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To understand the response of freshwater organisms to rising environmental salinity, it is essential to investigate their osmoionic regulatory physiology. Our laboratory experiment investigated the transfer of Palaemon argentinus (Nobili, 1901) from 2[per thousand] (control condition) to concentrated salinity (15 and 25[per thousand]) for short- (6 h), medium- (48 h), and long-term (>504 h) acclimation periods. We measured relevant parameters in the shrimp's haemolymph, the time course of the response of branchial V-[H.sup.+]-ATPase (VHA), [Na.sup.+], [K.sup.+]--ATPase (NKA), carbonic anhydrase (CA) activity, and muscle water content. Upon prolonged acclimation to 15[per thousand] (hyper-regulating condition), shrimp reached a new steady state of haemolymph osmolality by tightly adjusting ion concentrations to levels higher than the external medium. While NKA and CA activities recovered their pre-transfer levels, the downregulation of VHA suggests other functions rather than ion uptake after prolonged acclimation to 15[per thousand]. The activity of the three transport-related enzymes remained almost unchanged at the highest salinity (isosmotic condition), leading to increasing osmotic pressure and ion concentration after prolonged acclimation to 25[per thousand]. Although the freshwater shrimp studied here retains a certain degree of tolerance to high salinity, a common trait in palaemonid shrimps, our results highlight that 25[per thousand] represents a significant hypertonic challenge for this species. Keywords: carbonic anhydrase, gills, ionic regulation, [Na.sup.+], [K.sup.+]--ATPase, Palaemon argentinus, (V)-type H+--ATPase, Introduction Inland aquatic ecosystems from southern regions in South America as the ecoregion of the Pampas (Argentina), are vulnerable to increasing salinization due to human activities and global climate change [...]

Monthly variation in accumulation and release of phenolics in bryophytes, and their allelopathic effects are little known despite their ecological significance. We conducted a field transplant experiment to investigate Sphagnum growth and extracellular enzymes that modulate phenolic contents and release along water table level (WTL) gradient. Specifically, we assessed monthly changes in height increment, phenolic release, and allelopathic effect during the growing season, as well as extracellular enzymes at the end of the experiment, for the two Sphagnum species: Sphagnum angustifolium C.E.O. Jensen, 1896, a hollow dwelling species, and Sphagnum magellanicum Bridel, 1798, a hummock species. Both Sphagnum species showed season- and WTL-dependent height increment and released phenolics. Sphagnum angustifolium exhibited negative allelopathy regardless of WTL, while S. magellanicum demonstrated mainly positive allelopathy at high WTL and negative allelopathy at low WTL. The degree of allelopathy was not contingent on phenolic release, but phenolic production was negatively correlated with height growth of the two species at their atypical habitats. Moreover, phenoloxidase or peroxidase did not show high activity at low WTL compared to high WTL. Our findings demonstrate for the first time that the strength and direction of allelopathy in Sphagnum may vary depending on the growing precipitation and temperature, WTL, and inter- and intraspecific trait variability. Longer term experiments may further elucidate the seasonal dynamics and mechanism of Sphagnum allelopathy. Key words: allelopathy, extracellular enzymes, secondary metabolites, moss, peatland, Introduction Allelopathy, the release of chemicals by plants that affect other plants in their environment, can be a major driver in shaping natural plant communities (Wardle et al. 1997; Zackrisson [...]

A sub-ångström-resolution X-ray crystal structure of [NiFe] hydrogenase, with direct detection of the products of the heterolytic splitting of dihydrogen into a hydride bridging the Ni and Fe and a proton attached to the sulphur of a cysteine ligand. Hydrogens visualized in hydrogenase enzyme [NiFe] hydrogenases use nickel and iron to catalyse the reversible oxidation of molecular hydrogen. They are the focus of much research worldwide because of their potential in biotechnology and in serving as natural models for biomimetic catalysts in the energy sector for hydrogen production and conversion. In protein X-ray crystallography it is notoriously difficult to detect hydrogens, a particularly significant problem in hydrogenases where hydrogens are involved directly in the reaction. Hideaki Ogata et al. have succeeded in obtaining a sub-ångström resolution X-ray crystal structure of [NiFe] hydrogenase leading to detection of most of the hydrogens even close to the metal ions. Using their technique authors were able to detect the products of the heterolytic splitting of dihydrogen: a hydride that bridges the Ni and Fe ions, and a proton that is attached to the sulfur of a cysteine ligand. The enzyme hydrogenase reversibly converts dihydrogen to protons and electrons at a metal catalyst.sup.1. The location of the abundant hydrogens is of key importance for understanding structure and function of the protein.sup.2,3,4,5,6. However, in protein X-ray crystallography the detection of hydrogen atoms is one of the major problems, since they display only weak contributions to diffraction and the quality of the single crystals is often insufficient to obtain sub-ångström resolution.sup.7. Here we report the crystal structure of a standard [NiFe] hydrogenase (~91.3 kDa molecular mass) at 0.89 Å resolution. The strictly anoxically isolated hydrogenase has been obtained in a specific spectroscopic state, the active reduced Ni-R (subform Ni-R1) state. The high resolution, proper refinement strategy and careful modelling allow the positioning of a large part of the hydrogen atoms in the structure. This has led to the direct detection of the products of the heterolytic splitting of dihydrogen into a hydride (H.sup.-) bridging the Ni and Fe and a proton (H.sup.+) attached to the sulphur of a cysteine ligand. The Ni-H.sup.- and Fe-H.sup.- bond lengths are 1.58 Å and 1.78Å, respectively. Furthermore, we can assign the Fe-CO and Fe-CN.sup.- ligands at the active site, and can obtain the hydrogen-bond networks and the preferred proton transfer pathway in the hydrogenase. Our results demonstrate the precise comprehensive information available from ultra-high-resolution structures of proteins as an alternative to neutron diffraction and other methods such as NMR structural analysis., Author(s): Hideaki Ogata [sup.1] , Koji Nishikawa [sup.1] [sup.2] , Wolfgang Lubitz [sup.1] Author Affiliations: (1) Max Planck Institute for Chemical Energy Conversion, Stiftstrasse 34-36, D-45470 Mülheim an der Ruhr, [...]

