153 from the nuclear pore complex. Instead, nuclear envelope permeabilization was accompanied by hyperphosphorylation of Nup62 in cells infected with wild-type mengovirus, whereas both of these alterations were suppressed in L-deficient virus mutants. Since phosphorylation of Nup62 (although less prominent) did accompany permeabilization of the nuclear envelope prior to its mitotic disassembly in uninfected cells, we hypothesize that cardiovirus L alters the nucleocytoplasmic traffic by hijacking some components of the normal cell division machinery. The variability and biological significance of picornaviral interactions with the nucleocytoplasmic transport in the infected cells are discussed. PMID:19144712

Bardina, Maryana V.; Lidsky, Peter V.; Sheval, Eugene V.; Fominykh, Ksenia V.; van Kuppeveld, Frank J. M.; Polyakov, Vladimir Y.; Agol, Vadim I.



Molybdenum in enzymatic and heterogeneous catalysis.  


The chemistry common to molybdenum at the active centers of molybdoenzymes and at the surface of heterogeneous catalysts is described. Oxomolybdenum(VI) compounds catalyze selective oxidation of unsaturated hydrocarbons, e.g., propene to acrolein. Similarly, oxomolybdenum species take part in reactions catalyzed by molybdoenzymes, e.g., xanthine oxidase, sulfite oxidase, nitrate reductase. In these reactions H+, O2- or HO-, and electrons transfer between substrate molecules and molybdenum atoms and groups at the active centres. The chemistry involved is the acid-base and redox chemistry of molybdenum. Molybdenum disulfide catalyzes hydrogenation of unsaturated hydrocarbons, e.g., acetylene. The active site is a coordinately unsaturated molybdenum atom in a sulfur-ligand environment. The enzyme nitrogenase, which is a protein-bound iron-molybdenum sulfide, is also an excellent hydrogenation catalyst. Both catalysts exploit the chemistry of lower-valent molybdenum coordinated by sulfur. The extent to which understanding of the catalysis can be transferred between the two types of catalyst is assessed. PMID:3806088

Mitchell, P C



Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells  

NASA Astrophysics Data System (ADS)

Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2 min in cells seeded on low fibronectin concentration ([FN]) and 4.3 min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin ?v?3, but not in those by integrin ?5?1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.

Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-Chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y.-S.; Chien, Shu; Wang, Yingxiao



Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells  

PubMed Central

Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2?min in cells seeded on low fibronectin concentration ([FN]) and 4.3?min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin ?v?3, but not in those by integrin ?5?1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics. PMID:25056908

Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y.-S.; Chien, Shu; Wang, Yingxiao



Restoration of Gibberellin Production in Fusarium proliferatum by Functional Complementation of Enzymatic Blocks  

PubMed Central

Nine biological species, or mating populations (MPs), denoted by letters A to I, and at least 29 anamorphic Fusarium species have been identified within the Gibberella fujikuroi species complex. Members of this species complex are the only species of the genus Fusarium that contain the gibberellin (GA) biosynthetic gene cluster or at least parts of it. However, the ability of fusaria to produce GAs is so far restricted to Fusarium fujikuroi, although at least six other MPs contain all the genes of the GA biosynthetic gene cluster. Members of Fusarium proliferatum, the closest related species, have lost the ability to produce GAs as a result of the accumulation of several mutations in the coding and 5? noncoding regions of genes P450-4 and P450-1, both encoding cytochrome P450 monooxygenases, resulting in metabolic blocks at the early stages of GA biosynthesis. In this study, we have determined additional enzymatic blocks at the first specific steps in the GA biosynthesis pathway of F. proliferatum: the synthesis of geranylgeranyl diphosphate and the synthesis of ent-kaurene. Complementation of these enzymatic blocks by transferring the corresponding genes from GA-producing F. fujikuroi to F. proliferatum resulted in the restoration of GA production. We discuss the reasons for Fusarium species outside the G. fujikuroi species complex having no GA biosynthetic genes, whereas species distantly related to Fusarium, e.g., Sphaceloma spp. and Phaeosphaeria spp., produce GAs. PMID:16204516

Malonek, S.; Rojas, M. C.; Hedden, P.; Hopkins, P.; Tudzynski, B.



Restoration of gibberellin production in Fusarium proliferatum by functional complementation of enzymatic blocks.  


Nine biological species, or mating populations (MPs), denoted by letters A to I, and at least 29 anamorphic Fusarium species have been identified within the Gibberella fujikuroi species complex. Members of this species complex are the only species of the genus Fusarium that contain the gibberellin (GA) biosynthetic gene cluster or at least parts of it. However, the ability of fusaria to produce GAs is so far restricted to Fusarium fujikuroi, although at least six other MPs contain all the genes of the GA biosynthetic gene cluster. Members of Fusarium proliferatum, the closest related species, have lost the ability to produce GAs as a result of the accumulation of several mutations in the coding and 5' noncoding regions of genes P450-4 and P450-1, both encoding cytochrome P450 monooxygenases, resulting in metabolic blocks at the early stages of GA biosynthesis. In this study, we have determined additional enzymatic blocks at the first specific steps in the GA biosynthesis pathway of F. proliferatum: the synthesis of geranylgeranyl diphosphate and the synthesis of ent-kaurene. Complementation of these enzymatic blocks by transferring the corresponding genes from GA-producing F. fujikuroi to F. proliferatum resulted in the restoration of GA production. We discuss the reasons for Fusarium species outside the G. fujikuroi species complex having no GA biosynthetic genes, whereas species distantly related to Fusarium, e.g., Sphaceloma spp. and Phaeosphaeria spp., produce GAs. PMID:16204516

Malonek, S; Rojas, M C; Hedden, P; Hopkins, P; Tudzynski, B



Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells.  


Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2 min in cells seeded on low fibronectin concentration ([FN]) and 4.3 min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin ?v??, but not in those by integrin ????. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics. PMID:25056908

Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y-S; Chien, Shu; Wang, Yingxiao



Enzymatic specificity of three ribosome-inactivating proteins against fungal ribosomes, and correlation with antifungal activity.  


Ribosome-inactivating proteins (RIPs) are enzymes that cleave a specific adenine base from the highly conserved sarcin/ricin (S/R) loop of the large ribosomal RNA, thus arresting protein synthesis at the translocation step. In the present study, we employed three RIPs to dissect the antifungal activity of RIPs as plant defense proteins. We measured the catalytic activity of RAT (the catalytic A-chain of ricin from Ricinus communis L.), saporin-S6 (from Saponaria officinalis L.), and ME (RIP from Mirabilis expansa R&P) against intact ribosomal substrates isolated from various pathogenic fungi. We further determined the enzymatic specificity of these three RIPs against fungal ribosomes, from Rhizoctonia solani Kuhn, Alternaria solani Sorauer, Trichoderma reesei Simmons and Candida albicans Berkhout, and correlated the data with antifungal activity. RAT showed the strongest toxicity against all tested fungal ribosomes, except for the ribosomes isolated from C. albicans, which were most susceptible to saporin. RAT and saporin showed higher enzymatic activity than ME against ribosomes from all of the fungal species assayed, but did not show detectable antifungal activity. In contrast, ME showed substantial inhibitory activity against fungal growth. Using N-hydroxysuccinimide-fluorescein labeling of RIPs and fluorescence microscopy, we determined that ME was targeted to the surface of fungal cells and transferred into the cells. Thus, ME caused ribosome depurination and subsequent fungal mortality. In contrast, saporin did not interact with fungal cells, correlating with its lack of antifungal activity. PMID:12447536

Park, Sang-Wook; Stevens, Noah M; Vivanco, Jorge M



Synthesis of carborane-containing phosphates by catalytic phosphorylation  

Microsoft Academic Search

The catalytic phosphorylation ofo-carboranylmethanol with different phosphorus acid chlorides has been studied. This method makes it possible to obtain esters of phosphorus acids and esters of the corresponding phosphorus acid chlorides containing carboranylmethyl groups. The catalytic phosphorylation of 1,2-bis(hydroxymethyl)-o-carborane affords both acyclic and cyclic phosphate esters. The structure of the synthesizedo-carborano[1,2-e]-1,3,2-dioxaphosphepane was confirmed by X-ray diffraction analysis.

M. I. Kabachnik; L. S. Zakharov; E. L. Gefter; G. N. Molchanova; Yu. T. Struchkov; A. I. Yanovsky; A. V. Polyakov; P. V. Petrovskii



Fission Yeast Rad52 Phosphorylation Restrains Error Prone Recombination Pathways  

PubMed Central

Rad52 is a key protein in homologous recombination (HR), a DNA repair pathway dedicated to double strand breaks and recovery of blocked or collapsed replication forks. Rad52 allows Rad51 loading on single strand DNA, an event required for strand invasion and D-loop formation. In addition, Rad52 functions also in Rad51 independent pathways because of its ability to promote single strand annealing (SSA) that leads to loss of genetic material and to promote D-loops formation that are cleaved by Mus81 endonuclease. We have previously reported that fission yeast Rad52 is phosphorylated in a Sty1 dependent manner upon oxidative stress and in cells where the early step of HR is impaired because of lack of Rad51. Here we show that Rad52 is also constitutively phosphorylated in mus81 null cells and that Sty1 partially impinges on such phosphorylation. As upon oxidative stress, the Rad52 phosphorylation in rad51 and mus81 null cells appears to be independent of Tel1, Rad3 and Cdc2. Most importantly, we show that mutating serine 365 to glycine (S365G) in Rad52 leads to loss of the constitutive Rad52 phosphorylation observed in cells lacking Rad51 and to partial loss of Rad52 phosphorylation in cells lacking Mus81. Contrariwise, phosphorylation of Rad52-S365G protein is not affected upon oxidative stress. These results indicate that different Rad52 residues are phosphorylated in a Sty1 dependent manner in response to these distinct situations. Analysis of spontaneous HR at direct repeats shows that mutating serine 365 leads to an increase in spontaneous deletion-type recombinants issued from mitotic recombination that are Mus81 dependent. In addition, the recombination rate in the rad52-S365G mutant is further increased by hydroxyurea, a drug to which mutant cells are sensitive. PMID:24748152

Bellini, Angela; Girard, Pierre-Marie; Tessier, Ludovic; Sage, Evelyne; Francesconi, Stefania



Akt regulates growth by directly phosphorylating Tsc2  

Microsoft Academic Search

The direct mechanism by which the serine\\/threonine kinase Akt (also known as protein kinase B (PKB)) regulates cell growth is unknown. Here, we report that Drosophila melanogaster Akt\\/PKB stimulates growth by phosphorylating the tuberous sclerosis complex 2 (Tsc2) tumour suppressor and inhibiting formation of a Tsc1–Tsc2 complex. We show that Akt\\/PKB directly phosphorylates Drosophila Tsc2 in vitro at the conserved

Christopher J. Potter; Laura G. Pedraza; Tian Xu



Contractile protein phosphorylation predicts human heart disease phenotypes  

PubMed Central

Human heart failure has been associated with a low level of thin-filament protein phosphorylation and an increase in calcium sensitivity of contraction relative to both “control” human heart tissue and tissue from small animal models. However, diverse strategies of human tissue procurement and the reliance on tissue obtained from subjects with end-stage heart failure suggest this may be an incomplete characterization. Therefore, we evaluated cardiac left ventricular (LV) biopsy samples from patients with aortic stenosis undergoing valve replacement who presented either with LV hypertrophy and preserved systolic function (Hyp) or with LV dilation and reduced ejection fraction (Dil). In Hyp, total troponin I (TnI) phosphorylation was markedly increased and myosin light chain 2 (MLC2) phosphorylation was unchanged relative to a control group of patients with normal LV function. Conversely, in Dil, total TnI phosphorylation was significantly reduced compared with control subjects and MLC2 phosphorylation was increased. Site-specific analysis of TnI phosphorylation revealed phenotype-specific differences such that Hyp samples demonstrated significant increases in phosphorylation at serine 22/23 and Dil samples had significant decreases at serine 43. The ratio of phosphorylation at the two sites was biased toward serine 22/23 in Hyp and toward serine 43/45 in Dil. Western blot analysis showed that protein phosphatase-1 was reduced in Hyp and protein phosphatase-2 was reduced in Dil. These data suggest that posttranslational modifications of sarcomeric proteins, both singly and in combination, are stage specific. Defining these changes in progressive heart disease may provide important diagnostic and treatment information. PMID:23564307

Fullerton, David A.; Buttrick, Peter M.



Phospho.ELM: a database of phosphorylation sites - update 2008  

Microsoft Academic Search

Phospho.ELM is a manually curated database of eukaryotic phosphorylation sites. The resource includes data collected from published literature as well as high-throughput data sets. The current release of Phospho.ELM (version 7.0, July 2007) contains 4078 phospho-protein sequences covering 12025 phospho-serine, 2362 phospho-threonine and 2083 phospho-tyrosine sites. The entries provide information about the phosphorylated proteins and the exact position of known

Francesca Diella; Cathryn M. Gould; Claudia Chica; Allegra Via; Toby J. Gibson



In vivo phosphorylation of adaptors regulates their interaction with clathrin  

Microsoft Academic Search

The coat proteins of clathrin-coated vesicles (CCV) spontaneously self-assemble in vitro, but, in vivo, their self-assembly must be regulated. To deter- mine whether phosphorylation might influence coat formation in the cell, the in vivo phosphorylation state of CCV coat proteins was analyzed. Individual compo- nents of the CCV coat were isolated by immunoprecip- itation from Madin-Darby bovine kidney cells, labeled

Andrew Wilde; Frances M. Brodsky



Cell survival after UV radiation stress in the unicellular chlorophyte Dunaliella tertiolecta is mediated by DNA repair and MAPK phosphorylation  

PubMed Central

Ultraviolet radiation (UVR) induces damage in a variety of organisms, and cells may adapt by developing repair or tolerance mechanisms to counteract such damage; otherwise, the cellular fate is cell death. Here, the effect of UVR-induced cell damage and the associated signalling and repair mechanisms by which cells are able to survive was studied in Dunaliella tertiolecta. UVR did not cause cell death, as shown by the absence of SYTOX Green-positive labelling cells. Ultrastructure analysis by transmission electron microscopy demonstrated that the cells were alive but were subjected to morphological changes such as starch accumulation, chromatin disaggregation, and chloroplast degradation. This behaviour paralleled a decrease in F v/F m and the formation of cyclobutane–pyrimidine dimers, showing a 10-fold increase at the end of the time course. There was a high accumulation of the repressor of transcriptional gene silencing (ROS1), as well as the cell proliferation nuclear antigen (PCNA) in UVR-treated cells, revealing activation of DNA repair mechanisms. The degree of phosphorylation of c-Jun N-terminal kinase (JNK) and p38-like mitogen-activated protein kinases was higher in UVR-exposed cells; however, the opposite occurred with the phosphorylated extracellular signal-regulated kinase (ERK). This confirmed that both JNK and p38 need to be phosphorylated to trigger the stress response, as well as the fact that cell division is arrested when an ERK is dephosphorylated. In parallel, both DEVDase and WEHDase caspase-like enzymatic activities were active even though the cells were not dead, suggesting that these proteases must be considered within a wider frame of stress proteins, rather than specifically being involved in cell death in these organisms. PMID:22859678

García-Gómez, Candela; Segovia, María



Multisite phosphorylation of spinach leaf sucrose-phosphate synthase  

SciTech Connect

Spinach leaf sucrose-phosphate synthase is phosphorylated both in vivo and in vitro on serine residues. Phosphorylation of SPS in vivo yields twelve major phosphopeptides after a tryptic digest and two dimensional mapping. The in vivo labeling of three of these SPS P-peptides is reduced in illuminated leaves where the extracted enzyme is activated relative to that of dark leaves. Two of these inhibitory sites are phosphorylated as well when SPS is inactivated in vitro using ({sup 32}P)ATP. In vivo phosphorylation of two other sites is enhanced during mannose feeding of the leaves (in light or dark) which produces the highest activation state of SPS. Overall, the results confirm that light-dark regulation of SPS activity occurs as a result of regulatory seryl-phosphorylation and involves a balance between phosphorylation of sites which inhibit or stimulate activity. Regulation of the SPS protein kinase that inhibits activity is relatively unaffected by phosphate but inhibited by G1c 6-P (IC{sub 50}{approx}5 mM), which may explain the control of SPS activation state by light-dark signals.

