Search

The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiota. It is unclear, however, whether bacterial consortia or single organisms are required to depolymerize highly complex glycans. Here we show that the gut bacterium Bacteroides thetaiotaomicron uses the most structurally complex glycan known: the plant pectic polysaccharide rhamnogalacturonan-II, cleaving all but 1 of its 21 distinct glycosidic linkages. The deconstruction of rhamnogalacturonan-II side chains and backbone are coordinated to overcome steric constraints, and the degradation involves previously undiscovered enzyme families and catalytic activities. The degradation system informs revision of the current structural model of rhamnogalacturonan-II and highlights how individual gut bacteria orchestrate manifold enzymes to metabolize the most challenging glycan in the human diet., Author(s): Didier Ndeh [1]; Artur Rogowski [1]; Alan Cartmell [1]; Ana S. Luis [1]; Arnaud Basl [1]; Joseph Gray [1]; Immacolata Venditto [1]; Jonathon Briggs [1]; Xiaoyang Zhang [1]; Aurore [...]

The first crystallographic data for a ternary type II R67 DHFR.DHF.[NADP.sup.} (DHFR = dihyrofolate reductase) catalytic complex is presented. The structure provides insight into function of DHFR enzyme that lacks the active site carboxyl residue ubiquitous in type I DHFRs.

The method of voltage clamp fluorometry combined with site-directed fluorescence labeling was used to detect local protein motions of the fully active [Na.sup.+]/[K.sup.+]-ATPase in real time under physiological conditions. Because helix M5 extends from the cytoplasmic site of ATP hydrolysis into the cation binding region, we chose the extracellular M5-M6 loop of the sheep [[alpha].sub.1]-subunit for the insertion of cysteine residues to identify reporter positions for conformational rearrangements during the catalytic cycle. After expression of the single cysteine mutants in Xenopus oocytes and covalent attachment of tetramethylrhodamine-6-maleimide, only mutant N790C reported molecular rearrangements of the M5-M6 loop by showing large, ouabain-sensitive fluorescence changes ([approximately equal to] 5%) on addition of extracellular [K.sup.+]. When the enzyme was subjected to voltage jumps under [Na.sup.+]/[Na.sup.+]-exchange conditions, we observed fluorescence changes that directly correlated to transient charge movements originating from the [E.sub.1]P-[E.sub.2]P transition of the transport cycle. The voltage jump-induced fluorescence changes and transient currents were abolished after replacement of [Na.sup.+] by tetraethylammonium or on addition of ouabain, showing that conformational flexibility is impaired under these conditions. Voltage-dependent fluorescence changes could also be observed in the presence of subsaturating [K.sup.+] concentrations. This allowed to monitor the time course of voltage-dependent relaxations into a new stationary distribution of states under turnover conditions, showing the acceleration of relaxation kinetics with increasing [K.sup.+] concentrations. As a result, the stationary distribution between [E.sub.1] and [E.sub.2] states and voltage-dependent relaxation times can be determined at any time and membrane potential under [Na.sup.+]/[Na.sup.+] exchange as well as [Na.sup.+]/[K.sup.+] turnover conditions.

Seven secretory mammalian kexin-like subtilases have been identified that cleave a variety of precursor proteins at monobasic and dibasic residues. The recently characterized pyrolysin-like subtilase SKI-1 cleaves proproteins at nonbasic residues. In this work we describe the properties of a proteinase K-like subtilase, neural apoptosis-regulated convertase 1 (NARC-1), representing the ninth member of the secretory subtilase family. Biosynthetic and micro-sequencing analyses of WT and mutant enzyme revealed that human and mouse pro-NARC-1 are autocatalytically and intramolecularly processed into NARC-1 at the (Y,I)VV(V,L)(L,M) [down arrow] motif, a site that is representative of its enzymic specificity. In vitro peptide processing studies and/or Ala substitutions of the P1-P5 sites suggested that hydrophobic/aliphatic residues are more critical at P1, P3, and P5 than at P2 or P4. NARC-1 expression is highest in neuroepithelioma SK-N-MCIXC, hepatic BRL-3A, and in colon carcinoma LoVo-C5 cell lines. In situ hybridization and Northern blot analyses of NARC-1 expression during development in the adult and after partial hepatectomy revealed that it is expressed in cells that have the capacity to proliferate and differentiate. These include hepatocytes, kidney mesenchymal cells, intestinal ileum, and colon epithelia as well as embryonic brain telencephalon neurons. Accordingly, transfection of NARC-1 in primary cultures of embryonic day 13.5 telencephalon cells led to enhanced recruitment of undifferentiated neural progenitor cells into the neuronal lineage, suggesting that NARC-1 is implicated in the differentiation of cortical neurons. cleavage specificity | hypercholesterolemia | neurogenesis | hepatogenesis