Vibrio cholerae has caused epidemics around the world for centuries. Cholera has long been a companion of devastation and poverty, and epidemics occur in areas without clean water, sanitation, or [...]

The Diels-Alder reaction is a [4+2] cycloaddition reaction in which a cyclohexene ring is formed between a 1,3-diene and an electron-deficient alkene via a single pericyclic transition state (1). This reaction has been proposed as a key transformation in the biosynthesis of many cyclohexene-containing secondary metabolites (2-5). However, only four purified enzymes have thus far been implicated in bio-transformations that are consistent with a Diels-Alder reaction, namely solanapyrone synthase (6), LovB (7,8), macrophomate synthase (9,10), and riboflavin synthase (11,12). Although the stereochemical outcomes of these reactions indicate that the product formation could be enzyme-guided in each case, these enzymes typically demonstrate more than one catalytic activity, leaving their specific influence on the cycloaddition step uncertain. In our studies of the biosynthesis of spinosyn A, a tetracyclic polyketide-derived insecticide from Saccharopolyspora spinosa (13,14), we identified a cyclase, SpnF, that catalyses a transannular [4+2] cycloaddition to form the cyclohexene ring in spinosyn A. Kinetic analysis demonstrates that SpnF specifically accelerates the ring formation reaction with an estimated 500-fold rate enhancement. A second enzyme, SpnL, was also identified as responsible for the final cross-bridging step that completes the tetracyclic core of spinosyn A in a manner consistent with a Rauhut-Currier reaction (15). This work is significant because SpnF represents the first example characterized in vitro of a standalone enzyme solely committed to the catalysis of a [4+2] cycloaddition reaction. In addition, the mode of formation of the complex perhydro-as-indacene moiety in spinosyn A is now fully established., Spinosyn A (1), an active ingredient of several highly effective and environmentally benign commercial insecticides, has a complex aglycone structure comprising a perhydro-as-indacene moiety fused to a 12-membered macrolactone (13,14). [...]

All reactions are accelerated by an increase in temperature, but the magnitude of that effect on very slow reactions does not seem to have been fully appreciated. The hydrolysis of polysaccharides, for example, is accelerated 190,000-fold when the temperature is raised from 25 to 100[degrees]C, while the rate of hydrolysis of phosphate monoester dianions increases 10,300,000-fold. Moreover, the slowest reactions tend to be the most heat-sensitive. These tendencies collapse, by as many as five orders of magnitude, the time that would have been required for early chemical evolution in a warm environment. We propose, further, that if the catalytic effect of a 'proto-enzyme'--like that of modern enzymes--were mainly enthalpic, then the resulting rate enhancement would have increased automatically as the environment became cooler. Several powerful nonenzymatic catalysts of very slow biological reactions, notably pyridoxal phosphate and the ceric ion, are shown to meet that criterion. Taken together, these findings greatly reduce the time that would have been required for early chemical evolution, countering the view that not enough time has passed for life to have evolved to its present level of complexity. activation energy | thermophilic organisms | pyridoxal phosphate | phosphate ester hydrolysis | amino acid decarboxylation doi/ 10.1073/pnas.1013647107

Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and ate targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110+ predicted mammalian metabolic SHs, in large part because of a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems. We show here that the vast majority (>80%) of mammalian metabolic SHs can be labeled in proteomes by a single, active site-directed fluorophosphonate probe. We exploit this universal activity-based assay in a library-versus-library format to screen 70+ SHs against 140+ structurally diverse carbamates. Lead inhibitors were discovered for ~40% of the screened enzymes, including many poorly characterized SHs. Global profiles identified carbamate inhibitors that discriminate among highly sequence-related SHs and, conversely, enzymes that share inhibitor sensitivity profiles despite lacking sequence homology. These findings indicate that sequence relatedness is not a strong predictor of shared pharmacology within the SH superfamily. Finally, we show that lead carbamate inhibitors can be optimized into pharmacological probes that inactivate individual SHs with high specificity in vivo. enzymology | mass spectrometry | profiling | proteomics doi/10.1073/pnas.1011663107

The hydrolysis of biomass to fermentable sugars using glycosyl hydrolases such as cellulases and hemicellulases is a limiting and costly step in the conversion of biomass to biofuels. Enhancement in hydrolysis efficiency is necessary and requires improvement in both enzymes and processing strategies. Advances in both areas in turn strongly depend on the progress in developing high-throughput assays to rapidly and quantitatively screen a large number of enzymes and processing conditions. For example, the characterization of various cellodextrins and xylooligomers produced during the time course of saccharification is important in the design of suitable reactors, enzyme cocktail compositions, and biomass pretreatment schemes. We have developed a microfluidicchip-based assay for rapid and precise characterization of glycans and xylans resulting from biomass hydrolysis. The technique enables multiplexed separation of soluble cellodextrins and xylose olignmers in around 1 min (10fold faster than HPLC). The microfluidic device was used to elucidate the mode of action of Tm_Cel5A, a novel cellulase from hyperthermophile Thermotogn maritima. The results demonstrate that the cellulase is active at 80 [degrees]C and effectively hydrolyzes cellodextrins and ionic-liquid-pretreated switchgrass and Avicel to glucose, cellobiose, and cellotriose. The proposed microscale approach is ideal for quantitative large-scale screening of enzyme libraries for biomass hydrolysis, for development of energy feedstocks, and for polysaccharide sequencing. 10.1021/ac102243f