Huber, J.L.; Huber, S.C. (North Carolina State Univ., Raleigh (USA))



Phosphorylation of Mutant Huntingtin at Serine 116 Modulates Neuronal Toxicity  

PubMed Central

Phosphorylation has been shown to have a significant impact on expanded huntingtin-mediated cellular toxicity. Several phosphorylation sites have been identified on the huntingtin (Htt) protein. To find new potential therapeutic targets for Huntington's Disease (HD), we used mass spectrometry to identify novel phosphorylation sites on N-terminal Htt, expressed in HEK293 cells. Using site-directed mutagenesis we introduced alterations of phosphorylation sites in a N586 Htt construct containing 82 polyglutamine repeats. The effects of these alterations on expanded Htt toxicity were evaluated in primary neurons using a nuclear condensation assay and a direct time-lapse imaging of neuronal death. As a result of these studies, we identified several novel phosphorylation sites, validated several known sites, and discovered one phospho-null alteration, S116A, that had a protective effect against expanded polyglutamine-mediated cellular toxicity. The results suggest that S116 is a potential therapeutic target, and indicate that our screening method is useful for identifying candidate phosphorylation sites. PMID:24505464

Waldron-Roby, Elaine; O'Meally, Robert; Ratovitski, Tamara; Cole, Robert N.; Ross, Christopher A.



Synthetic Phosphorylation of p38? Recapitulates Protein Kinase Activity  

PubMed Central

Through a “tag-and-modify” protein chemical modification strategy, we site-selectively phosphorylated the activation loop of protein kinase p38?. Phosphorylation at natural (180) and unnatural (172) sites created two pure phospho-forms. p38? bearing only a single phosphocysteine (pCys) as a mimic of pThr at 180 was sufficient to switch the kinase to an active state, capable of processing natural protein substrate ATF2; 172 site phosphorylation did not. In this way, we chemically recapitulated triggering of a relevant segment of the MAPK-signaling pathway in vitro. This allowed detailed kinetic analysis of global and stoichiometric phosphorylation events catalyzed by p38? and revealed that site 180 is a sufficient activator alone and engenders dominant mono-phosphorylation activity. Moreover, a survey of kinase inhibition using inhibitors with different (Type I/II) modes (including therapeutically relevant) revealed unambiguously that Type II inhibitors inhibit phosphorylated p38? and allowed discovery of a predictive kinetic analysis based on cooperativity to distinguish Type I vs II. PMID:24393126



Protein phosphorylation differs significantly among ontogenetic phases in Malus seedlings  

PubMed Central

Background Although protein phosphorylation is an important post-translational modification affecting protein function and metabolism, dynamic changes in this process during ontogenesis remain unexplored in woody angiosperms. Methods Phosphorylated proteins from leaves of three apple seedlings at juvenile, adult vegetative and reproductive stages were extracted and subjected to alkaline phosphatase pre-treatment. After separating the proteins by two-dimensional gel electrophoresis and phosphoprotein-specific Pro-Q Diamond staining, differentially expressed phosphoproteins were identified by MALDI-TOF-TOF mass spectrometry. Results A total of 107 phosphorylated protein spots on nine gels (three ontogenetic phases?×?three seedlings) were identified by MALDI-TOF-TOF mass spectrometry. The 55 spots of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) large-chain fragments varied significantly in protein abundance and degree of phosphorylation among ontogenetic phases. Abundances of the 27 spots corresponding to Rubisco activase declined between juvenile and reproductive phases. More extensively, phosphorylated ?-tubulin chain spots with lower isoelectric points were most abundant during juvenile and adult vegetative phases. Conclusions Protein phosphorylation varied significantly during vegetative phase change and floral transition in apple seedlings. Most of the observed changes were consistent among seedlings and between hybrid populations. PMID:24904238



Dual regulation of SIRPalpha phosphorylation by integrins and CD47.  


Signal regulatory protein alpha (SIRPalpha, SHPS-1) is a plasma membrane receptor for CD47 and a key regulator of phagocytosis, growth factor signaling, and migration. Phosphorylation of immunoreceptor tyrosine-based inhibition motifs in its cytoplasmic tail is essential for the functional effects of SIRPalpha, at least in part, because the phosphorylated immunoreceptor tyrosine-based inhibition motifs recruit Src homology 2 domain-containing tyrosine phosphatases. Ligation by CD47 and integrin engagement both have been thought to regulate SIRPalpha phosphorylation. However, their distinct contributions have not been distinguished. Here, we show that the importance of CD47 varies with cell type, since ligation of CD47 is not necessary for SIRPalpha phosphorylation in myeloid cells, whereas it is required in endothelial cells. In contrast, integrin-mediated adhesion is required for SIRPalpha phosphorylation in both cell types. This shows that SIRPalpha phosphorylation is dually regulated and demonstrates a new mechanism for functional cooperation between integrins and the integrin-associated protein CD47. PMID:17584740

Johansen, Mette L; Brown, Eric J



Protein phosphorylation in isolated hepatocytes of septic and endotoxemic rats  

SciTech Connect

The purpose of this study was to investigate possible alterations induced by sepsis and endotoxicosis in the late phase of Ca2+-dependent signaling in rat liver. Hepatocytes isolated from septic or chronically endotoxin (ET)-treated rats were labeled with (32P)H3PO4 and stimulated with various agents. Proteins were resolved by one-dimensional polyacrylamide gel electrophoresis and autoradiographed. Vasopressin (VP)- and phenylephrine (PE)-induced responses were attenuated in both septic and ET-treated rats for cytosolic and membrane proteins compared with their respective controls. Glucagon and 12-O-myristate phorbol-13-acetate (TPA) affected only the phosphorylation of membrane proteins. Glucagon-induced changes in the phosphorylation of membrane proteins were affected by both sepsis and endotoxicosis, whereas TPA-stimulated phosphorylation was lowered only in endotoxicosis. Response to the Ca2+ ionophore A23187 was depressed in septic rats for cytosolic proteins. The phosphorylation of two cytosolic proteins, i.e., 93 and 61 kDa (previously identified as glycogen phosphorylase and pyruvate kinase, respectively), in response to VP, PE, and A23187 was severely impaired by endotoxicosis and sepsis. TPA did not affect the phosphorylation state of these two proteins. The results show that sepsis and endotoxicosis produce perturbations of the phosphorylation step in Ca2+ transmembrane signaling. Such changes can explain alterations of glycogenolysis and gluconeogenesis associated with sepsis and endotoxicosis.

Deaciuc, I.V.; Spitzer, J.A. (Louisiana State Univ. Medical Center, New Orleans (USA))



Identification of Phosphorylation Sites on Extracellular Corneal Epithelial Cell Maspin  

PubMed Central

Maspin, a 42-kDa non classical serine protease inhibitor (serpin) is expressed by epithelial cells of various tissues including the cornea. The protein localizes to the nucleus and cytosol, and is present in the extracellular space. While extracellular maspin regulates corneal stromal fibroblast adhesion and inhibits angiogenesis during wound healing in the cornea, the molecular mechanism of its extracellular functions is unclear. We hypothesized that identifying post-translational modifications of maspin, such as phosphorylation, may help decipher its mode of action. The focus of this study was on the identification of phosphorylation sites on extracellular maspin, since the extracellular form of the molecule is implicated in several functions. Multi-stage fragmentation mass spectrometry was used to identify sites of phosphorylation on extracellular corneal epithelial cell maspin. A total of eight serine and threonine phosphorylation sites (Thr50, Ser97, Thr118, Thr157, Ser240, Ser298, Thr310, Ser316) were identified on the extracellular forms of the molecule. Phosphorylation of tyrosine residues on extracellular maspin was not detected on extracellular maspin from corneal epithelial cell, in contrast to breast epithelial cells. This study provides the basis for further investigation into the functional role of phosphorylation of corneal epithelial maspin. PMID:21365746

Narayan, Malathi; Mirza, Shama P.; Twining, Sally S.



Enzymatic removal of dissolved aromatics from industrial aqueous effluents  

SciTech Connect

A new method has been elaborated for the removal of phenols and aromatic amines from industrial waste waters. It involves the treatment of solutions containing the pollutants with horseradish peroxidase and hydrogen peroxide. Such treatment results in precipitation of phenols and aromatic amines from water due to their enzymatic crosslinking. This approach was used to remove over 40 different phenols and aromatic amines (including known human carcinogens and mutagens) from water. For many pollutants, the efficiencies of the enzymatic removal are very high (exceeding 99%), whereas for others they are significantly lower. It was discovered that easily removed phenols and aromatic amines greatly enhance the enzymatic precipitation of those that have relatively low removal efficiencies. This finding is of great importance for the treatment of real industrial wastewaters, which always contain mixtures of pollutants. The described method appears to be economically feasible and has been successfully employed for treatment of wastewaters obtained from a chemical plant.

Alberti, B.N.; Klibanov, A.M.



Engineering aspects of enzymatic signal transduction: photoreceptors in the retina.  

PubMed Central

Identifying the basic module of enzymatic amplification as an irreversible cycle of messenger activation/deactivation by a "push-pull" pair of opposing enzymes, we analyze it in terms of gain, bandwidth, noise, and power consumption. The enzymatic signal transduction cascade is viewed as an information channel, the design of which is governed by the statistical properties of the input and the noise and dynamic range constraints of the output. With the example of vertebrate phototransduction cascade we demonstrate that all of the relevant engineering parameters are controlled by enzyme concentrations and, from functional considerations, derive bounds on the required protein numbers. Conversely, the ability of enzymatic networks to change their response characteristics by varying only the abundance of different enzymes illustrates how functional diversity may be built from nearly conserved molecular components. PMID:11106590

Detwiler, P B; Ramanathan, S; Sengupta, A; Shraiman, B I



Enzymatic colorimetry of lecithin and sphingomyelin in aqueous solution.  


A procedure for the enzymatic determination of lecithin and sphingomyelin in aqueous solution is described. The phospholipids are first dissolved in chloroform:methanol (2:1 by vol), the solvent is evaporated, and the residue is redissolved in an aqueous zwitterionic detergent solution. The enzymatic reaction sequences of both assays involve hydrolysis of the phospholipids to produce choline, which is then oxidized to betaine, thus generating hydrogen peroxide. The hydrogen peroxide is subsequently utilized in the enzymatic coupling of 4-aminoantipyrine and sodium 2-hydroxy-3,5-dichlorobenzenesulfonate, an intensely red color being formed. The presence of a non-reacting phospholipid enhances the hydrolysis of the reacting phospholipid. Thus we added lecithin to the sphingomyelin standards and sphingomyelin to the lecithin standards. This precise procedure may be applicable to determination of lecithin and sphingomyelin in amniotic fluid. PMID:6872211

McGowan, M W; Artiss, J D; Zak, B



High volumetric power density, non-enzymatic, glucose fuel cells  

PubMed Central

The development of new implantable medical devices has been limited in the past by slow advances in lithium battery technology. Non-enzymatic glucose fuel cells are promising replacement candidates for lithium batteries because of good long-term stability and adequate power density. The devices developed to date however use an “oxygen depletion design” whereby the electrodes are stacked on top of each other leading to low volumetric power density and complicated fabrication protocols. Here we have developed a novel single-layer fuel cell with good performance (2??W cm?2) and stability that can be integrated directly as a coating layer on large implantable devices, or stacked to obtain a high volumetric power density (over 16??W cm?3). This represents the first demonstration of a low volume non-enzymatic fuel cell stack with high power density, greatly increasing the range of applications for non-enzymatic glucose fuel cells. PMID:23390576

Oncescu, Vlad; Erickson, David



Plasmon-Enhanced Enzymatic Reactions: A Study of Nanoparticle-Enzyme Distance- and Nanoparticle Loading-Dependent Enzymatic Activity  

PubMed Central

A detailed investigation of the dependence of the efficiency of plasmon-enhanced enzymatic reactions on the distance between silver island films (SIFs) and horse radish peroxidase (HRP) enzyme and on the loading of SIFs on glass surfaces is presented. Three different extent of loading of SIFs on glass slides were used: 1) low, 2) medium and 3) high, which was characterized by using optical absorption spectroscopy and scanning electron microscopy. Streptavidin-linked HRP enzyme was deposited onto SIFs and glass slides by using three different strategies: strategy 1: biotin-avidin protein assay (distance between SIFs and HRP = 4–8 nm), strategy 2: self assembled monolayers (SAMs) (1–5 nm), strategy 3: polymer layer (1–5 nm). The efficiency of enzymatic conversion of O-phenylenediamine dihydrochloride (OPD) to a colored product by HRP on SIFs and glass surfaces was assessed by optical absorption spectroscopy. The distance between SIFs and HRP and the extent of loading of SIFs on the glass surfaces were shown to have significant effect on the efficiency of plasmon-enhanced enzymatic reactions. In this regard, up to an %250 increase in enzymatic conversion of OPD was observed from SIFs with high loading using strategy 1. In addition, we have studied the potential of repeated use of SIFs in plasmon-enhanced enzymatic reactions. PMID:21949594

Abel, Biebele; Akinsule, Alice; Andrews, Canisha; Aslan, Kadir



Enzymatic catalysis via liquid-liquid interfaces.  


Enzymes acting in a biphasic liquid of water and organic solvent may show catalytic activity dependent on the amount of phase interface available. Such an effect may be caused by several mechanisms. For example, for hydroxynitrile lyase from Prunus amygdalus, substrate mass transfer limitation has been advocated, but also adsorption of the enzyme on the interface. In this commentary it is shown that often these two mechanisms will have qualitatively similar consequences. The reaction rate will be influenced by the organic substrate concentration, by the initial enzyme concentration, and by the volume-specific interfacial area, and these influences will be linear at low values but reach a saturation level at high values. To rule out any of the models, their quantitative mathematical descriptions should be compared, taking into account that both models may be valid simultaneously. PMID:12800131

Straathof, Adrie J J



Technology transfer  

NASA Technical Reports Server (NTRS)

The topics are presented in viewgraph form and include the following: international comparison of R&D expenditures in 1989; NASA Technology Transfer Program; NASA Technology Utilization Program thrusts for FY 1992 and FY 1993; National Technology Transfer Network; and NTTC roles.

Penaranda, Frank E.



Integrated reactor concepts for the enzymatic kinetic synthesis of cephalexin.  


Integrated process concepts for enzymatic cephalexin synthesis were investigated by our group, and this article focuses on the integration of reactions and product removal during the reactions. The last step in cephalexin production is the enzymatic kinetic coupling of activated phenylglycine (phenylglycine amide or phenylglycine methyl ester) and 7-aminodeacetoxycephalosporanic acid (7-ADCA). The traditional production of 7-ADCA takes place via a chemical ring expansion step and an enzymatic hydrolysis step starting from penicillin G. However, 7-ADCA can also be produced by the enzymatic hydrolysis of adipyl-7-ADCA. In this work, this reaction was combined with the enzymatic synthesis reaction and performed simultaneously (i.e., one-pot synthesis). Furthermore, in situ product removal by adsorption and complexation were investigated as means of preventing enzymatic hydrolysis of cephalexin. We found that adipyl-7-ADCA hydrolysis and cephalexin synthesis could be performed simultaneously. The maximum yield on conversion (reaction) of the combined process was very similar to the yield of the separate processes performed under the same reaction conditions with the enzyme concentrations adjusted correctly. This implied that the number of reaction steps in the cephalexin process could be reduced significantly. The removal of cephalexin by adsorption was not specific enough to be applied in situ. The adsorbents also bound the substrates and therewith caused lower yields. Complexation with beta-naphthol proved to be an effective removal technique; however, it also showed a drawback in that the activity of the cephalexin-synthesizing enzyme was influenced negatively. Complexation with beta-naphthol rendered a 50% higher cephalexin yield and considerably less byproduct formation (reduction of 40%) as compared to cephalexin synthesis only. If adipyl-7-ADCA hydrolysis and cephalexin synthesis were performed simultaneously and in combination with complexation with beta-naphthol, higher cephalexin concentrations also were found. In conclusion, a highly integrated process (two reactions simultaneously combined with in situ product removal) was shown possible, although further optimization is necessary. PMID:12209770

Schroën, C G P H; Nierstrasz, V A; Bosma, R; Kroon, P J; Tjeerdsma, P S; DeVroom, E; VanderLaan, J M; Moody, H M; Beeftink, H H; Janssen, A E M; Tramper, J



In Vitro Phosphorylation Does not Influence the Aggregation Kinetics of WT ?-Synuclein in Contrast to Its Phosphorylation Mutants  

PubMed Central

The aggregation of alpha-synuclein (?-SYN) into fibrils is characteristic for several neurodegenerative diseases, including Parkinson’s disease (PD). Ninety percent of ?-SYN deposited in Lewy Bodies, a pathological hallmark of PD, is phosphorylated on serine129. ?-SYN can also be phosphorylated on tyrosine125, which is believed to regulate the membrane binding capacity and thus possibly its normal function. A better understanding of the effect of phosphorylation on the aggregation of ?-SYN might shed light on its role in the pathogenesis of PD. In this study we compare the aggregation properties of WT ?-SYN with the phospho-dead and phospho-mimic mutants S129A, S129D, Y125F and Y125E and in vitro phosphorylated ?-SYN using turbidity, thioflavin T and circular dichroism measurements as well as transmission electron microscopy. We show that the mutants S129A and S129D behave similarly compared to wild type (WT) ?-SYN, while the mutants Y125F and Y125E fibrillate significantly slower, although all mutants form fibrillar structures similar to the WT protein. In contrast, in vitro phosphorylation of ?-SYN on either S129 or Y125 does not significantly affect the fibrillization kinetics. Moreover, FK506 binding proteins (FKBPs), enzymes with peptidyl-prolyl cis-trans isomerase activity, still accelerate the aggregation of phosphorylated ?-SYN in vitro, as was shown previously for WT ?-SYN. In conclusion, our results illustrate that phosphorylation mutants can display different aggregation properties compared to the more biologically relevant phosphorylated form of ?-SYN. PMID:24434619

Schreurs, Sarah; Gerard, Melanie; Derua, Rita; Waelkens, Etienne; Taymans, Jean-Marc; Baekelandt, Veerle; Engelborghs, Yves



Three-dimensional graphene-carbon nanotube hybrid for high-performance enzymatic biofuel cells.  