Sulfotransferases are an important class of enzymes that catalyze the transfer of a sulfuryl group to a hydroxyl or amine moiety on various molecules including small-molecule drugs, steroids, hormones, carbohydrates, and proteins. They have been implicated in a number of disease states but remain poorly understood, complicating the design of specific, small-molecule inhibitors. A linear free-energy analysis in both the forward and reverse directions indicates that the transfer of a sulfuryl group to an aryl hydroxyl group catalyzed by [beta]-arylsulfotransferase IV likely proceeds by a dissociative (sulfotrioxide-like) mechanism. Values for the Bronsted coefficients ([[beta].sub.nuc] and [[beta].sub.lg]) are + 0.33 and -0.45, giving Leffler [alpha] values of 0.19 and 0.61 for the forward and reverse reactions, respectively. sulfotransferase | linear free energy | mechanism

Research has been conducted on P-glycoprotein which mediates drug transport. The number of nucleotides bound to this P-glycoprotein during different stages of catalytic cycles and nucleotide binding affinity have been measured via the use of fluorescence techniques, and the results are reported.

The role of the [Na.sup.+] pump [[alpha].sub.2]-subunit in [Ca.sup.2+] signaling was examined in primary cultured astrocytes from wild-type ([[alpha].sub.2.sup.+/+] = WT) mouse fetuses and those with a null mutation in one [[[alpha].sub.2.sup.+/-] = heterozygote (Het)] or both [[[alpha].sub.2.sup.-/-] = knockout (KO)] [[alpha].sub.2] genes. [Na.sup.+] pump catalytic ([alpha]) subunit expression was measured by immunoblot; cytosol [[Na.sup.+]] ([[[Na.sup.+]].sub.cyt]) and [[Ca.sup.2+]] ([[[Ca.sup.2+]].sub.cyt]) were measured with sodium-binding benzofuran isophthalate and fura 2 by using digital imaging. Astrocytes express [Na.sup.+] pumps with both [[alpha].sub.1-] ([approximately equal to] 80% of total [alpha]) and [[alpha].sub.2-] ([approximately equal to] 20% of total [alpha]) subunits. Het astrocytes express [approximately equal to] 50% of normal [[alpha].sub.2]; those from KO express none. Expression of [[alpha].sub.1] is normal in both Het and KO cells. Resting [[[Na.sup.+]].sub.cyt] = 6.5 mM in WT, 6.8 mM in Het (P > 0.05 vs. WT), and 8.0 mM in KO cells (P < 0.001); 500 nM ouabain (inhibits only [[alpha].sub.2]) equalized [[[Na.sup.+]].sub.cyt] at 8 mM in all three cell types. Resting [[[Ca.sup.2+]].sub.cyt] = 132 nM in WT, 162 nM in Het, and 196 nM in KO cells (both P < 0.001 vs. WT). Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER) [Ca.sup.2+] pumps and unloads the ER, induces transient (in [Ca.sup.2+]-free media) or sustained (in [Ca.sup.2+]-replete media) elevation of [[[Ca.sup.2+]].sub.cyt]. These [Ca.sup.2+] responses to 10 [micro]M CPA were augmented in Het as well as KO cells. When CPA was applied in [Ca.sup.2+]-free media, the reintroduction of [Ca.sup.2+] induced significantly larger transient rises in [[[Ca.sup.2+]].sub.cyt] (due to [Ca.sup.2+] entry through store-operated channels) in Het and KO cells than in WT cells. These results correlate with published evidence that [[alpha].sub.2] [Na.sup.+] pumps and [Na.sup.+]/[Ca.sup.2+] exchangers are confined to plasma membrane microdomains that overlie the ER. The data suggest that selective reduction of [[alpha].sub.2] [Na.sup.+] pump activity can elevate local [[Na.sup.+]] and, via [Na.sup.+]/[Ca.sup.2+] exchange, [[Ca.sup.2+]] in the tiny volume of cytosol between the plasma membrane and ER. This, in turn, augments adjacent ER [Ca.sup.2+] stores and thereby amplifies [Ca.sup.2+] signaling without elevating bulk [[[Na.sup.+]].sub.cyt]. astrocytes; catalytic subunit; fura 2; sodium-binding benzofuran isophthalate; sodium-potassium-adenosine 5'-triphosphatase isoforms; transgenic mice