Capillary electrophoresis (CE) on microfabricated structures has achieved impressive sample throughput by combining fast separation speed and parallel operations. One obstacle to further increasing throughput has been lack of methods for loading and injecting individual samples at a rate that matches analysis speed. To address this issue, we have developed a microfluidic device in which samples stored as nanoliter volume plugs segmented by a fluorocarbon oil are introduced sequentially to an array of three electrophoresis channels. A microfluidic interface consisting of patterned surface chemistry and geometric restriction was used to extract samples from each segmented flow channel and transfer to the respective electrophoresis channel for separation. Fluorescence detection was achieved by imaging the chip using a fluorescence microscope equipped with a charge-coupled device. Characterization of the system shows that injection volume is controlled by sample plug volume, flow rate during introduction, and voltage applied to the electrophoresis channel. The system was tested for a GTPase assay. Peak area ratios of enzyme product and internal standard had 6% relative standard deviations. Cross-contamination between peaks was 7%. Throughput of 120 samples in 10 rain was achieved. Further development of the system may allow application to high. throughput applications such as drug screening. 10.1021/ac101755y

In nonneuronopathic type 1 Gaucher disease (GD1), mutations in the glucocerebrosidase gene (GBA1) gene result in glucocerebrosidase deficiency and the accumulation of its substrate, glucocerebroside (GL-1), in the lysosomes of mononuclear phagocytes. This prevailing macrophage-centric view, however, does not explain emerging aspects of the disease, including malignancy, autoimmune disease, Parkinson disease, and osteoporosis. We conditionally deleted the GBA1 gene in hematopoietic and mesenchymal cell lineages using an Mx1 promoter. Although this mouse fully recapitulated human GD1, cytokine measurements, microarray analysis, and cellular immunophenotyping together revealed widespread dysfunction not only of macrophages, but also of thymic T cells, dendritic cells, and osteoblasts. The severe osteoporosis was caused by a defect in osteoblastic bone formation arising from an inhibitory effect of the accumulated lipids LysoGL-1 and GL-1 on protein kinase C. This study provides direct evidence for the involvement in GD1 of multiple cell lineages, suggesting that cells other than macrophages may be worthwhile therapeutic targets. osteopenia | osteoblast | glucosylsphingosine | T cells | PKC doi/ 10.1073/pnas.1003308107

Liquid chromatography-selected reaction monitoring (LCSRM) is a highly specific and sensitive mass spectrometry (MS) technique that is widely being applied to selectively qualify and validate candidate markers within complex biological samples. However, in order for LC-SRM methods to take on these attributes, target-specific optimization of sample processing is required, in order to reduce analyte complexity, prior to LC-SRM. In this study, we have developed a targeted platform consisting of protein immunoaffinity enrichment on magnetic beads and LCSRM for measuring carbonic anhydrase 12 (CA12) protein in a renal cell carcinoma (RCC) cell line (PRC3), a candidate biomarker for RCC whose expression at the protein level has not been previously reported. Sample processing and LC-SRM assay were optimized for signature peptides selected as surrogate markers of CA12 protein. Using LC-SRM coupled with stable isotope dilution, we achieved limits of quantitation in the low fmol range sufficient for measuring clinically relevant biomarkers with good intra-and interassay accuracy and precision ([less than or equal to] 17%). Our results show that using a quantitative immunoaffinity capture approach provides specific, accurate, and robust assays amenable to high-throughput verification of potential biomarkers. 10.1021/ac101981t

Folding analysis of the zinc protein, carbonic anhydrase 2 (CA2), was performed using electrospray ionization ion mobility spectrometry coupled with collision-induced dissociation (ESI IMS/C1D). Multiply protonated ions with a bimodal charge state distribution were observed indicating the presence of at least two folding states for gas-phase CA2 ions as was described in a previous study (Nabuchi, Y.; Murao, N.; Asoh, Y.; Takayama, M. Anal. Chem. 2007, 79, 8342-8349). in the IMS driftgram, several ions with different mobility were observed for each multiply charged ion, and this suggests that CA2 ions consist of several components with different folding states. IMS/CID spectra were acquired against precursor ions separated by mobility. The CID spectra gave several characteristic product ions including those from the N- and C-terminal region of CA2. A shift to larger charge number for the most abundant of the several product ions was observed for ions having a larger drift time. This charge number shift indicates that the folding state of the ion is more unfolded. Furthermore, differences in the production of an ion corresponding to the N-terminal side fragment gave information about the unfolding process of CA2. 10.1021/ac101482b

This study reports a simple calorimetric method for the sensitive detection of S-adenosylhomocysteine hydrolase (SAHH) activity and inhibition using fluorosurfactant-capped gold nanoparticles (FSN-AuNPs). FSN stabilizes the AuNPs against conditions of high ionic strength, and FSN-AuNPs are merely aggregated in the presence of homocysteine (HCys) and cysteine. Because of this feature, FSN-AuNPs were found to be dispersed in the presence of S-adenosylhomocysteine (SAH) that lacks a free thiol group. After SAHH catalyzed the hydrolysis of SAH, the produced HCys molecules were bound to the surface of AuNPs through the formation of Au--S bonds. As a result, the nanoparticle (NP) aggregation occurred through electrostatic attraction between each HCys-attached AuNP. This approach had a minimum detectable concentration of 100 units/L (~6 nM). Additionally, because adenosine analogs are capable of inhibiting SAHH activity, the addition of adenosine analogs to a solution containing SAIl and SAHH resulted in the suppression of hydrolyzed SAH-induced NP aggregation. Adenosine analogs exhibited the following trend in the half-maximal inhibitory concentrations: adenosine > adenosine monophosphate > adenosine diphosphate adenosine triphosphate. We have demonstrated that the combination of SAHH inhibition and FSN-AuNPs can be utilized for the selective detection of adenosine. 10.1021/ac102020n