Enzymatic biofuel cells (EBFCs) are promising renewable and implantable power sources. However, their power output is often limited by inefficient electron transfer between the enzyme molecules and the electrodes, hindered mass transport, low conductivity, and small active surface area of the electrodes. To tackle these issues, we herein demonstrated a novel EBFC equipped with enzyme-functionalized 3D graphene-single walled carbon nanotubes (SWCNTs) hybrid electrodes using the naturally abundant glucose as the fuel and oxygen as the oxidizer. Such EBFCs, with high stability, can nearly attain the theoretical limit of open circuit voltage (?1.2 V) and a high power density ever reported (2.27 ± 0.11 mW cm(-2)). PMID:24533856

Prasad, Kenath Priyanka; Chen, Yun; Chen, Peng



FEBS Letters 372 (1995)238-242 FEBS 16062 Histidine and tyrosine phosphorylation in pea mitochondria  

E-print Network

mitochondria: evidence for protein phosphorylation in respiratory redox signalling Gunilla H~kansson*, John F Abstract A 37 kDa protein in pea mitochondria was found to contain phosphorylated residues. Phosphorylation in plant mitochondria. We also describe the first example of tyrosine phosphorylation in plant organelles

Allen, John F.


Enzymatic Processing of Bioactive Glycosides from Natural Sources  

NASA Astrophysics Data System (ADS)

A number of biologically active natural products are glycosides. Often, the glycosidic residue is crucial for their activity. In other cases, glycosylation only improves their pharmacokinetic parameters. Enzymatic modification of these glycosides - both extension of the glycoside moiety and its selective trimming - is advantageous due to their selectivity and mildness of the reaction conditions in the presence of reactive and sensitive complex aglycones. Enzymatic reactions enable the resulting products to be used as "natural products", e.g., in nutraceuticals. This chapter concentrates on naturally occurring glycosides used in medicine but also in the food and flavor industry (e.g., sweeteners). Both "classical" and modern methods will be discussed.

Weignerová, Lenka; K?en, Vladimír


Structural Basis for Inactivation of the Human Pyruvate Dehydrogenase Complex by Phosphorylation: Role of Disordered Phosphorylation Loops  

SciTech Connect

We report the crystal structures of the phosporylated pyruvate dehydrogenase (E1p) component of the human pyruvate dehydrogenase complex (PDC). The complete phosphorylation at Ser264-{alpha} (site 1) of a variant E1p protein was achieved using robust pyruvate dehydrogenase kinase 4 free of the PDC core. We show that unlike its unmodified counterpart, the presence of a phosphoryl group at Ser264-{alpha} prevents the cofactor thiamine diphosphate-induced ordering of the two loops carrying the three phosphorylation sites. The disordering of these phosphorylation loops is caused by a previously unrecognized steric clash between the phosphoryl group at site 1 and a nearby Ser266-{alpha}, which nullifies a hydrogen-bonding network essential for maintaining the loop conformations. The disordered phosphorylation loops impede the binding of lipoyl domains of the PDC core to E1p, negating the reductive acetylation step. This results in the disruption of the substrate channeling in the PDC, leading to the inactivation of this catalytic machine.

Kato, Masato; Wynn, R. Max; Chuang, Jacinta L.; Tso, Shih-Chia; Machius, Mischa; Li, Jun; Chuang, David T. (UTSMC)



Tyrosine phosphorylation of NEDD4 activates its ubiquitin ligase activity.  


Ligand binding to the receptor tyrosine kinase fibroblast growth factor (FGF) receptor 1 (FGFR1) causes dimerization and activation by transphosphorylation of tyrosine residues in the kinase domain. FGFR1 is ubiquitylated by the E3 ligase NEDD4 (also known as NEDD4-1), which promotes FGFR1 internalization and degradation. Although phosphorylation of FGFR1 is required for NEDD4-dependent endocytosis, NEDD4 directly binds to a nonphosphorylated region of FGFR1. We found that activation of FGFR1 led to activation of c-Src kinase-dependent tyrosine phosphorylation of NEDD4, enhancing the ubiquitin ligase activity of NEDD4. Using mass spectrometry, we identified several FGF-dependent phosphorylated tyrosines in NEDD4, including Tyr(43) in the C2 domain and Tyr(585) in the HECT domain. Mutating these tyrosines to phenylalanine to prevent phosphorylation inhibited FGF-dependent NEDD4 activity and FGFR1 endocytosis and enhanced cell proliferation. Mutating the tyrosines to glutamic acid to mimic phosphorylation enhanced NEDD4 activity. Moreover, the NEDD4 C2 domain bound the HECT domain, and the presence of phosphomimetic mutations inhibited this interaction, suggesting that phosphorylation of NEDD4 relieves an inhibitory intra- or intermolecular interaction. Accordingly, activation of FGFR1 was not required for activation of NEDD4 that lacked its C2 domain. Activation of c-Src by epidermal growth factor (EGF) also promoted tyrosine phosphorylation and enhanced the activity of NEDD4. Thus, we identified a feedback mechanism by which receptor tyrosine kinases promote catalytic activation of NEDD4 and that may represent a mechanism of receptor crosstalk. PMID:25292214

Persaud, Avinash; Alberts, Philipp; Mari, Sara; Tong, Jiefei; Murchie, Ryan; Maspero, Elena; Safi, Frozan; Moran, Michael F; Polo, Simona; Rotin, Daniela



Effects of Phosphorylation in Chlamydomonas Centrin Ser 167  

PubMed Central

Centrin is a conserved calcium binding protein belonging to the EF-hand superfamily with two independent structural domains. This protein is found to be phosphorylated near the carboxyl terminal end. Our goal was to perform a novel comparative study of phosphorylated and unphosphorylated centrin by Fourier transform infrared (FT-IR) spectroscopy, two-dimensional correlation spectroscopy (2D-COS) analysis and differential scanning calorimetry (DSC). To achieve this goal, we have bacterially expressed, isolated, purified and phosphorylated centrin. We verified the extent of phosphorylation to be >97% for centrin by MALDI MS analysis and determined the absence of aggregated protein. The thermal denaturation temperature and ?Cp were determined to be Tm = 112.1 °C (?Cp = 7.8 Kcal/mole/?C) and Tm = 111.0°C (?Cp = 5.0 Kcal/mole/°C) for holo-centrin and phosphorylated centrin, respectively. We have also described the molecular dynamics leading up to the thermal denaturation of the protein: for holo-centrin the vibrational modes associated with the calcium binding sites aspartates and glutamates, loops then the arginines, followed by the structured backbone vibrational modes the ?-helix at 1635 cm?1 then ?-sheet and finally the more exposed ?-helix at 1650 cm?1; while for phosphorylated centrin aspartate, glutamate and arginine, followed by the backbone associated vibrational modes ?-helix (1650 cm?1), loop then the ?-sheet (1633 cm?1) and finally the ?-helix (1637 cm?1). Therefore, the effect on domain stability due to phosphorylation at Ser167 was observed in the loops as well as the ?-helix at 1650 cm?1. PMID:22162668

Sanoguet, Zuleika; Campbell, Muriel; Ramos, Sindia; Seda, Christina; Moreno, Luis Perez; Pastrana-Rios, Belinda



Cytokines Alter Glucocorticoid Receptor Phosphorylation in Airway Cells  

PubMed Central

Corticosteroid insensitivity (CSI) represents a profound challenge in managing patients with asthma. We recently demonstrated that short exposure of airway smooth muscle cells (ASMCs) to proasthmatic cytokines drastically reduced their responsiveness to glucocorticoids (GCs), an effect that was partially mediated via interferon regulatory factor-1, suggesting the involvement of additional mechanisms (Am J Respir Cell Mol Biol 2008;38:463–472). Although GC receptor (GR) can be phosphorylated at multiple serines in the N-terminal region, the major phosphorylation sites critical for GR transcriptional activity are serines 211 (Ser211) and 226 (Ser226). We tested the novel hypothesis that cytokine-induced CSI in ASMCs is due to an impaired GR phosphorylation. Cells were treated with TNF-? (10 ng/ml) and IFN-? (500 UI/ml) for 6 hours and/or fluticasone (100 nm) added 2 hours before. GR was constitutively phosphorylated at Ser226 but not at Ser211 residues. Cytokines dramatically suppressed fluticasone-induced phosphorylation of GR on Ser211 but not on Ser226 residues while increasing the expression of Ser/Thr protein phosphatase (PP)5 but not that of PP1 or PP2A. Transfection studies using a reporter construct containing GC responsive elements showed that the specific small interfering RNA–induced mRNA knockdown of PP5, but not that of PP1 or PP2A, partially prevented the cytokine suppressive effects on GR-meditated transactivation activity. Similarly, cytokines failed to inhibit GC-induced GR-Ser211 phosphorylation when expression of PP5 was suppressed. We propose that the novel mechanism that proasthmatic cytokine-induced CSI in ASMCs is due, in part, to PP5-mediated impairment of GR-Ser211 phosphorylation. PMID:22592921

Bouazza, Belaid; Krytska, Kateryna; Debba-Pavard, Manel; Amrani, Yassine; Honkanen, Richard E.; Tran, Jennifer



Systematic Analysis of Protein Phosphorylation Networks From Phosphoproteomic Data*  

PubMed Central

In eukaryotes, hundreds of protein kinases (PKs) specifically and precisely modify thousands of substrates at specific amino acid residues to faithfully orchestrate numerous biological processes, and reversibly determine the cellular dynamics and plasticity. Although over 100,000 phosphorylation sites (p-sites) have been experimentally identified from phosphoproteomic studies, the regulatory PKs for most of these sites still remain to be characterized. Here, we present a novel software package of iGPS for the prediction of in vivo site-specific kinase-substrate relations mainly from the phosphoproteomic data. By critical evaluations and comparisons, the performance of iGPS is satisfying and better than other existed tools. Based on the prediction results, we modeled protein phosphorylation networks and observed that the eukaryotic phospho-regulation is poorly conserved at the site and substrate levels. With an integrative procedure, we conducted a large-scale phosphorylation analysis of human liver and experimentally identified 9719 p-sites in 2998 proteins. Using iGPS, we predicted a human liver protein phosphorylation networks containing 12,819 potential site-specific kinase-substrate relations among 350 PKs and 962 substrates for 2633 p-sites. Further statistical analysis and comparison revealed that 127 PKs significantly modify more or fewer p-sites in the liver protein phosphorylation networks against the whole human protein phosphorylation network. The largest data set of the human liver phosphoproteome together with computational analyses can be useful for further experimental consideration. This work contributes to the understanding of phosphorylation mechanisms at the systemic level, and provides a powerful methodology for the general analysis of in vivo post-translational modifications regulating sub-proteomes. PMID:22798277

Song, Chunxia; Ye, Mingliang; Liu, Zexian; Cheng, Han; Jiang, Xinning; Han, Guanghui; Songyang, Zhou; Tan, Yexiong; Wang, Hongyang; Ren, Jian; Xue, Yu; Zou, Hanfa



Human Cytochrome P450c17: Single Step Purification and Phosphorylation of Serine 258 by Protein Kinase A  

PubMed Central

Cytochrome P450c17 (P450c17) is the single microsomal enzyme that catalyzes steroid 17?-hydroxylase and 17,20 lyase activities. The ratio of lyase to hydroxylase activity of human P450c17 determines whether steroidogenesis leads to the synthesis of cortisol or sex steroids. This ratio is regulated posttranslationally by factors that influence the efficiency of electron transfer from P450 oxidoreductase to P450c17. One factor favoring more efficient electron transfer and 17,20 lyase activity is cAMP-dependent serine/threonine phosphorylation of P450c17. Identifying the responsible kinase(s) and the P450c17 residues that undergo phosphorylation has been challenging, partly because of difficulties in preparing biochemically useful amounts of pure, catalytically active P450c17. We describe a modified strategy for preparing P450c17 in which the traditional carboxy-terminal 4×His tag is replaced by 3×Gly6×His. This construct permits more rotational freedom of the protein when bound to the nickel affinity column, reducing steric associations between the protein and the column, and permitting a single-step chromatographic purification to apparent homogeneity. Using this vector, we explored P450c17 phosphorylation by mutagenesis of Ser and/or Thr residues to Asp or Glu to mimic the approximate size and charge of phospho-Ser or phospho-Thr. This strategy did not identify Ser and/or Thr site(s) that increase the ratio of lyase to hydroxylase activity, suggesting that the regulatory phosphorylation strategy of human P450c17 is very complicated. Although previous work has excluded protein kinase A (PKA) as the responsible kinase, the cAMP-inducible nature of the phosphorylation-associated increase in lyase activity suggests that PKA may play a role, possibly as a priming kinase. Using our novel vector and a series of mutations, we identified the P450c17 site phosphorylated by PKA as Ser258. PMID:20160131

Wang, Yue-Hao; Tee, Meng Kian; Miller, Walter L.



"Transfer Shock" or "Transfer Ecstasy?"  

ERIC Educational Resources Information Center

The alleged characteristic drop in grade point average (GPA) of transfer students and the subsequent rise in GPA was investigated in this study. No statistically significant difference was found in first term junior year GPA between junior college transfers and native Florida State University students after the variance accounted for by the…

Nickens, John M.


Enzymatic Activity versus Structural Dynamics: The Case of Acetylcholinesterase Tetramer  

E-print Network

Enzymatic Activity versus Structural Dynamics: The Case of Acetylcholinesterase Tetramer Alemayehu of synaptic acetylcholine (ACh) by tetrameric acetylcholinesterase (AChEt). Despite decades of efforts acetylcholinesterase (AChE) (4,5). AChE is a serine hydrolase that exists as a monomer, dimer, or tetramer, the latter

Lu, Benzhuo


Enzymatic combinatorial nucleoside deletion scanning mutagenesis of functional RNA.  


We describe a general and simple method to identify catalytically and structurally important nucleotides in functional RNAs. Our approach is based on statistical replacement of each nucleoside with a non-nucleosidic spacer (C3 linker, ?), followed by separation of active library variants and readout of interference effects by analysis of enzymatic primer extension reactions. PMID:25097037

Wawrzyniak-Turek, Katarzyna; Höbartner, Claudia



Enzymatic synthesis of derivatives of vitamin A in organic media  

Microsoft Academic Search

The present article provides an enzymatic method of retinol esterification to reduce photodestruction and irritation problems characteristic of retinol. More specifically, it relates to a method of synthesising retinyl adipate, retinyl succinate, retinyl oleate and retinyl lactate greatly appreciated by cosmetic manufacturer. Desired compounds can be synthesised directly using Candida antarctica lipase and Rhizomucor miehei lipase in organic media.

Thierry Maugard; Marie Dominique Legoy



Soil solid materials affect the kinetics of extracellular enzymatic reactions  

Microsoft Academic Search

INTRODUCTION Soil solid materials affect the degradation processes of many organic compounds by decreasing the bioavailability of substrates and by interacting with degraders. The magnitude of this effect in the environment is shown by the fact that xenobiotics which are readily metabolized in aquatic environments can have long residence times in soil. Extracellular enzymatic hydrolysis of cellobiose (enzyme: beta-glucosidase from

C. Lammirato; A. Miltner; M. Kästner



Enzymatic reactivity of glucose oxidase confined in nanochannels.  