In enzyme catalysis, where exquisitely positioned functionality is the sine qua non, atomic coordinates for a Michaelis complex can provide powerful insights into activation of the substrate. We focus here on the initial proton transfer of the isomerization reaction catalyzed by triosephosphate isomerase and present the crystal structure of its Michaelis complex with the substrate dihydroxyacetone phosphate at near-atomic resolution. The active site is highly compact, with unusually short and bifurcated hydrogen bonds for both catalytic Glu-165 and His-95 residues. The carboxylate oxygen of the catalytic base Glu-165 is positioned in an unprecedented close interaction with the ketone and the [alpha]-hydroxy carbons of the substrate (C ... O 3.0 [approximately equal to] [Angstrom]), which is optimal for the proton transfer involving these centers. The electrophile that polarizes the substrate, His-95, has close contacts to the substrate's O1 and O2 (N ... O [less than or equal to] 3.0 and 2.6 [Angstrom], respectively). The substrate is conformationally relaxed in the Michaelis complex: the phosphate group is out of the plane of the ketone group, and the hydroxy and ketone oxygen atoms are not in the cisoid configuration. The [epsilon] ammonium group of the electrophilic Lys-12 is within hydrogen-bonding distance of the substrate's ketone oxygen, the bridging oxygen, and a terminal phosphate's oxygen, suggesting a role for this residue in both catalysis and in controlling the flexibility of active-site loop.

The x-ray crystal structure of dimeric (+)-bornyl diphosphate synthase, a metal-requiring monoterpene cyclase from Salvia officinalis, is reported at 2.0-[Angstrom] resolution. Each monomer contains two [alpha]-helical domains: the C-terminal domain catalyzes the cyclization of geranyl diphosphate, orienting and stabilizing multiple reactive carbocation intermediates; the N-terminal domain has no clearly defined function, although its N terminus caps the active site in the C-terminal domain during catalysis. Structures of complexes with aza analogues of substrate and carbocation intermediates, as well as complexes with pyrophosphate and bornyl diphosphate, provide 'snapshots' of the terpene cyclization cascade.

Research has been conducted on the neusteroid allopregnanolone. The kinetic mechanism of 5alpha-dihydroprogesterone reduction catalyzed by 3alpha-hydroxysteroid dehydrogenase type III has been investigated in studying the activation of 3alpha-hydroxysteroid dehydrogenase type III catalyzed allopregnanolone formation and the details are reported.

Research has been conducted on enoyl-CoA hydratase. The ability of this hydratase to catalyze Cr-CoA hydration to 3(S)-hydroxybutyryl with an exceptional degree of stereospecificity has been confirmed and the results indicate that the correct product enantiomer formation requires oxyanion hole and substrate's optimal positioning.

Research has been conducted on the translocation step of elongation. The rate constant of the translocation reactions catalyzed by elongation factor G in the presence of non-hydrolyzable GTP analogues has been determined providing contributions of elongation factor G binding and GTP hydrolysis to translocation catalysis.

Research has been conducted on Saccharomyces cerevisiae lysine 213 and histidine 233. The role of these residues in Mn (super)2+ binding and in catalysis has been investigated and the results demonstrate that mutation of lysine 213 and histidine 233 affects catalysis and that they are required for proper Mn (super)2+ binding.

Research has been conducted on the coupled protein motion role in enzymic catalysis. The relationship between protein motions and the hydride rate transfer has been investigated via the study of additional residues involved in the coupled network and the details are reported.