Clostridium thermocellum is a thermophilic anaerobic bacterium that rapidly solubilizes cellulose with the aid of a multienzyme cellulosome complex. Creation of knockout mutants for Cel48S (also known as CelS, [S.sub.s], and S8), the most abundant cellulosome subunit, was undertaken to gain insight into its role in enzymatic and microbial cellulose solubilization. Cultures of the Cel48S deletion mutant (S mutant) were able to completely solubilize 10 g/L crystalline cellulose. The cellulose hydrolysis rate of the S mutant strain was 60% lower than the parent strain, with the S mutant strain also exhibiting a 40% reduction in cell yield. The cellulosome produced by the S mutant strain was purified by affinity digestion, characterized enzymatically, and found to have a 35% lower specific activity on Avicel. The composition of the purified cellulosome was analyzed by tandem mass spectrometry with APEX quantification and no significant changes in abundance were observed in any of the major (>1% of cellulosomal protein) enzymatic subunits. Although most cellulolytic bacteria have one family 48 cellulase, C. thermocellum has two, Cel48S and Cel48Y. Cellulose solubilization by a Cel48S and Cel48Y double knockout was essentially the same as that of the Cel48S single knockout. Our results indicate that solubilization of crystalline cellulose by C. thermocellum can proceed to completion without expression of a family 48 cellulase. cellulosome | CelS | family 48 | exoglucanase doi/ 10.1073/pnas.1003584107

Thermolysin-like proteases (TLPs), a large group of zinc metalloproteases, are synthesized as inactive precursors. TLPs with a long propeptide (~200 residues) undergo maturation following autoprocessing through an elusive molecular mechanism. We report the first two crystal structures for the autoprocessed complexes of a typical TLP, MCP-02. In the autoprocessed complex, Ala205 shifts upward by 33 [Angstrom] from the previously covalently linked residue, His204, indicating that, following autocleavage of the peptide bond between His204 and Ala205, a large conformational change from the zymogen to the autoprocessed complex occurs. The eight N-terminal residues (residues Ala205-Gly212) of the catalytic domain form a new [beta]-strand, nestling into two other [beta]-strands. Simultaneously, the apparent [T.sub.m] of the autoprocessed complex increases 20[degrees]C compared to that of the zymogen. The stepwise degradation of the propeptide begins with two sequential cuttings at Ser49-Val50 and Gly57-Leu58, which lead to the disassembly of the propeptide and the formation of mature MCP-02. Our findings give new insights into the molecular mechanism of TLP maturation. zymogen conversion | zinc metalloproteases inhibition | intrarnolecular chaperone doi/ 10.1073/pnas.1005681107

Bacillus anthracis spores, the etiological agents of anthrax, possess a loosely fitting outer layer called the exosporium that is composed of a basal layer and an external hairlike nap. The filaments of the nap are formed by trimers of the collagenlike glycoprotein BclA. Multiple pentasaccharide and trisaccharide side chains are O linked to BclA. The nonreducing terminal residue of the pentasaccharide side chain is the unusual sugar anthrose. A plausible biosynthetic pathway for anthrose biosynthesis has been proposed, and an antABCD operon encoding four putative anthrose biosynthetic enzymes has been identified. In this study, we genetically and biochemically characterized the activities of these enzymes. We also used mutant B. anthracis strains to determine the effects on BclA glycosylation of individually inactivating the genes of the anthrose operon. The inactivation of antA resulted in the appearance of BclA pentasaccharides containing anthrose analogs possessing shorter side chains linked to the amino group of the sugar. The inactivation of antB resulted in BclA being replaced with only trisaccharides, suggesting that the enzyme encoded by the gene is a dTDP-[beta]-L-rhamnose [alpha]-l,3-L-rhamnosyi transferase that attaches the fourth residue of the pentasaccharide side chain. The inactivation of antC and antD resulted in the disappearance of BclA pentasaccharides and the appearance of a tetrasaccharide lacking anthrose. These phenotypes are entirely consistent with the proposed roles for the antABCD-encoded enzymes in anthrose biosynthesis. Purified AntA was then shown to exhibit [beta]-methylcrotonyl-coenzyme A (CoA) hydratase activity, as we predicted. Similarly, we confirmed that purified AntC had aminotransferase activity and that purified AntD displayed N-acyltransferase activity. doi: 10.1128/JB.00568-10

One of the fundamental challenges in biotechnology and in biochemistry is the ability to design effective enzymes. Doing so would be a convincing manifestation of a full understanding of the origin of enzyme catalysis. Despite an impressive progress, most of the advances on this front have been made by placing the reacting fragments in the proper places, rather than by optimizing the environment preorganization, which is the key factor in enzyme catalysis. Rational improvement of the preorganization would require approaches capable of evaluating reliably the actual catalytic effect. This work takes apreviously designed kemp eliminases as a benchmark for a computer aided enzyme design, using the empirical valence bond as the main screening tool. The observed absolute catalytic effect and the effect of directed evolution are reproduced and analyzed (assuming that the substrate is in the designed site). It is found that, in the case of kemp eliminases, the transition state charge distribution makes it hard to exploit the active site polarity, even with the ability to quantify the effect of different mutations. Unexpectedly, it is found that the directed evolution mutants lead to the reduction of solvation of the reactant state by water molecules rather that to the more common mode of transition state stabilization used by naturally evolved enzymes, Finally it is pointed out that our difficulties in improving Kemp eliminase are not due to overlooking exotic effect, but to the challenge in designing a preorganized environment that would exploit the small change it charge distribution during the formation of the transition state. computer aided enzyme design | empirical valence bond | directed evolution doi/ 10.1073/pnas.1010381107