The construction of nanodevices coupled with an integrated real-time detection system for evaluation of the function of biomolecules in biological processes, and enzymatic reaction kinetics occurring at the confined space or interface is a significant challenge. In this work, a nanochannel-enzyme system in which the enzymatic reaction could be investigated with an electrochemical method was constructed. The model system was established by covalently linking glucose oxidase (GOD) onto the inner wall of the nanochannels of the porous anodic alumina (PAA) membrane. An Au disc was attached at the end of the nanochannels of the PAA membrane as the working electrode for detection of H2O2 product of enzymatic reaction. The effects of ionic strength, amount of immobilized enzyme and pore diameter of the nanochannels on the enzymatic reaction kinetics were illustrated. The GOD confined in nanochannels showed high stability and reactivity. Upon addition of glucose to the nanochannel-enzyme system, the current response had a calibration range span from 0.005 to 2 mM of glucose concentration. The apparent Michaelis-Menten constant (K(m)(app)) of GOD confined in nanochannel was 0.4 mM. The presented work provided a platform for real-time monitoring of the enzyme reaction kinetics confined in nanospaces. Such a nanochannel-enzyme system could also help design future biosensors and enzyme reactors with high sensitivity and efficiency. PMID:24412427

Yu, Jiachao; Zhang, Yuanjian; Liu, Songqin



Toward Efficient Enzymatic Glycan Synthesis: Directed Evolution and Enzyme Engineering  

Microsoft Academic Search

Enzymatic synthesis of oligosaccharides has traditionally employed glycosyltransferases, or glycosidases run in transglycosylation mode. Glycosynthases, mutant glycosidases in which the catalytic nucleophile has been removed, function as efficient transferases with glycosyl fluoride donors, frequently giving stoicheometric yields. Glycoligases, in which the acid\\/base catalyst has been mutated, synthesise sulfur-linked oligosaccharides when an activated donor is used in conjunction with a thiosugar

David H. Kwan; Stephen G. Withers



Enzymatic properties and cholesterol content of mitochondrial outer mem-  

E-print Network

Enzymatic properties and cholesterol content of mitochondrial outer mem- branes in liver of obese and microsomes. Simultaneously microsomes were shown to be richer in cholesterol than mitochondrial outer, it was determined that outer membranes are extremely poor in cholesterol, far richer in to- tal fatty acids than

Boyer, Edmond


An improved competitive inhibition enzymatic immunoassay method for tetrodotoxin quantification  

PubMed Central

Quantifying tetrodotoxin (TTX) has been a challenge in both ecological and medical research due to the cost, time and training required of most quantification techniques. Here we present a modified Competitive Inhibition Enzymatic Immunoassay for the quantification of TTX, and to aid researchers in the optimization of this technique for widespread use with a high degree of accuracy and repeatability. PMID:22410273



Enzymatic activity of rodents acclimated to cold and long scotophase  

NASA Astrophysics Data System (ADS)

Rodents representative of a diurnal species ( Rhabdomys pumilio) as well as a nocturnal species ( Praomys natalensis) were acclimated to cold (Ta = 8°C) at a photoperiod of LD 12:12 and a long scotophase (LD 8; 16) at a temperature of 25° C(Ta). Control groups were kept for both species at Ta = 25° C and LD 12:12 and winter acclimated individuals were obtained during July and August to serve as further reference. Blood samples obtained from the tail were analysed for enzymes representative of three major biochemical pathways. The enzymatic activity of LDH (glycolytic pathway), MDH (Krebs cycle) and G6PDH (hexose monophosphate shunt, as an indicator of gonadal activity) were monitored to represent metabolic activity of the respective cycles. Cold acclimated as well as winter acclimatized mice revealed similar enzymatic patterns for both species and significant increases in LDH and MDH were recorded with a concurrent decrease in G6PDH activity. Specimens exposed to long scotophase exhibited similar enzymatic patterns for both species studied, but enzymatic activity was higher than those of cold acclimated individuals. From these results it is concluded that cold as well as long scotophase induce metabolic adaptations through biochemical activity in the experimental animals. The effect of long scotophase is assumed to be an important factor in the induction of winter acclimatization.

Fourie, F. Le R.; Haim, A.



Development of an oxygen-rich biosensor using enzymatic reaction  

Microsoft Academic Search

This work reports a novel strategy for the development of an O2-rich biosensor. The principle is based on an enzymatic reaction between catalase and H2O2 to release O2, thus to increase the O2 amount in the enzyme matrix. This method improves the determination reliability by alleviating the O2 dependence.

Yue Cui; John P. Barford; Reinhard Renneberg



BSA Treatment to Enhance Enzymatic Hydrolysis of Cellulose in Lignin  

E-print Network

) pretreated corn stover and Douglas fir treated by SO2 steam explosion and for simultaneous saccharification. Similar improve- ments were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX the saccharification rate of cellulose in Sigmacell 100 and steam exploded poplar by as much as a factor of 7 while

California at Riverside, University of


Steam explosion pretreatment and enzymatic hydrolysis of eucalyptus wood  

Microsoft Academic Search

Wood from Eucalyptus globulus Labill has been pretreated under various conditions of acid impregnation and steam explosion. The effects of these pretreatment conditions have been assessed by measuring monomer (xylose and glucose) and oligomer solubilization. Samples pretreated under conditions that yielded optimal hemicellulose solubilization for acid impregnated or non-impregnated wood have been further subjected to enzymatic hydrolysis, using a preparation

A. P. Nunes; J. Pourquie



Lime pretreatment and fermentation of enzymatically hydrolyzed sugarcane bagasse.  


Sugarcane bagasse was subjected to lime (calcium hydroxide) pretreatment and enzymatic hydrolysis for second-generation ethanol production. A central composite factorial design was performed to determine the best combination of pretreatment time, temperature, and lime loading, as well as to evaluate the influence of enzymatic loadings on hydrolysis conversion. The influence of increasing solids loading in the pretreatment and enzymatic hydrolysis stages was also determined. The hydrolysate was fermented using Saccharomyces cerevisiae in batch and continuous mode. In the continuous fermentation, the hydrolysates were concentrated with molasses. Lime pretreatment significantly increased the enzymatic digestibility of sugarcane bagasse without the need for prior particle size reduction. In the optimal pretreatment conditions (90 h, 90 °C, 0.47 glime/g bagasse) and industrially realistic conditions of hydrolysis (12.7 FPU/g of cellulase and 7.3 CBU/g of ?-glucosidase), 139.6 kglignin/ton raw bagasse and 126.0 kg hemicellulose in the pretreatment liquor per ton raw bagasse were obtained. The hydrolysate from lime pretreated sugarcane bagasse presented low amounts of inhibitors, leading to ethanol yield of 164.1 kgethanol/ton raw bagasse. PMID:23334836

Rabelo, Sarita C; Maciel Filho, Rubens; Costa, Aline C



Discovery of phosphorylation motif mixtures in phosphoproteomics data  

PubMed Central

Motivation: Modification of proteins via phosphorylation is a primary mechanism for signal transduction in cells. Phosphorylation sites on proteins are determined in part through particular patterns, or motifs, present in the amino acid sequence. Results: We describe an algorithm that simultaneously discovers multiple motifs in a set of peptides that were phosphorylated by several different kinases. Such sets of peptides are routinely produced in proteomics experiments.Our motif-finding algorithm uses the principle of minimum description length to determine a mixture of sequence motifs that distinguish a foreground set of phosphopeptides from a background set of unphosphorylated peptides. We show that our algorithm outperforms existing motif-finding algorithms on synthetic datasets consisting of mixtures of known phosphorylation sites. We also derive a motif specificity score that quantifies whether or not the phosphoproteins containing an instance of a motif have a significant number of known interactions. Application of our motif-finding algorithm to recently published human and mouse proteomic studies recovers several known phosphorylation motifs and reveals a number of novel motifs that are enriched for interactions with a particular kinase or phosphatase. Our tools provide a new approach for uncovering the sequence specificities of uncharacterized kinases or phosphatases. Availability: Software is available at http:/ Contact:; Supplementary information: Supplementary data are available at Bioinformatics online. PMID:18996944

Ritz, Anna; Shakhnarovich, Gregory; Salomon, Arthur R.; Raphael, Benjamin J.



Cellular Functions Regulated by Phosphorylation of EGFR on Tyr845  

PubMed Central

The Src gene product (Src) and the epidermal growth factor receptor (EGFR) are prototypes of oncogene products and function primarily as a cytoplasmic non-receptor tyrosine kinase and a transmembrane receptor tyrosine kinase, respectively. The identification of Src and EGFR, and the subsequent extensive investigations of these proteins have long provided cutting edge research in cancer and other molecular and cellular biological studies. In 1995, we reported that the human epidermoid carcinoma cells, A431, contain a small fraction of Src and EGFR in which these two kinase were in physical association with each other, and that Src phosphorylates EGFR on tyrosine 845 (Y845) in the Src-EGFR complex. Y845 of EGFR is located in the activation segment of the kinase domain, where many protein kinases contain kinase-activating autophosphorylation sites (e.g., cAMP-dependent protein kinase, Src family kinases, transmembrane receptor type tyrosine kinases) or trans-phosphorylation sites (e.g., cyclin-dependent protein kinase, mitogen-activated protein kinase, Akt protein kinase). A number of studies have demonstrated that Y845 phosphorylation serves an important role in cancer as well as normal cells. Here we compile the experimental facts involving Src phosphorylation of EGFR on Y845, by which cell proliferation, cell cycle control, mitochondrial regulation of cell metabolism, gamete activation and other cellular functions are regulated. We also discuss the physiological relevance, as well as structural insights of the Y845 phosphorylation. PMID:23702846

Sato, Ken-ichi



A Phosphorylated Pseudokinase Complex Controls Cell Wall Synthesis in Mycobacteria  

PubMed Central

Prokaryotic cell wall biosynthesis is coordinated with cell growth and division, but the mechanisms regulating this dynamic process remain obscure. Here, we describe a phosphorylation-dependent regulatory complex that controls peptidoglycan (PG) biosynthesis in Mycobacterium tuberculosis. We found that PknB, a PG-responsive Ser-Thr protein kinase (STPK), initiates complex assembly by phosphorylating a kinase-like domain in the essential PG biosynthetic protein, MviN. This domain was structurally diverged from active kinases and did not mediate phosphotransfer. Threonine phosphorylation of the pseudokinase domain recruited the FhaA protein through its forkhead-associated (FHA) domain. The crystal structure of this phosphorylated pseudokinase–FHA domain complex revealed the basis of FHA domain recognition, which included unexpected contacts distal to the phosphorylated threonine. Conditional degradation of these proteins in mycobacteria demonstrated that MviN was essential for growth and PG biosynthesis and that FhaA regulated these processes at the cell poles and septum. Controlling this spatially localized PG regulatory complex is only one of several cellular roles ascribed to PknB, suggesting that the capacity to coordinate signaling across multiple processes is an important feature conserved between eukaryotic and prokaryotic STPK networks. PMID:22275220

Gee, Christine L.; Papavinasasundaram, Kadamba G.; Blair, Sloane R.; Baer, Christina E.; Falick, Arnold M.; King, David S.; Griffin, Jennifer E.; Venghatakrishnan, Harene; Zukauskas, Andrew; Wei, Jun-Rong; Dhiman, Rakesh K.; Crick, Dean C.; Rubin, Eric J.; Sassetti, Christopher M.; Alber, Tom



Interplay between Sumoylation and Phosphorylation for Protection against ?-Synuclein Inclusions.  


Parkinson disease is associated with the progressive loss of dopaminergic neurons from the substantia nigra. The pathological hallmark of the disease is the accumulation of intracytoplasmic inclusions known as Lewy bodies that consist mainly of post-translationally modified forms of ?-synuclein. Whereas phosphorylation is one of the major modifications of ?-synuclein in Lewy bodies, sumoylation has recently been described. The interplay between ?-synuclein phosphorylation and sumoylation is poorly understood. Here, we examined the interplay between these modifications as well as their impact on cell growth and inclusion formation in yeast. We found that ?-synuclein is sumoylated in vivo at the same sites in yeast as in human cells. Impaired sumoylation resulted in reduced yeast growth combined with an increased number of cells with inclusions, suggesting that this modification plays a protective role. In addition, inhibition of sumoylation prevented autophagy-mediated aggregate clearance. A defect in ?-synuclein sumoylation could be suppressed by serine 129 phosphorylation by the human G protein-coupled receptor kinase 5 (GRK5) in yeast. Phosphorylation reduced foci formation, alleviated yeast growth inhibition, and partially rescued autophagic ?-synuclein degradation along with the promotion of proteasomal degradation, resulting in aggregate clearance in the absence of a small ubiquitin-like modifier. These findings suggest a complex interplay between sumoylation and phosphorylation in ?-synuclein aggregate clearance, which may open new horizons for the development of therapeutic strategies for Parkinson disease. PMID:25231978

Shahpasandzadeh, Hedieh; Popova, Blagovesta; Kleinknecht, Alexandra; Fraser, Paul E; Outeiro, Tiago F; Braus, Gerhard H



Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication  

PubMed Central

Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells. PMID:25006834

Diaz, Jason; Wang, Xin; Tsang, Sabrina H.; Jiao, Jing; You, Jianxin



Phosphorylation of large T antigen regulates merkel cell polyomavirus replication.  


Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT's ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells. PMID:25006834

Diaz, Jason; Wang, Xin; Tsang, Sabrina H; Jiao, Jing; You, Jianxin



Regulation of ABC Transporter Function Via Phosphorylation by Protein Kinases  

PubMed Central

ATP-binding cassette (ABC) transporters are multispanning membrane proteins that utilize ATP to move a broad range of substrates across cellular membranes. ABC transporters are involved in a number of human disorders and diseases [1]. Overexpression of a subset of the transporters has been closely linked to multidrug resistance in both bacteria and viruses and in cancer. A poorly understood and important aspect of ABC transporter biology is the role of phosphorylation as a mechanism to regulate transporter function. In this review, we summarize the current literature addressing the role of phosphorylation in regulating ABC transporter function. A comprehensive list of all the phosphorylation sites that have been identified for the human ABC transporters is presented, and we discuss the role of individual kinases in regulating transporter function. We address the potential pitfalls and difficulties associated with identifying phosphorylation sites and the corresponding kinase(s), and we discuss novel techniques that may circumvent these problems. We conclude by providing a brief perspective on studying ABC transporter phosphorylation. PMID:21118091

Stolarczyk, Elzbieta I.; Reiling, Cassandra J.; Paumi, Christian M.



Highly efficient production of phosphorylated hepatitis B core particles in yeast Pichia pastoris.  


Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are routinely used in HBV diagnostics worldwide and are of potential interest as carriers of foreign peptides (e.g., immunological epitopes and targeting addresses, and/or as vessels for packaged diagnostic and therapeutic nanomaterials). Despite numerous reports exploiting different expression systems, a rapid and comprehensive large-scale methodology for purification of HBc VLPs from yeast is still lacking. Here, we present a convenient protocol for highly efficient production and rapid purification of endotoxin-free ayw subtype HBc VLPs from the methylotrophic yeast Pichia pastoris. The HBc gene expression cassette along with the geneticin resistance gene was transferred to the P. pastoris genome via homologous recombination. A producer clone was selected among 2000 transformants for the optimal synthesis of the target protein. Fermentation conditions were established ensuring biomass accumulation of 163g/L. A simple combination of pH/heat and salt treatment followed by a single anion-exchange chromatography step resulted in a more than 90% pure preparation of HBc VLPs, with a yield of about 3.0mg per 1g of wet cells. Purification is performed within a day and may be easily scaled up if necessary. The quality of HBc VLPs was verified by electron microscopy. Mass spectrometry analysis and direct polyacrylamide gel staining revealed phosphorylation of HBc at at least two sites. To our knowledge, this is the first report of HBc phosphorylation in yeast. PMID:20854910

Freivalds, Janis; Dislers, Andris; Ose, Velta; Pumpens, Paul; Tars, Kaspars; Kazaks, Andris



Functional characterization of alpha-glucan,water dikinase, the starch phosphorylating enzyme.  