Research has been conducted on the tartrate dehydrogenase mechanism. The analysis of this mechanism has been carried out via the multiple isotope effect studies in investigating the hypothesis that the change in mechanism is a phenomenon of the divalent metal ion-dependent oxidative decarboxylase.

Research has been conducted on the protein tyrosine kinase Csk. The structural properties and intrinsic fluorescence of Csk have been investigated via site-specific muitagenesis and urea denaturation and the role of the Csk conserved signature residues has been examined and described.

Research has been conducted on the lysozyme from the silkworm. The three-dimensional structure of this lysozyme has been investigated via the use of X-ray crystallography and the results demonstrate the similarity between the silkworm lysozyme and the psychrophilic enzymes which explains its effective activity at low temperatures.

Research has been conducted on the Sorghum bicolor hydroxynitrile lyase. The crystal structure of this lyase has been investigated in complex with the inhibitor benzoic acid and the effect of this structure on the catalysis evolution in the hydroxynitrile lyase super family has been analyzed and discussed.

Research has been conducted on the hepatitis delta virus ribozyme. Results demonstrate that the trans-acting hepatitis delta virus ribozyme is positioned along the trajectory from the precursor to product for the reaction chemistry.

This review gives a synopsis of the meeting cosponsored by the American Society for Microbiology and the Society for General Microbiology on the title topic. The B3 topics are discussed with respect to their biological, chemical, and biotechnological aspects and applications.

Research has been conducted on the potent mammalian tachykinin substance P receptor antagonist, aprepitant. The synthesis of this antagonist is described.

Research has been conducted on the vanadium oxyacetylacetonate catalytic system. The use of this system for the aerobic oxidation of propargylic alcohols and the mechanistic aspects of this catalytic reaction are discussed.

Enzyme-catalyzed [beta]-elimination of sugar uronic acids, exemplified by the degradation of plant cell wall pectins, plays an important role in a wide spectrum of biological processes ranging from the recycling of plant biomass through to pathogen virulence. The three-dimensional crystal structure of the catalytic module of a 'family PL-10' polysaccharide lyase, Pel10Acm from Cellvibrio japonicus, solved at a resolution of 1.3 [Angstrom], reveals a new polysaccharide lyase fold and is the first example of a polygalacturonic acid lyase that does not exhibit the 'parallel [beta]-helix' topology. The 'Michaelis' complex of an inactive mutant in association with the substrate trigalacturonate/[Ca.sup.2+] reveals the catalytic machinery harnessed by this polygalacturonate lyase, which displays a stunning resemblance, presumably through convergent evolution, to the tetragalacturonic acid complex observed for a structurally unrelated polygalacturonate lyase from family PL-1. Common coordination of the -1 and +1 subsite saccharide carboxylate groups by a protein-liganded [Ca.sup.2+] ion, the positioning of an arginine catalytic base in close proximity to the [alpha]-carbon hydrogen and numerous other conserved enzyme-substrate interactions, considered in light of mutagenesis data for both families, suggest a generic polysaccharide anti-[beta]-elimination mechanism.

Calcineurin, a [Ca.sup.2+]/calmodulin-dependent protein phosphatase, is the common target for two immunophilin--immunosuppressant complexes, cyclophilin A--cyclosporin A (CyPA-CsA) and FKBP--FK506. How the two structurally distinct immunophilin--drug complexes bind the same target has remained unknown. We report the crystal structure of calcineurin (CN) in complex with CyPA-CsA at 2.8-[Angstrom] resolution. The CyPA-CsA complex binds to a composite surface formed by the catalytic and regulatory subunits of CN, where the complex of FK506 and its binding protein FKBP also binds. While the majority of the CN residues involved in the binding are common for both immunophilin-immunosuppressant complexes, a significant number of the residues are distinct. Unlike FKBP-FK506, CyPA-CsA interacts with Arg-122 at the active site of CN, implying direct involvement of CyPA-CsA in the regulation of CN catalysis. The simultaneous interaction of CyPA with both the composite surface and the active site of CN suggests that the composite surface may serve as a substrate recognition site responsible for the narrow substrate specificity of CN. The comparison of CyPA-CsA-CN with FKBP-FK506-CN significantly contributes to understanding the molecular basis of regulation of CN activity by the immunophilin--immunosuppressant. immunosuppressants | FK506 | FK506-binding protein | protein phosphatase | T cell

Research has been conducted on trimesityliridium(III). The reaction of this compound with O (sub)2 carried out in producing oxotrimesityliridium(V) has been investigated and the results indicate that no evidence of substantial accumulation of an intermediate has been found.