Animal cells have two tRNA splicing pathways: (i) a 5'-P ligation mechanism, where the 5'-phosphate of the 3' tRNA half becomes the junction phosphate of the new phosphodiester linkage, and (ii) a 3'-P ligation process, in which the 3'-phosphate of the 5' tRNA half turns into the junction phosphate. Although both activities are known to exist in animals, in almost three decades of investigation, neither of the two RNA ligases has been identified. Here we describe a gene from the chordate Branchiostoma floridae that encodes an RNA ligase (Bf RNL) with a strict requirement for RNA substrates with a 2'-phosphate terminus for the ligation of RNAs with 5'-phosphate and 3'-hydroxyl ends. Unlike the yeast and plant tRNA ligases involved in tRNA splicing, Bf RNL lacks healing activities and requires the action of a polynucleotide kinase (PNK) and a cyclic phosphodiesterase (CDPase) in trans. The activities of these two enzymes were identified in a single B. floridae protein (Bf PNK/ CPDase). The combined activities of Bf RNL and Bf PNK/CPDase are sufficient for the joining of tRNA splicing intermediates in vitro, and for the functional complementation of a tRNA ligase-deficient Saccharomyces cerevisiae strain in vivo. Hence, these two proteins constitute the 5'-P RNA ligation pathway in an animal organism. doi/ 10.1073/pnas.1011703107

[Fe.sup.III]-[O.sub.2.sup.*-] intermediates are well known in heme enzymes, but none have been characterized in the nonheme mononuclear [Fe.sup.II] enzyme family. Many steps in the [O.sub.2] activation and reaction cycle of [Fe.sup.II]-containing homoprotocatechuate 2,3-dioxygenase are made detectable by using the alternative substrate 4-nitrocatechol (4NC) and mutation of the active site His200 to Asn (H200N). Here, the first intermediate (Int-1) observed after adding [O.sub.2] to the H200N-4NC complex is trapped and characterized using EPR and Mossbauer (MB) spectroscopies. Int-1 is a high-spin ([S.sub.1] = 5/2) [Fe.sup.III] antiferromagnetically (AF) coupled to an [S.sub.2] = 1/2 radical (J [approximately equal to] 6 [cm.sup.-1] in H = [JS.sub.1]*[S.sub.2]). It exhibits parallel-mode EPR signals at g = 8.17 from the S = 2 multiplet, and g = 8.8 and 11.6 from the S = 3 multiplet. These signals are broadened significantly by [sup.17][O.sub.2] hyperfine interactions ([A17.sub.O] [approximately equal to] 180 MHz). Thus, Int-1 is an AF-coupled [Fe.sup.III]-[O.sub.2.sup.*-] species. The experimental observations are supported by density functional theory calculations that show nearly complete transfer of spin density to the bound [O.sub.2]. Int-1 decays to form a second intermediate (Int-2). MB spectra show that it is also an AF-coupled [Fe.sup.III]-radical complex. Int-2 exhibits an EPR signal at g = 8.05 arising from an S = 2 state. The signal is only slightly broadened by [sup.17][O.sub.2] ( oxygen activation | oxygenase | spectroscopy | superoxide doi/ 10.1073/pnas.1010015107

Global analysis of glycoproteins shows great promise for the discovery of therapeutic targets and clinical biomarkers. Selective capture of glycopeptides by hydrazide resin followed by mass spectrometric identification of the peptides released by PNGaseF treatment has been most widely used. However, the majority of the reports using this approach focus on global profiling, rather than relative quantitation of glycoprotein alternations in pathological states. We describe an integrated strategy allowing for relative quantitstian of glycoproteins in complex biological mixtures using this approach. The strategy includes periodate oxidation of tryptic digests, solid-phase enrichment of glycopeptides via hydrazide-eoupled magnetic beads, in conjunction with [sup.18]O stable isotope labeling catalyzed by both trypsin and PNGaseF, and subsequent identification and quantitation by LC-MS/MS analysis. Three [sup.18]O atoms ([sup.18][O.sub.3]) are incorporated into N-linked glycopeptides for samples treated in [sup.18]O-water, two at the carboxyl terminus by trypsin during hydrazide coupling and the third at the N-glycosylation site through PNGaseF-mediated deglycosylation. Thus, mass shifts of 6 and 8 Da are indicative of singly and doubly glycosylated peptides, respectively. Experimental conditions were optimized to promote the trypsin-mediated [sup.18][O.sub.2] incorporation and prevent backbone exchange. The accuracy, reproducibility, and linearity of relative quuntitation were evaluated by using 15 glycoproteins spiked into mouse serum at different concentration ratios. Using this approach, we were able to identify and quantitate 224 N-glycopeptides representing 130 unique glycoproteins from 20 [micro]L of the undepleted mouse serum samples. The strategy can be easily adapted to the analysis of glycoproteins in tissues, cell lines, and other sample origins. 10.1021/ac101564t

The time it takes an enzyme to complete its reaction is a stochastic quantity governed by thermal fluctuations. With the advent of high-resolution methods of single-molecule manipulation and detection, it is now possible to observe directly this natural variation in the enzymatic cycle completion time and extract kinetic information from the statistics of its fluctuations. To this end, the inverse of the squared coefficient of variation, which we term [n.sub.min], is a useful measure of fluctuations because it places a strict lower limit on the number of kinetic states in the enzymatic mechanism. Here we show that there is a single general expression for the substrate dependence of [n.sub.min] for a wide range of kinetic models, This expression is governed by three kinetic parameters, which we term [N.sub.L], [N.sub.S], and [alpha]. These parameters have simple geometric interpretations and provide clear constraints on possible kinetic mechanisms. As a demonstration of this analysis, we fit the fluctuations in the dwell times of the packaging motor of the bacteriophage [phi]29, revealing additional features of the nucleotide loading process in this motor. Because a diverse set of kinetic models display the same substrate dependence for their fluctuations, the expression for this general dependence may prove of use in the characterization and study of the dynamics of a wide range of enzymes. statistical kinetics | enzyme dynamics | single molecule | molecular motors doi/ 10.1073/pnas.1006997107