PubMed Central

GWD (alpha-glucan,water dikinase) is the enzyme that catalyses the phosphorylation of starch by a dikinase-type reaction in which the beta-phosphate of ATP is transferred to either the C-6 or the C-3 position of the glycosyl residue of amylopectin. GWD shows similarity in both sequence and reaction mechanism to bacterial PPS (pyruvate,water dikinase) and PPDK (pyruvate,phosphate dikinase). Amino acid sequence alignments identified a conserved histidine residue located in the putative phosphohistidine domain of potato GWD. Site-directed mutagenesis of this histidine residue resulted in an inactive enzyme and loss of autophosphorylation. Native GWD is a homodimer and shows a strict requirement for the presence of alpha-1,6 branch points in its polyglucan substrate, and exhibits a sharp 20-fold increase in activity when the degree of polymerization is increased from 27.8 to 29.5. In spite of the high variability in the degree of starch phosphorylation, GWD proteins are ubiquitous in plants. The overall reaction mechanism of GWD is similar to that of PPS and PPDK, but the GWD family appears to have arisen after divergence of the plant kingdom. The nucleotide-binding domain of GWD exhibits a closer phylogenetic relationship to prokaryotic PPSs than to PPDKs. PMID:14525539

Mikkelsen, Rene; Baunsgaard, Lone; Blennow, Andreas



Mechanochemical Phosphorylation and Solubilisation of ?-D-Glucan from Yeast Saccharomyces cerevisiae and Its Biological Activities  

PubMed Central

To obtain a water-soluble ?-D-glucan derivative cleanly and conveniently, a highly efficient mechanochemical method, planetary ball milling, was used to phosphorylate ?-D-glucan isolated from yeast Saccharomyces cerevisiae in solid state. Soluble ?-D-glucan phosphate (GP) with a high degree of substitution (0.77–2.09) and an apparent PEAK molecular weight of 6.6–10.0 kDa was produced when ?-D-glucan was co-milled with sodium hexametaphosphate at 139.5–186.0 rad/s for 12–20 min. The energy transferred was 3.03–11.98 KJ/g. The phosphorylation of GPs was demonstrated by Fourier transform infrared spectroscopy and 13C and 31P Nuclear magnetic resonance spectroscopy. Three GP products with different degree of substitution (DS) and degree of polymerisation (DP) were able to upregulate the functional events mediated by activated murine macrophage RAW264.7 cells, among which GP-2 with a DS of 1.24 and DP of 30.5 exerted the highest immunostimulating activity. Our results indicate that mechanochemical processing is an efficient method for preparing water-soluble and biologically active GP with high DS. PMID:25075740

Shi, Feng; Shi, Jikui; Li, Yongfu



Molecular dynamics study of enhanced Man5B enzymatic activity  

PubMed Central

Background Biofuels are a well-known alternative to the largely used fossil-derived fuels, however the competition with food production is an ethical dilemma. Fortunately a solution is offered by second-generation biofuels which can be produced from agricultural waste or, more specifically, from plant cell wall polysaccharides. The conversion process involves typically enzymatic hydrolysis of lignocellulosic biomass and then separation of its constituent sugars that are further fermented to produce ethanol. Over the years several technologies have been developed that allow this conversion process to occur and the objective is now to make this process cost-competitive in today’s markets. Results We observe that reduction of enzymatic efficiency in the presence of gluco-oligosaccharides is associated with a loss of the enzyme’s flexibility, the latter being required to bind new substrate, while the presence of manno-oligosaccharides does not pose this problem. Molecular dynamics simulations identify key contacts between substrates and the enzyme catalytic pocket that might be modified through site-directed mutagenesis to prevent loss of enzymatic efficiency. Conclusions Based on previous experimental studies and the new molecular dynamics data, we suggest that cellohexaose in the active site pocket slows down or even inhibits Man5B enzymatic activity. The assumption of such a mechanism is reasonable since when the gluco-oligosaccharide substrate is attached to the catalytic pocket it takes much longer to leave the pocket and thus prevents other substrates from reaching the active site. The insight is of crucial importance since the inhibition of enzymes by the enzymatic product or by an unsuitable substrate is a major technological problem in reducing the competitiveness of second-generation biofuel production. PMID:24976862



Sensing core histone phosphorylation -- A matter of perfect timing?  

PubMed Central

Systematic analysis of histone modifications has revealed a plethora of posttranslational modifications that mediate changes in chromatin structure and gene expression. Histone phosphorylation is a transient histone modification that becomes induced by extracellular signals, DNA damage or entry into mitosis. Importantly, phosphorylation of histone proteins does lead not only to the binding of specific reader proteins but also to changes in the affinity for readers or writers of other histone modifications. This induces a cross-talk between different chromatin modifications that allows the spatio-temporal control of chromatin-associated events. In this review we will summarize the progress in our current knowledge of factors sensing reversible histone phosphorylation in different biological scenarios. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function. PMID:24747175

Sawicka, Anna; Seiser, Christian



Phosphorylation of unique domains of Src family kinases  

PubMed Central

Members of the Src family of kinases (SFKs) are non-receptor tyrosine kinases involved in numerous signal transduction pathways. The catalytic, SH3 and SH2 domains are attached to the membrane-anchoring SH4 domain through the intrinsically disordered “Unique” domains, which exhibit strong sequence divergence among SFK members. In the last decade, structural and biochemical studies have begun to uncover the crucial role of the Unique domain in the regulation of SFK activity. This mini-review discusses what is known about the phosphorylation events taking place on the SFK Unique domains, and their biological relevance. The modulation by phosphorylation of biologically relevant inter- and intra- molecular interactions of Src, as well as the existence of complex phosphorylation/dephosphorylation patterns observed for the Unique domain of Src, reinforces the important functional role of the Unique domain in the regulation mechanisms of the Src kinases and, in a wider context, of intrinsically disordered regions in cellular processes. PMID:25071818

Amata, Irene; Maffei, Mariano; Pons, Miquel



Phosphorylation: a Fundamental Regulator of Steroid Receptor Action  

PubMed Central

Steroid hormone receptors (SHRs) are hormone-activated transcription factors involved in numerous cellular functions and in health and disease. Their activities depend on the cellular level of the receptor, the presence of coregulator proteins and the cell signaling pathways that are active in the cell. SHRs and their coregulators are phosphorylated on multiple sites by a wide variety of kinases. Each site may contribute to multiple functions and the net effect of an individual phosphorylation depends on the activating kinase. Here we discuss functions of known SHR phosphorylation sites, kinase regulation, evidence of translational relevance, and cross-talk between SHRs and cell signaling pathways. Understanding how cell signaling pathways regulate SHRs might yield novel therapeutic targets for multiple human diseases. PMID:23838532

Trevino, Lindsey S.; Weigel, Nancy L.



KAT5 tyrosine phosphorylation couples chromatin sensing to ATM signalling.  


The detection of DNA lesions within chromatin represents a critical step in cellular responses to DNA damage. However, the regulatory mechanisms that couple chromatin sensing to DNA-damage signalling in mammalian cells are not well understood. Here we show that tyrosine phosphorylation of the protein acetyltransferase KAT5 (also known as TIP60) increases after DNA damage in a manner that promotes KAT5 binding to the histone mark H3K9me3. This triggers KAT5-mediated acetylation of the ATM kinase, promoting DNA-damage-checkpoint activation and cell survival. We also establish that chromatin alterations can themselves enhance KAT5 tyrosine phosphorylation and ATM-dependent signalling, and identify the proto-oncogene c-Abl as a mediator of this modification. These findings define KAT5 tyrosine phosphorylation as a key event in the sensing of genomic and chromatin perturbations, and highlight a key role for c-Abl in such processes. PMID:23708966

Kaidi, Abderrahmane; Jackson, Stephen P



Regulation of CDK9 Activity by Phosphorylation and Dephosphorylation  

PubMed Central

HIV-1 transcription is regulated by CDK9/cyclin T1, which, unlike a typical cell cycle-dependent kinase, is regulated by associating with 7SK small nuclear ribonuclear protein complex (snRNP). While the protein components of this complex are well studied, the mechanism of the complex formation is still not fully understood. The association of CDK9/cyclin T1 with 7SK snRNP is, in part, regulated by a reversible CDK9 phosphorylation. Here, we present a comprehensive review of the kinases and phosphatases involved in CDK9 phosphorylation and discuss their role in regulation of HIV-1 replication and potential for being targeted for drug development. We propose a novel pathway of HIV-1 transcription regulation via CDK9 Ser-90 phosphorylation by CDK2 and CDK9 Ser-175 dephosphorylation by protein phosphatase-1. PMID:24524087



Regulatory Phosphorylation of Ikaros by Bruton's Tyrosine Kinase  

PubMed Central

Diminished Ikaros function has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL), the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros is of paramount importance for normal lymphocyte ontogeny. Here we provide genetic and biochemical evidence for a previously unknown function of Bruton's tyrosine kinase (BTK) as a partner and posttranslational regulator of Ikaros, a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis. We demonstrate that BTK phosphorylates Ikaros at unique phosphorylation sites S214 and S215 in the close vicinity of its zinc finger 4 (ZF4) within the DNA binding domain, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Our results further demonstrate that BTK-induced activating phosphorylation is critical for the optimal transcription factor function of Ikaros. PMID:23977012

Zhang, Jian; Ishkhanian, Rita; Uckun, Fatih M.



Dopamine D1 receptors mediate CREB phosphorylation via phosphorylation of the NMDA receptor at Ser897-NR1  

PubMed Central

Addictive drugs such as amphetamine and cocaine stimulate the dopaminergic system, activate dopamine receptors and induce gene expression throughout the striatum. The signal transduction pathway leading from dopamine receptor stimulation at the synapse to gene expression in the nucleus has not been fully elucidated. Here, we present evidence that D1 receptor stimulation leads to phosphorylation of the transcription factor Ca2+ and cyclic AMP response element binding protein (CREB) in the nucleus by means of NMDA receptor-mediated Ca2+ signaling. Stimulation of D1 receptors induces the phosphorylation of Ser897 on the NR1 subunit by protein kinase A (PKA). This phosphorylation event is crucial for D1 receptor-mediated CREB phosphorylation. Dopamine cannot induce CRE-mediated gene expression in neurons transfected with a phosphorylation-deficient NR1 construct. Moreover, stimulation of D1 receptors or increase in cyclic AMP levels leads to an increase in cytosolic Ca2+ in the presence of glutamate, but not in the absence of glutamate, indicating the ability of dopamine and cyclic AMP to facilitate NMDA channel activity. The recruitment of the NMDA receptor signal transduction pathway by D1 receptors may provide a general mechanism for gene regulation that is fundamental for mechanisms of drug addiction and long-term memory. PMID:14622123

Dudman, Joshua T.; Eaton, Molly E.; Rajadhyaksha, Anjali; Macias, Wendy; Taher, Muffadal; Barczak, Amy; Kameyama, Kimihiko; Huganir, Richard; Konradi, Christine



Study of the Enzymatic Hydrolysis of Cellulose for Production of Fuel Ethanol  

E-print Network

Study of the Enzymatic Hydrolysis of Cellulose for Production of Fuel Ethanol by the Simultaneous to ethanol, a promising alternative fuel, can be carried out efficiently and economically using are presented in light of the impact of enzymatic hydrolysis on fuel ethanol production. Key words: enzymatic

California at Riverside, University of


Enzymatic Resolution and Separation of Secondary Alcohols Based on Fatty Esters as Acylating Agents  

ERIC Educational Resources Information Center

The enzymatic resolution of "rac"-1-phenylethanol using ethyl myristate as acylating agent and solvent and "Candida antarctica" lipase B (CAL-B) as biocatalyst was demonstrated with catalyst and medium reuse. Both enantiomers of 1-phenylethanol were isolated by sequential enzymatic reactions and product distillations. From the first enzymatic

Monteiro, Carlos M.; Afonso, Carlos A. M.; Lourenco, Nuno M. T.



Application of Uncertainty and Sensitivity Analysis to a Kinetic Model for Enzymatic Biodiesel Production  

E-print Network

Application of Uncertainty and Sensitivity Analysis to a Kinetic Model for Enzymatic Biodiesel benefits of using uncertainty and sensitivity analysis in the kinetics of enzymatic biodiesel production, Monte-Carlo Simulations, Enzymatic Biodiesel 1. INTRODUCTION In order to determine the optimal

Mosegaard, Klaus


?-catenin tyrosine 654 phosphorylation increases Wnt signalling and intestinal tumorigenesis  

PubMed Central

Objective Deregulation of the Wnt signalling pathway by mutations in the Apc or ?-catenin genes underlies colorectal carcinogenesis. As a result, ?-catenin stabilises, translocates to the nucleus, and activates gene transcription. Intestinal tumours show a heterogeneous pattern of nuclear ?-catenin, with the highest levels observed at the invasion front. Activation of receptor tyrosine kinases in these tumour areas by growth factors expressed by surrounding stromal cells phosphorylate ?-catenin at tyrosine residues, which is thought to increase ?-catenin nuclear translocation and tumour invasiveness. This study investigates the relevance of ?-catenin tyrosine phosphorylation for Wnt signalling and intestinal tumorigenesis in vivo. Design A conditional knock-in mouse model was generated into which the phospho-mimicking Y654E modification in the endogenous ?-catenin gene was introduced. Results This study provided in vivo evidence that ?-cateninE654 is characterised by reduced affinity for cadherins, increased signalling and strongly increased phosphorylation at serine 675 by protein kinase A (PKA). In addition, homozygosity for the ?-cateninE654 targeted allele caused embryonic lethality, whereas heterozygosity predisposed to intestinal tumour development, and strongly enhanced Apc-driven intestinal tumour initiation associated with increased nuclear accumulation of ?catenin. Surprisingly, the expression of ?-cateninE654 did not affect histological grade or induce tumour invasiveness. Conclusions A thus far unknown mechanism was uncovered in which Y654 phosphorylation of ?-catenin facilitates additional phosphorylation at serine 675 by PKA. In addition, in contrast to the current belief that ?-catenin Y654 phosphorylation increases tumour progression to a more invasive phenotype, these results show that it rather increases tumour initiation by enhancing Wnt signalling. PMID:21307168

Le, Ngoc H; Helvensteijn, Werner; Blonden, Lau; Theeuwes, Myrte; Bakker, Elvira R M; Franken, Patrick F; van Gurp, Leon; Meijlink, Frits; van der Valk, Martin A; Kuipers, Ernst J; Fodde, Riccardo; Smits, Ron



Liprin phosphorylation regulates binding to LAR: evidence for liprin autophosphorylation.  


The LAR transmembrane tyrosine phosphatase associates with liprin-alpha proteins and colocalizes with liprin-alpha1 at focal adhesions. LAR has been implicated in axon guidance, and liprins are involved in synapse formation and synapse protein trafficking. Several liprin mutants have weaker binding to LAR as assessed by yeast interaction trap assays, and the extents of in vitro and in vivo phosphorylation of these mutants were reduced relative to that of wild-type liprin-alpha1. Treatment of liprin-alpha1 with calf intestinal phosphatase weakened its interaction with the recombinant GST-LAR protein. A liprin LH region mutant that inhibited liprin phosphorylation did not bind to LAR as assessed by coprecipitation studies. Endogenous LAR was shown to bind phosphorylated liprin-alpha1 from MDA-486 cells labeled in vivo with [32P]orthophosphate. In further characterizing the phosphorylation of liprin, we found immunoprecipitates of liprin-alpha1 expressed in COS-7 cells to incorporate phosphate after washes of up to 4 M NaCl. Additionally, purified liprin-alpha1 derived from Sf-9 insect cells retained the ability to incorporate phosphate in in vitro phosphorylation assays, and a liprin-alpha1 truncation mutant incorporated phosphate after denaturation and/or renaturation in SDS gels. Finally, binding assays showed that liprin binds to ATP-agarose and that the interaction is challenged by free ATP, but not by free GTP. Moreover, liprin LH region mutations that inhibit liprin phosphorylation stabilized the association of liprin with ATP-agarose. Taken together, our results suggest that liprin autophosphorylation regulates its association with LAR. PMID:16313174

Serra-Pagès, Carles; Streuli, Michel; Medley, Quintus G



Characterization and Prediction of Protein Phosphorylation Hotspots in Arabidopsis thaliana  

PubMed Central

The regulation of protein function by modulating the surface charge status via sequence-locally enriched phosphorylation sites (P-sites) in so called phosphorylation “hotspots” has gained increased attention in recent years. We set out to identify P-hotspots in the model plant Arabidopsis thaliana. We analyzed the spacing of experimentally detected P-sites within peptide-covered regions along Arabidopsis protein sequences as available from the PhosPhAt database. Confirming earlier reports (Schweiger and Linial, 2010), we found that, indeed, P-sites tend to cluster and that distributions between serine and threonine P-sites to their respected closest next P-site differ significantly from those for tyrosine P-sites. The ability to predict P-hotspots by applying available computational P-site prediction programs that focus on identifying single P-sites was observed to be severely compromised by the inevitable interference of nearby P-sites. We devised a new approach, named HotSPotter, for the prediction of phosphorylation hotspots. HotSPotter is based primarily on local amino acid compositional preferences rather than sequence position-specific motifs and uses support vector machines as the underlying classification engine. HotSPotter correctly identified experimentally determined phosphorylation hotspots in A. thaliana with high accuracy. Applied to the Arabidopsis proteome, HotSPotter-predicted 13,677 candidate P-hotspots in 9,599 proteins corresponding to 7,847 unique genes. Hotspot containing proteins are involved predominantly in signaling processes confirming the surmised modulating role of hotspots in signaling and interaction events. Our study provides new bioinformatics means to identify phosphorylation hotspots and lays the basis for further investigating novel candidate P-hotspots. All phosphorylation hotspot annotations and predictions have been made available as part of the PhosPhAt database at PMID:22973286

Christian, Jan-Ole; Braginets, Rostyslav; Schulze, Waltraud X.; Walther, Dirk



Bioinformatics Study of Cancer-Related Mutations within p53 Phosphorylation Site Motifs  

PubMed Central

p53 protein has about thirty phosphorylation sites located at the N- and C-termini and in the core domain. The phosphorylation sites are relatively less mutated than other residues in p53. To understand why and how p53 phosphorylation sites are rarely mutated in human cancer, using a bioinformatics approaches, we examined the phosphorylation site and its nearby flanking residues, focusing on the consensus phosphorylation motif pattern, amino-acid correlations within the phosphorylation motifs, the propensity of structural disorder of the phosphorylation motifs, and cancer mutations observed within the phosphorylation motifs. Many p53 phosphorylation sites are targets for several kinases. The phosphorylation sites match 17 consensus sequence motifs out of the 29 classified. In addition to proline, which is common in kinase specificity-determining sites, we found high propensity of acidic residues to be adjacent to phosphorylation sites. Analysis of human cancer mutations in the phosphorylation motifs revealed that motifs with adjacent acidic residues generally have fewer mutations, in contrast to phosphorylation sites near proline residues. p53 phosphorylation motifs are mostly disordered. However, human cancer mutations within phosphorylation motifs tend to decrease the disorder propensity. Our results suggest that combination of acidic residues Asp and Glu with phosphorylation sites provide charge redundancy which may safe guard against loss-of-function mutations, and that the natively disordered nature of p53 phosphorylation motifs may help reduce mutational damage. Our results further suggest that engineering acidic amino acids adjacent to potential phosphorylation sites could be a p53 gene therapy strategy. PMID:25075982

Ji, Xiaona; Huang, Qiang; Yu, Long; Nussinov, Ruth; Ma, Buyong



Enzymatic fuel cells: integrating flow-through anode and air-breathing cathode into a membrane-less biofuel cell design.  