Research has been conducted on the rhenium tricarbonyl bipyridine complexes. The spectroscopic characterization of these complexes has been carried out and the results demonstrate that they catalyze benzylideneaniline aziridination with ethyl diazoacetate.

Research has been conducted on the aryl halides. Results demonstrate that the amination of these compounds carried out in the presence of air-stable alkali metal hydroxide bases and a palladium catalyst has produced arylamines.

Research has been conducted on the cuscohygrine and (+)-dihydrocuscohygrine. The results of the enantioselective synthesis of these compounds indicate that cuscohygrine has been obtained via Jones oxidation and (+)-dihydrocuscohygrine has been obtained via ruthenium-catalyzed tandem ring rearrangement metathesis.

Research has been conducted on the Tet(L) and Tet(K) proteins. Results indicate that these proteins couple tetracycline-divalent metal efflux to K (super)+ uptake and use this K (super)+ as a coupling ion when external pH increases.

Research has been conducted on the homologous tungsten(VI) derivatives. The reactivity, epoxidation mechanism and applicability of these compounds have been investigated and compared to those of the molybdenum(VI) compounds and the details are presented.

Research has been conducted on the biphenyl derivatives with the alkyne unit. The conversion of these derivatives into substituted phenanthrenes after the exposure to the catalytic amount of PtCl (sub)2 in toluene is described.

Research has been conducted on tryptophan regioisomers. The catalytic asymmetric syntheses of these regioisomers have been carried out in expanding the tryptophan-like amino acid toolbox and the details are reported.

Research has been conducted on the bicyclo[3.1.0]hexane scaffold. The generation of the complex molecular scaffold from the simple starting material and with chemical diastereoselectivity and the ability to combine this chemistry with the lipase-catalyzed resolution step are discussed.

Research has been conducted on peptide deformylase. The function of the conserved glutamate in Escherichia coli peptide deformylase has been investigated via the difference FTIR spectroscopic analysis and the results indicate that it functions as a general acid during the tetrahedral intermediate decomposition.

Aurora-A is a serine-threonine kinase implicated in the assembly and maintenance of the mitotic spindle. Here we show that human Aurora-A binds to TPX2, a prominent component of the spindle apparatus. TPX2 was identified by mass spectrometry as a major protein coimmunoprecipitating specifically with Aurora-A from mitotic HeLa cell extracts. Conversely, Aurora-A could be detected in TPX2 immunoprecipitates. This indicates that subpopulations of these two proteins undergo complex formation in vivo. Binding studies demonstrated that the N[H.sub.2] terminus of TPX2 can directly interact with the COOH-terminal catalytic domain of Aurora-A. Although kinase activity was not required for this interaction, TPX2 was readily phosphorylated by Aurora-A. Upon siRNA-mediated elimination of TPX2 from cells, the association of Aurora-A with the spindle microtubules was abolished, although its association with spindle poles was unaffected. Conversely, depletion of Aurora-A by siRNA had no detectable influence on the localization of TPX2. We propose that human TPX2 is required for targeting Aurora-A kinase to the spindle apparatus. In turn, Aurora-A might regulate the function of TPX2 during spindle assembly.

Research has been conducted on Mytilus edulis PEP mutase. The crystal structure of this mutase in complex with the substrate-analogue inhibitor has been investigated and the results indicate that the phosphoryl group transfer occurs on PEP Si-face with the configuration retention at phosphorus.

Research has been conducted on the 2'-substituted 4-bromo-2,4'-bithiazoles. The synthesis of these compounds has been achieved in two steps starting from 2,4-dibromothiazole and the results of the regioselective palladium(0)-catalyzed cross-coupling step converting 2,4-dibromothiazole into 2-substituted 4-bromothiazoles are reported.