Sphingomyelinase (SMase) hydrolyzes membrane sphingomyelin into ceramide, which increases oxidants in nonmuscle cells. Serum SMase activity is elevated in sepsis and heart failure, conditions where muscle oxidants are increased, maximal muscle force is diminished, and fatigue is accelerated. We tested the hypotheses that exogenous SMase and accumulation of ceramide in muscle increases oxidants in muscle cells, depresses specific force of unfatigued muscle, and accelerates the fatigue process. We also anticipated that the antioxidant N-acetylcysteine (NAC) would prevent SMase effects on muscle function. We studied the responses of C2C12 myotubes and mouse diaphragm to SMase treatment in vitro. We observed that SMase caused a 2.8-fold increase in total ceramide levels in myotubes. Exogenous ceramide and SMase elevated oxidant activity in C2C12 myotubes by 15-35% (P < 0.05) and in diaphragm muscle fiber bundles by 58-120% (P < 0.05). The SMase-induced increase in diaphragm oxidant activity was prevented by NAC. Exogenous ceramide depressed diaphragm force by 55% (P < 0.05), while SMase depressed maximal force by 30% (P < 0.05) and accelerated fatigue--effects opposed by treatment with NAC. In conclusion, our findings suggest that SMase stimulates a ceramide-oxidant signaling pathway that results in muscle weakness and fatigue. exercise; oxidative stress; ceramide; diaphragm; inflammation doi: 10.1152/ajpcell.00065.2010.

Protective effects of blueberries (BB) against atherosclerosis and potential underlying mechanisms in reducing oxidative stress were examined in apoE-deficient ([apo.sup.E-/-]) mice. [apo.sup.E-/-] mice were fed an AIN-93G diet (CD) or CD formulated to contain 1% freeze-dried whole BB for 20 wk. The mean lesion area for [apo.sup.E-/-] mice fed BB was reduced by 39% (P < 0.001) in the aorta sinus and 58% (P < 0.001) in the descending aorta compared with CD-fed mice. These atheroprotective effects were independent of the serum lipid profile or total antioxidant capacity (as measured by oxygen radical absorbance capacity). The concentration of a biomarker of lipid peroxidation, [F.sub.2]-isoprostane, was lower in liver of BB-fed mice (P < 0.05). Genes analyzed by RT-PCR array showed that 4 major antioxidant enzymes in aorta [superoxide dismutase (SOD) 1, SOD2, glutathione reductase (GSR), and thioredoxin reductase 1] were upregulated in BB-fed mice. Enzyme activities of SOD and GSR were greater (P < 0.05) in liver and/or serum of BB-fed mice than those of CD-fed mice. In addition, serum paraoxonase 1 activity in serum of BB-fed mice was also greater than that of CD-fed mice (P< 0.05) at the end of the study. These results suggest a protective effectiveness of 8B against atherosclerosis in this [apoE.sup.-/-] mouse model. The potential mechanisms may involve reduction in oxidative stress by both inhibition of lipid peroxidation and enhancement of antioxidant defense. J. Nutr. 140: 1628-1632, 2010. doi: 10.3945/jn.110.123927.

Metagenomic and bioinformatic approaches were used to characterize plant biomass conversion within the foregut microbiome of Australia's 'model' marsupial, the Tammar wallaby (Macropus eugenii). Like the termite hindgut and bovine rumen, key enzymes and modular structures characteristic of the 'free enzyme' and 'cellulosome' paradigms of cellulose solubilization remain either poorly represented or elusive to capture by shotgun sequencing methods. Instead, multigene polysaccharide utilization loci-like systems coupled with genes encoding [beta]-1,4-endoglucanases and [beta]-1,4-endoxylanases--which have not been previously encountered in metagenomic datasets--were identified, as were a diverse set of glycoside hydrolases targeting noncellulosic polysaccharides. Furthermore, both rrs gene and other phylogenetic analyses confirmed that unique clades of the Lachnospiraceae, Bacteroidales, and Gammaproteobacteria are predominant in the Tammar foregut microbiome. Nucleotide composition-based sequence binning facilitated the assemblage of more than two megabase pairs of genomic sequence for one of the novel Lachnospiraceae clades (WG-2). These analyses show that WG-2 possesses numerous glycoside hydrolases targeting noncellulosic polysaccharides. These collective data demonstrate that Australian macropods not only harbor unique bacterial lineages underpinning plant biomass conversion, but their repertoire of glycoside hydrolases is distinct from those of the microbiomes of higher termites and the bovine rumen. cellulases | marsupials | metagenomics | plant biomass conversion | polysaccharide utilization loci doi/ 10.1073/pnas.1005297107

Nucleic acids studies use 7-deazagnanine ([G.sup.*]) and 7-deazaadenine ([A.sup.*]) as analogues of natural purine bases incapable of forming Hoogsteen base pairs, which prevents them from being involved in DNA triplexes and tetraplexes. Reduced propensity of the [G.sup.*]- and/or [A.sup.*]-modified DNA to form alternative DNA structures is utilized, for example, in PCR amplification of guanine-rich sequences. Both [G.sup.*] and [A.sup.*] exhibit significantly lower potentials of their oxidation, compared to the respective natural nucleobases. At carbon electrodes, [A.sup.*] yields an oxidation peak which is by about 200-250 mV less positive than the peak due to adenine, but coincides with oxidation peak produced by natural guanine residues. On the other hand, oxidation signal of [G.sup.*] occurs at a potential by about 300 mV less positive than the peak due to guanine, being well separated from electrochemical signals of any natural DNA component. We show that enzymatic incorporation of [G.sup.*] and [A.sup.*] can easily be monitored by simple ex situ voltammetric analysis of the modified DNA at carbon electrodes. Particularly G* is shown as an attractive electroactlve marker for DNA, efficiently incorporable by PCR. While densely [G.sup.*]-modified DNA fragments exhibit strong quenching of fluorescence of SYBR dyes, commonly used as fluorescent indicators in both gel staining and real time PCR applications, the electrochemical detection provides [G.sup.*]-specific signal suitable for the quantltation of the amplified DNA as well as for the determination of the DNA modification extent. Determination of DNA amplicons based on the measurement of peak [G.sup.*ox] is not affected by signals produced by residual oligonucleotide primers or primary templates containing natural purines. 10.1021/ac100757v