One of the key goals of enzymatic biofuel cells research has been the development of a fully enzymatic biofuel cell that operates under a continuous flow-through regime. Here, we present our work on achieving this task. Two NAD(+)-dependent dehydrogenase enzymes; malate dehydrogenase (MDH) and alcohol dehydrogenase (ADH) were independently coupled with poly-methylene green (poly-MG) catalyst for biofuel cell anode fabrication. A fungal laccase that catalyzes oxygen reduction via direct electron transfer (DET) was used as an air-breathing cathode. This completes a fully enzymatic biofuel cell that operates in a flow-through mode of fuel supply polarized against an air-breathing bio-cathode. The combined, enzymatic, MDH-laccase biofuel cell operated with an open circuit voltage (OCV) of 0.584 V, whereas the ADH-laccase biofuel cell sustained an OCV of 0.618 V. Maximum volumetric power densities approaching 20 ?W cm(-3) are reported, and characterization criteria that will aid in future optimization are discussed. PMID:21775124

Rincón, Rosalba A; Lau, Carolin; Luckarift, Heather R; Garcia, Kristen E; Adkins, Emily; Johnson, Glenn R; Atanassov, Plamen



Ferredoxin:NADP+ oxidoreductase in junction with CdSe/ZnS quantum dots: characteristics of an enzymatically active nanohybrid  

NASA Astrophysics Data System (ADS)

Ferredoxin:NADP+ oxidoreductase (FNR) is a plant and cyanobacterial photosynthetic enzyme, also found in non-photosynthetic tissues, where it is involved in redox reactions of biosynthetic pathways. In vivo it transfers electrons to nicotinamide adenine dinucleotide phosphate (NADP+), forming its reduced version, NADPH, while in vitro it can also use NADPH to reduce several substrates, such as ferricyanide, various quinones and nitriles. As an oxidoreductase catalyzing reaction of a broad range of substrates, FNR may be used in biotechnological processes. Quantum dots are semiconductor nanocrystals of a few to several nanometers diameter, having very useful luminescent properties. We present the spectroscopic and functional characteristics of a covalent conjugation of FNR and CdSe/ZnS quantum dots. Two types of quantum dots, of different diameter and emission maximum (550 and 650 nm), were used for comparison. Steady-state fluorescence and gel electrophoresis confirmed efficient conjugation, while fluorescence correlation spectroscopy (FCS) allowed for determination of the conjugates’ radii. The nanohybrids sustained enzymatic activity; however, changes in maximal reaction rates and Michaelis constant were found. Detailed analysis of the kinetic parameters showed that the changes in the enzyme activity depend on the substrate used for activity measurement but also on the size of the quantum dots. The presented nanohybrids, as the first example using plant and photosynthetic enzyme as a protein partner, may became a tool to study photosynthesis as well as other biosynthetic and biotechnological processes, involving enzymatically catalyzed electron transfer.

Szczepaniak, Krzysztof; Worch, Remigiusz; Grzyb, Joanna



Regulation of pig heart pyruvate dehydrogenase by phosphorylation. Studies on the subunit and phosphorylation stoicheiometries  

PubMed Central

1. The molecular weights of the subunits of purified pig heart pyruvate dehydrogenase complex were determined by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis and were: pyruvate decarboxylase, ?-subunit 40600, ?-subunit 35100; dihydrolipoyl acetyltransferase 76100; dihydrolipoyl dehydrogenase 58200. 2. Inactivation of the pyruvate dehydrogenase complex by its integral kinase corresponded to the incorporation of 0.46nmol of P/unit of complex activity inactivated. 3. Further incorporation of phosphate into the complex occurred to a limit of 1.27nmol of P/unit of complex inactivated (approx. 3 times that required for inactivation). 4. Phosphate was incorporated only into the ?-subunit of the decarboxylase. 5. The molar ratio of phosphate to ?-subunits of the decarboxylase was estimated by radioamidination of amino groups of pyruvate dehydrogenase [32P]phosphate complex by using methyl [1-14C]acetimidate, followed by separation of ?-subunits by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis. Inactivation of the complex (0.46nmol of P/unit of complex inactivated) corresponded to a molar ratio of one phosphate group per two ?-chains (i.e. one phosphate group/?2?2 tetramer). Complete phosphorylation corresponded to three phosphate groups per ?2?2 tetramer. 6. Subunit molar ratios in the complex were also estimated by the radioamidination technique. Results corresponded most closely to molar ratios of 4 ?-subunits:4 ?-subunits:2 dihydrolipoyl acetyltransferase subunits:1 dihydrolipoyl dehydrogenase subunit. PMID:697742

Sugden, Peter H.; Randle, Philip J.



Encephalomyocarditis Virus Leader Is Phosphorylated by CK2 and Syk as a Requirement for Subsequent Phosphorylation of Cellular Nucleoporins  

PubMed Central

ABSTRACT Encephalomyocarditis virus and Theilovirus are species in the Cardiovirus genus of the Picornaviridae family. For all cardioviruses, the viral polyprotein is initiated with a short Leader (L) protein unique to this genus. The nuclear magnetic resonance (NMR) structure of LE from encephalomyocarditis virus (EMCV) has been determined. The protein has an NH2-proximal CHCC zinc finger, a central linker, and a contiguous, highly acidic motif. The theiloviruses encode the same domains, with one or two additional, COOH-proximal domains, characteristic of the human Saffold viruses (SafV) and Theiler's murine encephalomyelitis viruses (TMEV), respectively. The expression of a cardiovirus L, in recombinant form, or during infection/transfection, triggers an extensive, cell-dependent, antihost phosphorylation cascade, targeting nucleoporins (Nups) that form the hydrophobic core of nuclear pore complexes (NPC). The consequent inhibition of active nucleocytoplasmic trafficking is potent and prevents the host from mounting an effective antiviral response. For this inhibition, the L proteins themselves must be phosphorylated. In cells (extracts or recombinant form), LE was shown to be phosphorylated at Thr47 and Tyr41. The first reaction (Thr47), catalyzed by casein kinase 2 (CK2), is an obligatory precedent to the second event (Tyr41), catalyzed by spleen tyrosine kinase (Syk). Site mutations in LE, or kinase-specific inhibitors, prevented LE phosphorylation and subsequent Nup phosphorylation. Parallel experiments with LS (SafV-2) and LT (TMEV BeAn) proteins confirmed the general cardiovirus requirement for L phosphorylation, but CK2 was not the culpable kinase. It is likely that LS and LT are both activated by alternative kinases in different cell types, probably reactive within the Theilo-specific domains. IMPORTANCE PMID:24335301

Basta, Holly A.; Bacot-Davis, Valjean R.; Ciomperlik, Jessica J.



Increased leukocyte survival and accelerated onset of lymphoma in the absence of MCL-1 S159-phosphorylation.  


The antiapoptotic BCL-2 protein MCL-1, which opposes mitochondrial outer membrane permeabilization, was shown to have a crucial role in the survival of hematopoietic cells. We have previously shown that, upon loss of phosphatidylinositol 3-kinase signaling, S159 of MCL-1 is phosphorylated by glycogen synthase kinase-3 (GSK-3), earmarking MCL-1 for enhanced ubiquitylation and degradation. In this study, we introduced MCL-1(wt) or the phosphorylation-deficient mutant MCL-1(S159A) in mouse BM cells, followed by adoptive transfer to recipient mice. Mice expressing MCL-1(S159A) exhibited significantly elevated white blood cell and lymphocyte counts, whereas no effect was observed on the distribution of T and B lymphocyte subsets or the numbers of monocytes, red blood cells or platelets. Expression of MCL-1(S159A) in E?-Myc transgenic bone marrow significantly accelerated the onset of disease, and these mice displayed increased spleen weights compared with E?-Myc/MCL-1(wt) mice. Our data demonstrate that the absence of MCL-1 S159 phosphorylation provides a survival advantage for hematopoietic cells in vivo and facilitates oncogenesis. PMID:24213575

Lindner, S E; Wissler, M; Gründer, A; Aumann, K; Ottina, E; Peintner, L; Brauns-Schubert, P; Preiss, F; Herzog, S; Borner, C; Charvet, C; Villunger, A; Pahl, H L; Maurer, U



Rho GTPase\\/Rho Kinase Negatively Regulates Endothelial Nitric Oxide Synthase Phosphorylation through the Inhibition of Protein Kinase B\\/Akt in Human Endothelial Cells  

Microsoft Academic Search

Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)\\/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase\\/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human

Xiu-Fen Ming; Hema Viswambharan; Christine Barandier; Jean Ruffieux; Kozo Kaibuchi; Sandro Rusconi; Zhihong Yang



Enzymatic hydrolysis of steryl glycosides for their analysis in foods.  


Steryl glycosides (SG) contribute significantly to the total intake of phytosterols. The standard analytical procedure involving acid hydrolysis fails to reflect the correct sterol profile of SG due to isomerization of some of the labile sterols. Therefore, various glycosylases were evaluated for their ability to hydrolyse SG under milder conditions. Using a pure SG mixture in aqueous solution, the highest glycolytic activity, as demonstrated by the decrease in SG and increase in free sterols was achieved using inulinase preparations (decrease of >95%). High glycolytic activity was also demonstrated using hemicellulase (63%). The applicability of enzymatic hydrolysis using inulinase preparations was further verified on SG extracted from foods. For example in potato peel ?(5)-avenasteryl glucoside, a labile SG, was well preserved and contributed 26.9% of the total SG. Therefore, enzymatic hydrolysis is suitable for replacing acid hydrolysis of SG in food lipid extracts to accurately determine the sterol profile of SG. PMID:24912717

Münger, Linda H; Nyström, Laura



Enzymatic synthesis of biodiesel via alcoholysis of palm oil.  


The enzymatic alcoholysis of crude palm oil with methanol and ethanol was investigated using commercial immobilized lipases (Lipozyme RM IM, Lipozyme TL IM). The effect of alcohol (methanol or ethanol), molar ratio of alcohol to crude palm oil, and temperature on biodiesel production was determined. The best ethyl ester yield was about 25 wt.% and was obtained with ethanol/oil molar ratio of 3.0, temperature of 50 degrees C, enzyme concentration of 3.0 wt.%, and stepwise addition of the alcohol after 4 h of reaction. Experiments with 1 and 3 wt.% of KOH and 3 wt.% of MgO were carried out to compare their catalytic behavior with the enzymatic transesterification results. The commercial immobilized lipase, Lipozyme TL IM, showed the best catalytic performance. PMID:19023524

Matassoli, André L F; Corrêa, Igor N S; Portilho, Márcio F; Veloso, Cláudia O; Langone, Marta A P



Pretreatment of Agave americana stalk for enzymatic saccharification.  


Agave americana is one of commonly grown agave species but currently less valuable because its large flower stalk cannot be used for producing alcoholic beverage. In the present study, the stalk was pretreated with dilute acid (DA), sulfite (SPORL), and sodium hydroxide (NaOH) to preliminarily assess its potential as feedstock for bioethanol production. The changes of cell wall components during the pretreatments, enzymatic digestibility of the pretreated stalks, and the adsorption of cellulases on the substrates were investigated. Results indicated that the pretreatments significantly improved the enzymatic digestibility of the agave stalk. SPORL pretreatment gave higher substrate and sugar yields, while NaOH pretreated stalk had better digestibility under the investigated conditions. The better hydrolysability of NaOH-pretreated stalk was attributed to low lignin and hemicellulose content and high affinity to cellulases. PMID:23122484

Yang, Qiang; Pan, Xuejun



Peripheral enzymatic deacetylation of chitin and reprecipitated chitin particles.  


The enzymatic deacetylation of various chitin preparations was investigated using the fungal chitin deacetylase (CDA) isolated from Rhizopus oryzae growth medium. Specific extracellular enzyme activity after solid state fermentation was 10 times higher than that after submerged fermentation. Natural crystalline chitin is a very poor substrate for the enzyme, but showed a five-time better deacetylation after dissolution and reprecipitation. Chitin particles, enzymatically deacetylated for only 1% exhibited a strongly increased binding capacity towards ovalbumin, while maintaining the rigidity and insolubility of chitin in a moderate acidic environment. Because of the unique combination of properties, these CDA treated chitin materials were named "chit-in-osan". Chitinosan was shown to be an attractive matrix for column chromatography because no hydrogel formation was observed, that impaired the flow of eluent. Under the same conditions, partially deacetylated chitosan swelled and blocked the flow in the column. PMID:15919204

Aye, Kyaw Nyein; Karuppuswamy, Renuka; Ahamed, Tangir; Stevens, Willem F



Bioremediation of uranium contamination with enzymatic uranium reduction  

USGS Publications Warehouse

Enzymatic uranium reduction by Desulfovibrio desulfuricans readily removed uranium from solution in a batch system or when D. desulfuricans was separated from the bulk of the uranium-containing water by a semipermeable membrane. Uranium reduction continued at concentrations as high as 24 mM. Of a variety of potentially inhibiting anions and metals evaluated, only high concentrations of copper inhibited uranium reduction. Freeze-dried cells, stored aerobically, reduced uranium as fast as fresh cells. D. desulfuricans reduced uranium in pH 4 and pH 7.4 mine drainage waters and in uraniumcontaining groundwaters from a contaminated Department of Energy site. Enzymatic uranium reduction has several potential advantages over other bioprocessing techniques for uranium removal, the most important of which are as follows: the ability to precipitate uranium that is in the form of a uranyl carbonate complex; high capacity for uranium removal per cell; the formation of a compact, relatively pure, uranium precipitate.

Lovley, D. R.; Phillips, E. J. P.



Coulometric titration of D(+)-glucose using its enzymatic oxidation.  


A definitive method is described for the indirect assay of milligram quantities of D(+)-glucose by coulometric titration. D(+)-Glucose was aerobically oxidized by glucose oxidase in an acetate buffer solution (pH 5.1). Subsequently, the enzymatically formed hydrogen peroxide was titrated coulometrically with electrogenerated hypobromite in sodium bromide-sodium tetraborate medium of pH 8.6, with biamperometric end-point detection. Parameters affecting the enzymatically catalyzed oxidation and coulometric titration were evaluated. The optimized conditions for the oxidation of up to 20 mg of D(+)-glucose include the addition of 4500 U of glucose oxidase and stirring over a 10-min interval at 25 degrees C. Under proposed conditions, the assay values of several commercial D(+)-glucose reagents were somewhat lower than the guaranteed minimum values, with RSDs (n = 5) of 0.071 - 0.106%. PMID:11990540

Tanaka, T; Shutto, E; Mizoguchi, T; Fukushima, K



Correlation Analysis of Enzymatic Reaction of a Single Protein Molecule  

PubMed Central

New advances in nano sciences open the door for scientists to study biological processes on a microscopic molecule-by-molecule basis. Recent single-molecule biophysical experiments on enzyme systems, in particular, reveal that enzyme molecules behave fundamentally differently from what classical model predicts. A stochastic network model was previously proposed to explain the experimental discovery. This paper conducts detailed theoretical and data analyses of the stochastic network model, focusing on the correlation structure of the successive reaction times of a single enzyme molecule. We investigate the correlation of experimental fluorescence intensity and the correlation of enzymatic reaction times, and examine the role of substrate concentration in enzymatic reactions. Our study shows that the stochastic network model is capable of explaining the experimental data in depth. PMID:23408514

Du, Chao; Kou, S. C.