The development of continuous glucose monitoring systems is a major trend in diabetes-related research. Small, easy-to-wear systems which are robust enough to function over many days without maintenance are the goal. We present a new sensing system for continuous glucose monitoring based on a micrereactor incorporating chaotic mixing channels. Two different types of chaotic mixing channels with arrays of either slanted or herringbone grooves were fabricated in poly(dimethylsiloxane) (PDMS) and compared to channels containing no grooves. Mixing in channels with slanted grooves was characterized using a fluorescence method as a function of distance and at different flow rates, and compared to the mixing behavior observed in channels with no grooves. For electrochemical detection, a thin-film Pt electrode was positioned at the end of the fluidic channel as an on-chip detector of the reaction product, [H.sub.2][O.sub.2]. Glucose determination was performed by rapidly mixing glucose and glucose oxidase (GOx) in solution at a flow rate of 0.5 [micro]L/min and 1.5 [micro]L/min, respectively. A 150 U/mL GOx solution was selected as the optimum concentration of enzyme. In order to investigate the dependence of device response on flow rate, experiments with a premixed solution of glucose and GOx were compared to experiments in which glucose and GOx were reacted on-chip. Calibration curves for glucose (0-20 mM, in the clinical range of interest) were obtained in channels with and without grooves, using amperometdc detection and a 150 U/mL GOx solution for in-chip reaction. 10.1021/ac1000509

Alkylglycerol monooxygenase (glyceryl-ether monooxygenase, EC 1.14.16.5) is the only enzyme known to cleave the O-alkyl bond of ether lipids which are essential components of brain membranes, protect the eye from cataract, interfere or mediate signalling processes, and are required for spermatogenesis. Along with phenylalanine hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase, and nitric oxide synthase, alkylglycerol monooxygenase is one of five known enzymatic reactions which depend on tetrahydrobiopterin. Although first described in 1964, no sequence had been assigned to this enzyme so far since it lost activity upon protein purification attempts. A functional library screen using pools of plasmids of a rat liver expression library transfected to CHO cells was also unsuccessful. We therefore selected human candidate genes by bioinformatic approaches and by proteomic analysis of partially purified enzyme and tested alkylglycerol monooxygenase activity in CHO cells transfected with expression plasmids. Transmembrane protein 195, a predicted membrane protein with unassigned function which occurs in bilateral animals, was found to encode for tetrahydrobiopterin-dependent alkylglycerol monooxygenase. This sequence assignment was confirmed by injection of transmembrane protein 195 cRNA into Xenopus laevis oocytes. Transmembrane protein 195 shows no sequence homology to aromatic amino acid hydroxylases or nitric oxide synthases, but contains the fatty acid hydroxylase motif. This motif is found in enzymes which contain a diiron center and which carry out hydroxylations of lipids at aliphatic carbon atoms like alkylglycerol monooxygenase. This sequence assignment suggests that alkylglycerol monooxygenase forms a distinct third group among tetrahydrobiopterin-dependent enzymes. EC 1.14.16.5 | glyceryl-ether monooxygenase | nitric oxide synthase | phenylalanine hydroxylase | transmembrane protein 195 doi/ 10.1073/pnas.1002404107

The hydrolysis of polysaccharides containing mannan requires endo-l,4-[beta]-mannanase and 1,4-[beta]-mannosidase activities. In the current report, the biochemical properties of two endo-[beta]-1,4-mannanases (Man5A and Man5B) from Caldanaerobius polysaccharolyticus were studied. Man5A is composed of an N-terminal signal peptide (SP), a catalytic domain, two carbohydrate-binding modules (CBMs), and three surface layer homology (SLH) repeats, whereas Man5B lacks the SP, CBMs, and SLH repeats. To gain insights into how the two glycoside hydrolase family 5 (GH5) enzymes may aid the bacterium in energy acquisition and also the potential application of the two enzymes in the biofuel industry, two derivatives of Man5A (Man5A-TMI [TM1 stands for truncational mutant 1], which lacks the SP and SLH repeats, and Man5A-TM2, which lacks the SP, CBMs, and SLH repeats) and the wild-type Man5B were biochemically analyzed. The Man5A derivatives displayed endo-l,4-[beta]-mannanase and endo-l,4-[beta]-glucanase activities and hydrolyzed oligosaccharides with a degree of polymerization (DP) of 4 or higher. Man5B exhibited endo-l,4-[beta]-mannanase activity and little endo-l,4-[beta]- glucanase activity; however, this enzyme also exhibited 1,4-[beta]-mannosidase and cellodextrinase activities. Man5A-TM1, compared to either Man5A-TM2 or Man5B, had higher catalytic activity with soluble and insoluble polysaccharides, indicating that the CBMs enhance catalysis of Man5A. Furthermore, Man5A-TM1 acted synergistically with Man5B in the hydrolysis of [beta]-mannan and carboxymethyl cellulose. The versatility of the two enzymes, therefore, makes them a resource for depolymerization of mannan-containing polysaccharides in the biofuel industry. Furthermore, on the basis of the biochemical and genomic data, a molecular mechanism for utilization of mannan-containing nutrients by C. polysaccharolyticus is proposed. doi: 10.1128/JB.00257-10