Enzymatic fabrication of high-density RNA arrays.  


A powerful new strategy for the fabrication of high-density RNA arrays is described. A high-density DNA array is fabricated by standard photolithographic methods, the surface-bound DNA molecules are enzymatically copied into their RNA complements from a surface-bound RNA primer, and the DNA templates are enzymatically destroyed, leaving behind the desired RNA array. The strategy is compatible with 2'-fluoro-modified (2'F) ribonucleoside triphosphates (rNTPs), which may be included in the polymerase extension reaction to impart nuclease resistance and other desirable characteristics to the synthesized RNAs. The use and fidelity of the arrays are explored with DNA hybridization, DNAzyme cleavage, and nuclease digestion experiments. PMID:25339581

Wu, Cheng-Hsien; Holden, Matthew T; Smith, Lloyd M



Mass transfer andMass transfer and Mass transfer andMass transfer and  

E-print Network

eknik Mass transfer andMass transfer and arationste Mass transfer andMass transfer and separation / mass transfer analogy7. Heat / mass transfer analogy Massöve Ron Zevenhoven �bo Akademi Universityy.1 Heat / mass transfer analogy7.1 Heat / mass transfer analogy 4 erföringo gygy Massöve RoNzfebruari 2012

Zevenhoven, Ron


Stress induces pain transition by potentiation of AMPA receptor phosphorylation.  


Chronic postsurgical pain is a serious issue in clinical practice. After surgery, patients experience ongoing pain or become sensitive to incident, normally nonpainful stimulation. The intensity and duration of postsurgical pain vary. However, it is unclear how the transition from acute to chronic pain occurs. Here we showed that social defeat stress enhanced plantar incision-induced AMPA receptor GluA1 phosphorylation at the Ser831 site in the spinal cord and greatly prolonged plantar incision-induced pain. Interestingly, targeted mutation of the GluA1 phosphorylation site Ser831 significantly inhibited stress-induced prolongation of incisional pain. In addition, stress hormones enhanced GluA1 phosphorylation and AMPA receptor-mediated electrical activity in the spinal cord. Subthreshold stimulation induced spinal long-term potentiation in GluA1 phosphomimetic mutant mice, but not in wild-type mice. Therefore, spinal AMPA receptor phosphorylation contributes to the mechanisms underlying stress-induced pain transition. PMID:25297100

Li, Changsheng; Yang, Ya; Liu, Sufang; Fang, Huaqiang; Zhang, Yong; Furmanski, Orion; Skinner, John; Xing, Ying; Johns, Roger A; Huganir, Richard L; Tao, Feng



Maximal entropy inference of oncogenicity from phosphorylation signaling  

PubMed Central

Point mutations in the phosphorylation domain of the Bcr-Abl fusion oncogene give rise to drug resistance in chronic myelogenous leukemia patients. These mutations alter kinase-mediated signaling function and phenotypic outcome. An information theoretic analysis of the correlation of phosphoproteomic profiling and transformation potency of the oncogene in different mutants is presented. The theory seeks to predict the leukemic transformation potency from the observed signaling by constructing a distribution of maximal entropy of site-specific phosphorylation events. The theory is developed with special reference to systems biology where high throughput measurements are typical. We seek sets of phosphorylation events most contributory to predicting the phenotype by determining the constraints on the signaling system. The relevance of a constraint is measured by how much it reduces the value of the entropy from its global maximum, where all events are equally likely. Application to experimental phospho-proteomics data for kinase inhibitor-resistant mutants shows that there is one dominant constraint and that other constraints are not relevant to a similar extent. This single constraint accounts for much of the correlation of phosphorylation events with the oncogenic potency and thereby usefully predicts the trends in the phenotypic output. An additional constraint possibly accounts for biological fine structure. PMID:20224037

Graeber, T. G.; Heath, J. R.; Skaggs, B. J.; Phelps, M. E.; Remacle, F.; Levine, R. D.



Regulation of neuronal cytoskeletal protein phosphorylation in neurodegenerative diseases.  


Neuronal cytoskeletal proteins such as neurofilaments (NFs) and tau are aberrantly and hyperphosphorylated in neurodegeneration. Under normal physiological conditions, NFs are synthesized in the cell bodies and phosphorylated and transported in the axonal compartment. However, under neurodegenerative disorders such as Alzheimer's disease (AD), spinal cord motor neuron inclusions of amyotrophic lateral sclerosis, Lewy bodies of Parkinson's disease, Pick's disease, Charcot-Marie-Tooth disease, and diabetic neuropathy, NFs are aberrantly and hyperphosphorylated in cell bodies. The proline directed protein kinases, such as cyclin-dependent protein kinase 5, mitogen activated protein kinase, and glycogen synthase kinase 3?, and the non proline-directed kinases, such as casein kinase 1, are deregulated in AD. Moreover, the reversible phosphorylation by protein phosphatase, PP2A, which mainly carries out the dephosphorylation of tau and NFs, is down regulated in AD brain. The aberrant phosphorylation of cytoskeletal proteins such as tau and NFs results in the axonal transport defects in neurodegeneration. The peptidyl-prolyl isomerase Pin1 plays a regulatory role in the post-phosphorylation mechanism of neuronal cytoskeletal proteins in AD brain. Possible therapeutic interventions for neurodegenerative disorders are (1) inhibition of proline-directed kinases, (2) activation of protein phosphatases such as PP2A, and (3) modulation of peptidyl-prolyl isomerases such as Pin1. Here, I discuss the regulation of neuronal cytoskeletal proteins under physiology and pathology. PMID:24748119

Rudrabhatla, Parvathi



Michael Lampson, Research Proposal Measuring phosphorylation dynamics in living cells  

E-print Network

, and then track the fate of the cells' progeny in the same animal. These studies will begin to bridge the gap will measure phosphorylation dynamics with high temporal and spatial resolution in living cells to test in the context of a vertebrate disease model. Aim 1: Test the hypothesis that local signaling networks operate

Kim, Junhyong


Phosphorylation Controls Autoinhibition of Cytoplasmic Linker Protein-170  

PubMed Central

Cytoplasmic linker protein (CLIP)-170 is a microtubule (MT) plus-end-tracking protein that regulates MT dynamics and links MT plus ends to different intracellular structures. We have shown previously that intramolecular association between the N and C termini results in autoinhibition of CLIP-170, thus altering its binding to MTs and the dynactin subunit p150Glued (J. Cell Biol. 2004: 166, 1003–1014). In this study, we demonstrate that conformational changes in CLIP-170 are regulated by phosphorylation that enhances the affinity between the N- and C-terminal domains. By using site-directed mutagenesis and phosphoproteomic analysis, we mapped the phosphorylation sites in the third serine-rich region of CLIP-170. A phosphorylation-deficient mutant of CLIP-170 displays an “open” conformation and a higher binding affinity for growing MT ends and p150Glued as compared with nonmutated protein, whereas a phosphomimetic mutant confined to the “folded back” conformation shows decreased MT association and does not interact with p150Glued. We conclude that phosphorylation regulates CLIP-170 conformational changes resulting in its autoinhibition. PMID:20519438

Lee, Ho-Sup; Nadezhdina, Elena S.; Anjum, Rana; Peloquin, John G.; Schober, Joseph M.; Danciu, Oana; van Haren, Jeffrey; Galjart, Niels; Gygi, Steven P.; Akhmanova, Anna; Borisy, Gary G.



Regulation of Tau Phosphorylation in Microtubule Fractions by Apolipoprotein E  

E-print Network

pathology correlates with the clinical severity of AD. Certain apolipoprotein E (apoE) isoforms have been an interaction and potentially modulatory effects on tau hyperphosphorylation by the different apoE isoforms. In these studies, we directly tested the effects of apoE, E2 ,E3, and E4 on AD-like phosphorylation of tau in brain

Wood, John G.


Phosphorylation at Tyrosine 262 Promotes GADD34 Protein Turnover*  

PubMed Central

In mammalian cells, metabolic and environmental stress increases the phosphorylation of the eukaryotic translational initiation factor, eIF2?, and attenuates global protein synthesis. Subsequent transcriptional activation of GADD34 assembles an eIF2? phosphatase that feeds back to restore mRNA translation. Active proteasomal degradation of GADD34 protein then reestablishes the sensitivity of cells to subsequent bouts of stress. Mass spectrometry established GADD34 phosphorylation on multiple serines, threonines, and tyrosines. Phosphorylation at tyrosine 262 enhanced the rate of the GADD34 protein turnover. Substrate-trapping studies identified TC-PTP (PTPN2) as a potential GADD34 phosphatase, recognizing phosphotyrosine 262. Reduced GADD34 protein levels in TC-PTP-null MEFs following ER stress emphasized the importance of TC-PTP in determining the cellular levels of GADD34 protein. The susceptibility of TC-PTP-null MEFs to ER stress-induced apoptosis was significantly ameliorated by ectopic expression of GADD34. The data suggested that GADD34 phosphorylation on tyrosine 262 modulates endoplasmic reticulum stress signaling and cell fate. PMID:24092754

Zhou, Wei; Jeyaraman, Krishna; Yusoff, Permeen; Shenolikar, Shirish



Circadian KaiC Phosphorylation: A Multi-Layer Network  

PubMed Central

Circadian KaiC phosphorylation in cyanobacteria reconstituted in vitro recently initiates a series of studies experimentally and theoretically to explore its mechanism. In this paper, we report a dynamic diversity in hexameric KaiC phosphoforms using a multi-layer reaction network based on the nonequivalence of the dual phosphorylation sites (S431 and T432) in each KaiC subunit. These diverse oscillatory profiles can generate a kaleidoscopic phase modulation pattern probably responsible for the genome-wide transcription rhythms directly and/or indirectly in cyanobacteria. Particularly, our model reveals that a single KaiC hexamer is an energy-based, phosphorylation-dependent and self-regulated circadian oscillator modulated by KaiA and KaiB. We suggest that T432 is the main regulator for the oscillation amplitude, while S431 is the major phase regulator. S431 and T432 coordinately control the phosphorylation period. Robustness of the Kai network was examined by mixing samples in different phases, and varying protein concentrations and temperature. Similar results were obtained regardless of the deterministic or stochastic method employed. Therefore, the dynamic diversities and robustness of Kai oscillator make it a qualified core pacemaker that controls the cellular processes in cyanobacteria pervasively and accurately. PMID:19936045

Li, Congxin; Chen, Xiaofang; Wang, Pengye; Wang, Weichi



CGGBP1 phosphorylation constitutes a telomere-protection signal  

PubMed Central

The shelterin proteins are required for telomere integrity. Shelterin dysfunction can lead to initiation of unwarranted DNA damage and repair pathways at chromosomal termini. Interestingly, many shelterin accessory proteins are involved in DNA damage signaling and repair. We demonstrate here that in normal human fibroblasts, telomeric ends are protected by phosphorylation of CGG triplet repeat-binding protein 1 (CGGBP1) at serine 164 (S164). We show that serine 164 is a major phosphorylation site on CGGBP1 with important functions. We provide evidence that one of the kinases that can phosphorylate S164 CGGBP1 is ATR. Overexpression of S164A phospho-deficient CGGBP1 exerted a dominant-negative effect, causing telomeric dysfunction, accelerated telomere shortening, enhanced fusion of telomeres, and crisis. However, overexpression of wild-type or phospho-mimicking S164E CGGBP1 did not cause these effects. This telomere damage was associated with reduced binding of the shelterin protein POT1 to telomeric DNA. Our results suggest that CGGBP1 phosphorylation at S164 is a novel telomere protection signal, which can affect telomere-protective function of the shelterin complex. PMID:24196442

Singh, Umashankar; Maturi, Varun; Jones, Rhiannon E; Paulsson, Ylva; Baird, Duncan M; Westermark, Bengt



Role of tyrosine phosphorylation in sperm capacitation \\/ acrosome reaction  

Microsoft Academic Search

Capacitation is an important physiological pre-requisite before the sperm cell can acrosome react and fertilize the oocyte. Recent reports from several laboratories have amply documented that the protein phosphorylation especially at tyrosine residues is one of the most important events that occur during capacitation. In this article, we have reviewed the data from our and other laboratories, and have constructed

Rajesh K Naz; Preeti B Rajesh



Cancer Cell Phosphorylation by Casein Kinase I Promotes  

E-print Network

.06.015 SUMMARY Mdm2 is the major negative regulator of the p53 pathway. Here, we report that Mdm2 is rapidly into the signaling pathways controlling Mdm2 destruction and further suggest that compromised regulation of Mdm2Cancer Cell Article Phosphorylation by Casein Kinase I Promotes the Turnover of the Mdm2

Lahav, Galit


Phosphorylation of Animal Virus Proteins by a Virion Protein Kinase  

PubMed Central

Compared with several other enveloped viruses, purified virions of frog virus 3 contained a relatively high activity of a protein kinase which catalyzed the phosphorylation of endogenous polypeptides or added substrate proteins. Virions also contained a phosphoprotein phosphatase activity which released phosphate covalently linked to proteins. It was possible to select reaction conditions where turnover of protein phosphoesters was minimal, as the phosphatase required Mn2+ ions for activity whereas the protein kinase was active in the presence of Mg2+ ions. Electrophoretic studies in polyacrylamide gels containing sodium dodecyl sulfate indicated that at least 10 of the virion polypeptides were phosphorylated in the in vitro protein kinase reaction. Characterization of these phosphoproteins demonstrated that the phosphate was incorporated predominantly in a phosphoester linkage with serine residues. The protein kinase was solubilized by disrupting purified virions with a nonionic detergent in a high-ionic-strength buffer and was separated from many of the virion substrate proteins by zonal centrifugation in glycerol gradients. The partially purified protein kinase would phosphorylate polypeptides of many different animal viruses, and maximal activity was not dependent on added cyclic nucleotides. These properties distinguished the virion protein kinase from a well characterized cyclic AMP-dependent protein kinase which phosphorylated viral proteins only to a small extent. Images PMID:4355852

Silberstein, Harvey; August, J. Thomas



Phosphorylation of connexin43 on S279/282 may contribute to laminopathy-associated conduction defects  

SciTech Connect

An understanding of the molecular mechanism behind the arrhythmic phenotype associated with laminopathies has yet to emerge. A-type lamins have been shown to interact and sequester activated phospho-ERK1/2(pERK1/2) at the nucleus. The gap junction protein connexin43 (Cx43) can be phosphorylated by pERK1/2 on S279/282 (pS279/282), inhibiting intercellular communication. We hypothesized that without A-type lamins, pS279/282 Cx43 will increase due to inappropriate phosphorylation by pERK1/2, resulting in decreased gap junction function. We observed a 1.6-fold increase in pS279/282 Cx43 levels in Lmna{sup ?/?} mouse embryonic fibroblasts (MEFs) compared to Lmna{sup +/+}, and 1.8-fold more pERK1/2 co-precipitated from Lmna{sup ?/?} MEFs with Cx43 antibodies. We found a 3-fold increase in the fraction of non-nuclear pERK1/2 and a concomitant 2-fold increase in the fraction of pS279/282 Cx43 in Lmna{sup ?/?} MEFs by immunofluorescence. In an assay of gap junctional function, Lmna{sup ?/?} MEFs transferred dye to 60% fewer partners compared to Lmna{sup +/+} controls. These results are mirrored in 5–6 week-old Lmna{sup ?/?} mice compared to their Lmna{sup +/+} littermates as we detect increased pS279/282 Cx43 in gap junctions by immunofluorescence and 1.7-fold increased levels by immunoblot. We conclude that increased pS279/282 Cx43 in the Lmna{sup ?/?} background results in decreased cell communication and may contribute to the arrhythmic pathology in vivo. - Highlights: ? Connexin43 phosphorylation plays a role in laminopathy-associated conduction defects. ? Loss of A-type lamin activity results in release of pERK1/2 from the nucleus. ? Increased cytoplasmic localization of pERK1/2 acts to phosphorylate S279/282 of Cx43. ? Phosphorylation of S279/282 on Cx43 decreases gap junction activity in cell culture. ? Mice lacking A-type lamins have increased phosphorylation on S279/282 of Cx43.