A novel DNA detection platform based on a hairpin DNA switch, nanoparticles, and enzyme signal amplification for ultrasensitive detection of DNA hybridization has been developed in this work. In this DNA assay, a 'stem--loop' DNA probe dually labeled with a thiol at its 5' end and a biotin at its 3' end, respectively, was used. This probe was immobilized on the gold nanoparticles (AuNPs) anchored by a protein, [gamma]-globulin, on a 96-well microplate. In the absence of target DNA, the immobilized probe with the stem-loop structure shields the biotin from being approached by a bulky horseradish peroxidase linked streptavidin (streptavidin--HRP) conjugate due to the steric hindrance. However, in the presence of target DNA, the hybridization between the hairpin DNA probe and the target DNA causes significant conformational change of the probe, which forces biotin away from the surface of AuNPs. As a result, the biotin becomes accessible by the streptavidin--HRP, and the target hybridization event can be sensitively detected via the HRP catalyzed substrate 3,3',5,5'-tetramethylbenzidine using a spectrophometric method. Some experimental parameters governing the performance of the assay have been optimized. At optimal conditions, this DNA assay can detect DNA at the concentration of femtemolar level by means of a signal amplification strategy based on the combination of enzymes and nanoparticles. This approach also has shown excellent specificity to distinguish single-base mismatches of DNA targets because of the intrinsic high selectivity of the hairpin DNA probe. 10.1021/ac1006238

A novel, simple, sensitive, and precise spectrofluorimetric method was developed for measuring the activity of the enzyme [alpha]-L-fucosidase (AFU). The method was based upon measuring the quenching of the luminescence intensity of the produced yellow colored complex ion associate of 2-chloro-4-nitrophenol [2-CNP] and a nano composite optical sensor samarium(III)--doxycycline [[[Sm.sup.3+]-DC].sup.+] complex in a sol--gel matrix at 645 nm. The remarkable quenching of the luminescence intensity of the [[[Sm.sup.3+]-DC].sup.+] complex doped in a sol--gel matrix by various concentrdtions of the reagent [2-CNP] was successfully used as an optical sensor for the assessment of AFU activity. The calibration plot was achieved over the concentration range 3.4 x [10.sup.9]-1.0 x [10.sup.-6] mol [L.sup.-1] [2-CNP] with a correlation coefficient of 0.99 and a detection limit of 6.0 x [10.sup.-10] mol [L.sup.-1]. The method was used satisfactorily for the assessment of the AFU activity in a number of serum samples collected from various patients. A significant correlation between the luminescence activity of the enzyme AFU measured by the proposed procedure and the standard method was applied to patients and controls. The method proceeds without practical artifacts compared to the standard method. 10.1021/ac101033j

TNK-tPA products from the innovator and follow-on manufacturers were characterized and compared. All tryptic peptides including N-terminal, C-terminal, and mutated peptides as well as the disulfide-linked peptides were identified, with the demonstration of the same primary sequence and disulfide linkages between the innovator and follow-on products. The three N-linked and one O-linked fucose glycosylation sites were identified. The two N-linked fucose sites (N103 and N448) and one O-linked fucose site (T61) were fully glycosylated in both innovator and follow-on products. The other N-linked site (N184) was partially giycosylated and exhibited a ~2.5-fold difference between the innovator (60% occupancy) and follow-on (25% occupancy) products. Since the glycosylation occupancy at this site is known to affect biological activity in the clot lysis assay, this observed difference could cause a concern as to their bioequivalence. The cleavage site for the conversion of the zymogen form to active enzyme was also identified between R275 and I276, with a cleavage of 40% for the innovator and 10% for the follow-on products. Both the giycosylation occupancy (%) and the chain cleavage (%) were determined by two independent approaches, starting from either the peptide or intact protein separation, with consistent results by both methods. Subtle differences of modifications such as deamidation and oxidation between the innovator and biosimilar products were shown at M207, M445, M490 and N58, N184. The observation of different extents of oxidation at M207 and deamidation at N 184, which could influence the clot lysis activity, were also of potential concern in drug efficacy between the follow-on and innovator products. 10.1021/ac100956x

A study was conducted to examine the patients in two unrelated pedigrees with combined OXPHOS deficiencies associated with a decrease in the synthesis of all mitochondrially encoded polypeptides. The identified 1 bp deletion in C12orf65 in one patient which does not exhibit peptidyl-tRNA hydrolase activity in an in vitro assay with bacterial ribosomes might play significant role in recycling abortive peptidyl-tRNA species, released from the ribosome during the elongation phase of translation.

Fusobacterium nucleatum produces a large amount of the toxic metabolite hydrogen sulfide in the oral cavity. Here, we report the molecular basis of F. nucleatum [H.sub.2]S production, which is associated with two different enzymes: the previously reported Cdl (Fn1220) and the newly identified Lcd (Fn0625). SDS-PAGE analysis with activity staining revealed that crude enzyme extracts from F. nucleatum ATCC 25586 contained three major [H.sub.2]S-producing proteins. Two of the proteins with low molecular masses migrated similarly to purified Fn0625 and Fn1220. Their kinetic values suggested that Fn0625 had a lower enzymic capacity to produce [H.sub.2]S from L-cysteine (~30%) than Fn1220. The Fn0625 protein degraded a variety of substrates containing [beta]C-S linkages to produce ammonia, pyruvate and sulfur-containing products. Unlike Fn0625, Fn1220 produced neither pyruvate nor ammonia from L-cysteine. Reversed-phase HPLC separation and mass spectrometry showed that incubation of L-cysteine with Fn1220 produced [H.sub.2]S and an uncommon amino acid, lanthionine, which is a natural constituent of the peptidoglycans of F. nucleatum ATCC 25586. In contrast, most of the sulfur-containing substrates tested, except L-cysteine, were not used by Fn1220. Real-time PCR analysis demonstrated that the fn 1220 gene showed several-fold higher expression than fn0625 and housekeeping genes in exponential-phase cultures of F. nucleatum. Thus, we conclude that Fn0625 and Fn1220 produce [H.sub.2]S in distinct manners: Fn0625 carries out [beta]-elimination of L-cysteine to produce [H.sub.2]S, pyruvate and ammonia, whereas Fn1220 catalyses the [beta]-replacement of L-cysteine to produce [H.sub.2]S and lanthionine, the latter of which may be used for peptidoglycan formation in F. nucleatum. DOI 10.1099/mic.0.039180-0