Chen, Steven C., E-mail: [Fred Hutchinson Cancer Research Center (FHCRC), Public Health Sciences Division, 1100 Fairview Ave. N., Seattle, WA 98109 (United States); University of Washington Department of Biochemistry, 1959 NE Pacific St., Seattle, WA 98195 (United States); Kennedy, Brian K., E-mail: [University of Washington Department of Biochemistry, 1959 NE Pacific St., Seattle, WA 98195 (United States); Buck Institute for Research on Aging, 8001 Redwood Boulevard, Novato, CA 94945 (United States); Lampe, Paul D., E-mail: [Fred Hutchinson Cancer Research Center (FHCRC), Public Health Sciences Division, 1100 Fairview Ave. N., Seattle, WA 98109 (United States)



[Technology Transfer.  

ERIC Educational Resources Information Center

Some authorities on technolgoy transfer feel that industry is not fully capitalizing on the inventive output of universities and nonprofit organizations. From the point of view of the government, the stakes are high. The magnitude of federal support of research and development in these organizations demands evidence of useful results if it is to…

Latker, Norman J.


The non-enzymatic microbicidal activity of lysozymes  

Microsoft Academic Search

T4 lysozyme was thought to destroy bacteria by its muramidase activity. However, we demonstrate here that amphipathic helix stretches in the C-terminus of T4 lysozyme mediate its bactericidal and fungistatic activities. In heat-denatured T4 lysozyme, the enzymatic activity is completely abolished but unexpectedly, the antimicrobial functions remain preserved. Small synthetic peptides corresponding to amphipathic C-terminal domains of T4 lysozyme show

Petra Porsch; Andreas Mahn; Olaf Brinkmann; Werner Gieffers



Biotechnological production of enantiopure epoxides by enzymatic kinetic resolution  

Microsoft Academic Search

Enantiopure epoxides are high value-added synthons for the production of pharmaceuticals, agrochemicals, as well as versatile\\u000a fine chemicals and have broad scope of market demand for their applications. A major challenge in conventional organic synthesis\\u000a is to generate such compounds in high enantiopurity with reasonable yield. Among possible chemical and biological technologies\\u000a for enantiopure epoxide preparation, enzymatic kinetic resolution has

Won Jae Choi



Enzymatic hydrolysis of defatted mackerel protein with low bitter taste  

Microsoft Academic Search

Ultrasound-assisted solvent extraction was confirmed as a novel, effective method for separating lipid from mackerel protein,\\u000a resulting in a degreasing rate (DR) of 95% and a nitrogen recovery (NR) of 88.6%. To obtain protein hydrolysates with high\\u000a nitrogen recovery and low bitter taste, enzymatic hydrolysis was performed using eight commercially available proteases. It\\u000a turned out that the optimum enzyme was

Hu Hou; Bafang Li; Xue Zhao



Spectrophotometric detection of histidine and lysine using combined enzymatic reactions.  


An amino acid-sensing system with absorption spectrophotometric detection was developed. To ensure specific recognition of each amino acid, aminoacyl-tRNA synthetases were employed and the concentration of NADH produced by way of several enzymatic reactions was measured. Using this detection system, from 1.5 to 55 ?M of histidine and from 15 to 95 ?M of lysine could be measured selectively in HEPES-KOH buffer (pH 8.0). PMID:24094198

Kugimiya, Akimitsu; Takamitsu, Emi



Hydroxypropylation of cellulose as a pretreatment for enzymatic hydrolysis  

E-print Network

Dr. E. J. Soltes Cellulose derivatives such as carboxymethylcellulose and water soluble cellulose acetate have shown increased ability to be converted to reducing sugars, including glucose, compared to pure cellulose. Hydroxypropylcellulose (HPC... of substituted AHG. Fermentation of enzymatic hydrolyzates, obtained from HPC samples, confirmed that the presence of hydroxypropyl derivatives does not inhibit the production of ethyl alcohol. DEDICATION This thesis is dedicated to Kim, Ryan, and Holly...

Brix, Scott Tyson



Enzymatic Determination of the Protein Quality of Individual Rumen Bacteria  

Microsoft Academic Search

Protein quality of individual rumen bacteria was studied using an in vitro enzymatic digest system, and a protein quality index (NPU,ni) was computed from egg ratios. Depending upon the individual strain the NPU«n,values ranged from 37 to 80, whereas control digests with casein gave NPU.�,values of 86.5 and 73.6. The proportion of the essential amino acids being released as free




Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis  

Microsoft Academic Search

Production of ethanol by bioconversion of lignocellulosic biomass has attracted much interest in recent years. However, the\\u000a pretreatment process for increasing the enzymatic digestibility of cellulose has become a key step in commercialized production\\u000a of cellulosic ethanol. During the last decades, many pretreatment processes have been developed for decreasing the biomass\\u000a recalcitrance, but only a few of them seem to

Xuebing Zhao; Keke Cheng; Dehua Liu



Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate  

Microsoft Academic Search

Enzymatic hydrolysis of corncob and ethanol fermentation from cellulosic hydrolysate were investigated. After corncob was pretreated by 1% H2SO4 at 108°C for 3h, the cellulosic residue was hydrolyzed by cellulase from Trichoderma reesei ZU-02 and the hydrolysis yield was 67.5%. Poor cellobiase activity in T. reesei cellulase restricted the conversion of cellobiose to glucose, and the accumulation of cellobiose caused

Ming Chen; Liming Xia; Peijian Xue



Enzymatic activity preservation and protection through entrapment within degradable hydrogels.  


This work aims to develop a repeatable enzyme entrapment method that preserves activity within an amicable environment while resisting activity reduction in the presence of environmental challenges. Advances in such methods have wide potential use in biosensor applications. In this work ?-galactosidase (lactase) enzyme was entrapped within hydrogel matrices of acrylamide (ACR) crosslinked with N,N'-methylenebisacrylamide (BIS, non-degradable) or poly(ethylene glycol) diacrylate (PEGDA, degradable) to create "biogels." Diffusivity studies of control, enzyme free, hydrogel constructs showed near-Fickian swelling behavior in PBS regardless of crosslinker type or density. As expected, the swelling rate, Ks , decreased when increasing the crosslink density from 78.6 to 14.7?min?¹ over a range of 1-20?mol% PEGDA indicating that diffusivity into the matrix is dependent on crosslink density. Fabricated biogels were evaluated for maintained enzyme activity in the 7 and 8 pH range. PEGDA crosslinked gels consistently showed improved enzymatic activity retention as compared to BIS crosslinked gels. As PEGDA crosslink density increased from 5 to 10?mol%, enzymatic activity retention post-initial entrapment increased. Higher PEGDA crosslink densities between 15% and 40% decreased enzymatic activity due to assumed steric hindrance of the entrapped enzyme and also decreased substrate and product diffusion. Increased enzymatic stability was observed in 40?mol% PEGDA crosslinked gels. The biogels were pH challenged to 8.0 and stability, measured as retention of activity, was observed to be 91%. Free, non-entrapped, solution based enzyme conversion only retained 23% activity under the same pH challenge conditions. No significant loss of active enzyme was determined to elute out of the biogels during storage in PBS or during biogel wash and recycling. This entrapment method illustrates the potential to sterically hinder and diffusively impede enzymes from performing their function. Degradation of the network crosslinks can then potentially enable the reactivation of the enzyme at a site and time dictated by the user. PMID:23744741

Mariani, Angela M; Natoli, Mary E; Kofinas, Peter



Composite aromatic boxes for enzymatic transformations of quaternary ammonium substrates.  


Cation-? interactions to cognate ligands in enzymes have key roles in ligand binding and enzymatic catalysis. We have deciphered the key functional role of both charged and aromatic residues within the choline binding subsite of CTP:phosphocholine cytidylyltransferase and choline kinase from Plasmodium falciparum. Comparison of quaternary ammonium binding site structures revealed a general composite aromatic box pattern of enzyme recognition sites, well distinguished from the aromatic box recognition site of receptors. PMID:25283789

Nagy, Gergely N; Marton, Lívia; Contet, Alicia; Ozohanics, Olivér; Ardelean, Laura-Mihaela; Révész, Agnes; Vékey, Károly; Irimie, Florin Dan; Vial, Henri; Cerdan, Rachel; Vértessy, Beáta G



Enzymatic activity of allergenic house dust and storage mite extracts.  


Proteases are involved in the pathogenicity of allergy, increasing epithelial permeability and acting as adjuvants. Enzymatic activity is therefore important for the allergenicity of an extract and also affects its stability and safety. However, the enzymatic activity of extracts is not usually evaluated. The objective of this study was to evaluate the enzymatic activity of the most allergenic mite extracts and to investigate their allergenic properties. Extracts from nine allergenic mite species (Dermatophagoides pteronyssinus, Dermatophagoides farinae Hughes, Euroglyphus maynei, Lepidoglyphus destructor, Tyrophagus putrescentiae (Schrank), Glycyphagus domesticus (DeGeer), Acarus siro L., Chortoglyphus arcuatus, and Blomia tropicalis) were characterized. Protein and allergen profiles were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western-blot, respectively. Gelatinolytic activity was evaluated with a zymogram and the activity of other enzymes (cysteine, serine proteases, and esterases) was evaluated individually or with the API-ZYM system. The main differences in protease activity were found between house dust mites and storage mites. House dust mites presented higher cysteine protease activity while storage mites presented higher serine protease activity. These differences are in line with their trophic specialization. A wide range of different activities was found in all the extracts analyzed, reflecting the fact that the extracts preserve the activity of many enzymes, this being necessary for a correct diagnosis. However, enzymes may act as adjuvants and, therefore, could lead to undesirable effects in immunotherapies, making this activity not suitable for treatment products. Modified extracts with lower enzymatic activity could be more appropriate for immunotherapy. PMID:23427664

Morales, Maria; Iraola, Víctor; Leonor, Jose R; Carnés, Jerónimo



Extrusion pretreatment of sugarcane bagasse for enzymatic hydrolysis  

E-print Network

milling one day and 18. 5% after six days. Enzymatic hydrolysis with cellulase from Trichoderma viride gave 38. 2% saccharification after the one-day milling treatment and 58% after the six-day treatment. Both alkali solubility and susceptibility... into the two roll mill, masticated for one minute intervals, and scraped off between intervals. Treated and untreated samples were hydrolyzed by cellulases of Trlchoderma viride QM 9414 and hydrolysates analyzed for total reducing sugar. Results indicated...

Ocana Camacho, Ronay



Immobilization and enzymatic properties of Bacillus megaterium glucose dehydrogenase  

SciTech Connect

The enzymatic production of hydrogen gas from renewable sources of energy; e.g., cellulose, starch, lactose, can be obtained by coupling the reactions catalyzed by glucose dehydrogenase (GDH) and hydrogenase. In order to enhance the thermostability of GDH from Bacillus megaterium, the enzyme was immobilized by ionic adsorption using the polycationic polymer DEAE-(dextran)Sephadex. The effect of enzyme concentration on immobilization showed a tendency to increase the activity of the immobilized enzyme with the increase of the amount of added GDH. When the enzyme: support ratio was 15.97 U: 100 mg, the immobilization yield was 84.76%. The enzymatic profiles for the immobilized GDH were a little different when compared to those for free enzyme with respect to the effects of pH and temperature. Concerning the effect of incubation time carried at pH 7.5 and at 40{degrees}C, the maximum production of reduced coenzyme by the immobilized enzyme was reached within 4 h and it was maintained up to 16 h without loss of enzymatic activity. The coupling of the immobilized GDH activity with that for free alkaline cellulose (Novozym. 342) demonstrated the possibility for obtaining reduced coenzyme from the cellulose hydrolysis and the immobilized GDH could be reassayed 10 times maintaining its enzyme activity.

Baron, M.; Fontana, J.D.; Guimaraes, M.F. [Federal Univ. of Parana, Curitiba, PR (Brazil); Woodward, J. [Oak Ridge National Lab., TN (United States)



Enzymatic hydrolysis of cellulose and various pretreated wood fractions  

SciTech Connect

Three strains of Trichoderma-Trichoderma reesei C30, Trichoderma reesei QM9414, and Trichoderma species E58-were used to study the enzymatic hydrolysis of pretreated wood substrates. Each of the culture filtrates was incubated with a variety of commercially prepared cellulose substrates and pretreated wood substrates. Solka floc was the most easily degraded commercial cellulose. The enzyme accessibility of steam-exploded samples which has been alkali extracted and then stored wet decreased with the duration of the steam treatment. Air drying reduced the extent of hydrolysis of all the samples but had a greater effect on the samples which had previously shown the greatest hydrolysis. Mild pulping using 2% chlorite increased the enzymatic hydrolysis of all the samples. Steam explosion was shown to be an excellent pretreatment method for aspen wood and was much superior to dilute nitric acid pretreatment. The results indicate that the distribution of the lignin as well as the surface area of the cellulosic substrate are important features in enzymatic hydrolysis. (Refs 17).

Saddler, J.N.; Brownell, H.H.; Clermont, L.P.; Levitin, N.



Enzymatic hydrolysis of organic phosphates adsorbed on mineral surfaces.  


Esters of phosphoric acid constitute a sizable fraction of the total phosphorus supply in the environment and thus play an important role in the global phosphorus cycle. Enzymatic hydrolysis of these esters to produce orthophosphate is often a required reaction preceding phosphorus uptake by plants and microorganisms. Generally, adsorption to environmental particles is assumed to limit this process. Here we show, however, that the rate of enzymatic hydrolysis of glucose-1-phosphate (G1P) adsorbed on goethite by acid phosphatase (AcPase) can be of the same order of magnitude as in aqueous solution. The surface process releases carbon to the solution whereas orthophosphate remains adsorbed on goethite. This hydrolysis reaction is strictly an interfacial process governed by the properties of the interface. A high surface concentration of substrate mediates the formation of a catalytically active layer of AcPase, and although adsorption likely reduces the catalytic efficiency of the enzyme, this reduction is almost balanced by the fact that enzyme and substrate are concentrated at the mineral surfaces. Our results suggest that mineral surfaces with appropriate surface properties can be very effective in concentrating substrates and enzymes thereby creating microchemical environments of high enzymatic activity. Hence, also strongly adsorbed molecules in soils and aquatic environments may be subjected to biodegradation by extracellular enzymes. PMID:22103404

Olsson, Rickard; Giesler, Reiner; Loring, John S; Persson, Per



Enzymatic function of cytochrome b559 in photosystem II.  


Cytochrome b(559) (cyt b(559)) is a heme-bridged protein heterodimer in photosystem II (PSII) of all oxygenic photosynthetic organisms. In spite of the fact that cyt b(559) is strictly required for proper function of PSII, it is not involved in the linear electron transport chain from water to plastoquinone. Instead of that the participation of cyt b(559) in the cyclic electron transport around PSII has been proposed mainly based on the ability of the heme iron to accept and donate an electron form the electron acceptor and to the electron donor side of PSII, respectively. In addition to the involvement of cyt b(559) in the cyclic electron transport around PSII, several lines of evidence have been provided on the enzymatic function of cyt b(559). The ability of oxygenic photosynthetic organisms to oxidize water and reduce plastoquinone is connected to the formation of reactive oxygen species (ROS) and thus required to develop an effective antioxidant defense system against ROS. The review attempts to summarize a recent progress on the role of cyt b(559) as oxygen reductase, superoxide reductase, superoxide oxidase and plastoquinol oxidase. The focus is mainly given on the characterization of redox, redox potential and acid-base properties of the heme iron in the putative enzymatic cycles. The possible oxidase and reductase enzymatic activity of cyt b(559) in protection from photoinhibition is discussed. PMID:21377371

Pospíšil, Pavel



Angling for Uniqueness in Enzymatic Preparation of Glycosides  

PubMed Central

In the early days of biocatalysis, limitations of an enzyme modeled the enzymatic applications; nowadays the enzyme can be engineered to be suitable for the process requirements. This is a general bird’s-eye view and as such cannot be specific for articulated situations found in different classes of enzymes or for selected enzymatic processes. As far as the enzymatic preparation of glycosides is concerned, recent scientific literature is awash with examples of uniqueness related to the features of the biocatalyst (yield, substrate specificity, regioselectivity, and resistance to a particular reaction condition). The invention of glycosynthases is just one of the aspects that has thrust forward the research in this field. Protein engineering, metagenomics and reaction engineering have led to the discovery of an expanding number of novel enzymes and to the setting up of new bio-based processes for the preparation of glycosides. In this review, new examples from the last decade are compiled with attention both to cases in which naturally present, as well as genetically inserted, characteristics of the catalysts make them attractive for biocatalysis. PMID:24970171

Trincone, Antonio