Journal articles on the topic 'Complex substrates'
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Kim, Ikjin, Jungmi Ahn, Chang Liu, Kaori Tanabe, Jennifer Apodaca, Tadashi Suzuki, and Hai Rao. "The Png1–Rad23 complex regulates glycoprotein turnover." Journal of Cell Biology 172, no. 2 (January 9, 2006): 211–19. http://dx.doi.org/10.1083/jcb.200507149.
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Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by a pathway termed ER-associated protein degradation (ERAD). Glycans are often removed from glycosylated ERAD substrates in the cytosol before substrate degradation, which maintains the efficiency of the proteasome. Png1, a deglycosylating enzyme, has long been suspected, but not proven, to be crucial in this process. We demonstrate that the efficient degradation of glycosylated ricin A chain requires the Png1–Rad23 complex, suggesting that this complex couples protein deglycosylation and degradation. Rad23 is a ubiquitin (Ub) binding protein involved in the transfer of ubiquitylated substrates to the proteasome. How Rad23 achieves its substrate specificity is unknown. We show that Rad23 binds various regulators of proteolysis to facilitate the degradation of distinct substrates. We propose that the substrate specificity of Rad23 and other Ub binding proteins is determined by their interactions with various cofactors involved in specific degradation pathways.
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Wu, Xi, Lanlan Li, and Hui Jiang. "Doa1 targets ubiquitinated substrates for mitochondria-associated degradation." Journal of Cell Biology 213, no. 1 (April 4, 2016): 49–63. http://dx.doi.org/10.1083/jcb.201510098.
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Mitochondria-associated degradation (MAD) mediated by the Cdc48 complex and proteasome degrades ubiquitinated mitochondrial outer-membrane proteins. MAD is critical for mitochondrial proteostasis, but it remains poorly characterized. We identified several mitochondrial Cdc48 substrates and developed a genetic screen assay to uncover regulators of the Cdc48-dependent MAD pathway. Surprisingly, we identified Doa1, a substrate-processing factor of Cdc48 that inhibits the degradation of some Cdc48 substrates, as a critical mediator of the turnover of mitochondrial Cdc48 substrates. Deletion of DOA1 causes the accumulation and mislocalization of substrates on mitochondria. Profiling of Cdc48 cofactors shows that Doa1 and Cdc48-Ufd1-Npl4 form a functional complex mediating MAD. Biochemically, Doa1 interacts with ubiquitinated substrates and facilitates substrate recruitment to the Cdc48-Ufd1-Npl4 complex. Functionally, Doa1 is critical for cell survival under mitochondrial oxidative stress, but not ER stress, conditions. Collectively, our results demonstrate the essential role of the Doa1–Cdc48-Ufd1-Npl4 complex in mitochondrial proteostasis and suggest that Doa1 plays dual roles on the Cdc48 complex.
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Dayan, Peter. "Simple substrates for complex cognition." frontiers in Neuroscience 2, no. 2 (December 15, 2008): 255–63. http://dx.doi.org/10.3389/neuro.01.031.2008.
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Kanehara, Kazue, Wei Xie, and Davis T. W. Ng. "Modularity of the Hrd1 ERAD complex underlies its diverse client range." Journal of Cell Biology 188, no. 5 (March 8, 2010): 707–16. http://dx.doi.org/10.1083/jcb.200907055.
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Secretory protein folding is monitored by endoplasmic reticulum (ER) quality control mechanisms. Misfolded proteins are retained and targeted to ER-associated degradation (ERAD) pathways. At their core are E3 ubiquitin ligases, which organize factors that recognize, ubiquitinate, and translocate substrates. Of these, we report that the Hrd1 complex manages three distinct substrate classes. A core complex is required for all classes and is sufficient for some membrane proteins. The accessory factors Usa1p and Der1p adapt the complex to process luminal substrates. Their integration is sufficient to process molecules bearing glycan-independent degradation signals. The presence of Yos9p extends the substrate range by mediating the recognition of glycan-based degradation signals. This modular organization enables the Hrd1 complex to recognize topologically diverse substrates. The Hrd1 system does not directly evaluate the folding state of polypeptides. Instead, it does so indirectly, by recognizing specific embedded signals displayed upon misfolding.
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Min, Mingwei, Ugo Mayor, and Catherine Lindon. "Ubiquitination site preferences in anaphase promoting complex/cyclosome (APC/C) substrates." Open Biology 3, no. 9 (September 2013): 130097. http://dx.doi.org/10.1098/rsob.130097.
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Ordered progression of mitosis requires precise control in abundance of mitotic regulators. The anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase plays a key role by directing ubiquitin-mediated destruction of targets in a temporally and spatially defined manner. Specificity in APC/C targeting is conferred through recognition of substrate D-box and KEN degrons, while the specificity of ubiquitination sites, as another possible regulated dimension, has not yet been explored. Here, we present the first analysis of ubiquitination sites in the APC/C substrate ubiquitome. We show that KEN is a preferred ubiquitin acceptor in APC/C substrates and that acceptor sites are enriched in predicted disordered regions and flanked by serine residues. Our experimental data confirm a role for the KEN lysine as an ubiquitin acceptor contributing to substrate destruction during mitotic progression. Using Aurora A and Nek2 kinases as examples, we show that phosphorylation on the flanking serine residue could directly regulate ubiquitination and subsequent degradation of substrates. We propose a novel layer of regulation in substrate ubiquitination, via phosphorylation adjacent to the KEN motif, in APC/C-mediated targeting.
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Knape, Matthias J., Maximilian Wallbott, Nicole C. G. Burghardt, Daniela Bertinetti, Jan Hornung, Sven H. Schmidt, Robin Lorenz, and Friedrich W. Herberg. "Molecular Basis for Ser/Thr Specificity in PKA Signaling." Cells 9, no. 6 (June 25, 2020): 1548. http://dx.doi.org/10.3390/cells9061548.
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cAMP-dependent protein kinase (PKA) is the major receptor of the second messenger cAMP and a prototype for Ser/Thr-specific protein kinases. Although PKA strongly prefers serine over threonine substrates, little is known about the molecular basis of this substrate specificity. We employ classical enzyme kinetics and a surface plasmon resonance (SPR)-based method to analyze each step of the kinase reaction. In the absence of divalent metal ions and nucleotides, PKA binds serine (PKS) and threonine (PKT) substrates, derived from the heat-stable protein kinase inhibitor (PKI), with similar affinities. However, in the presence of metal ions and adenine nucleotides, the Michaelis complex for PKT is unstable. PKA phosphorylates PKT with a higher turnover due to a faster dissociation of the product complex. Thus, threonine substrates are not necessarily poor substrates of PKA. Mutation of the DFG+1 phenylalanine to β-branched amino acids increases the catalytic efficiency of PKA for a threonine peptide substrate up to 200-fold. The PKA Cα mutant F187V forms a stable Michaelis complex with PKT and shows no preference for serine versus threonine substrates. Disease-associated mutations of the DFG+1 position in other protein kinases underline the importance of substrate specificity for keeping signaling pathways segregated and precisely regulated.
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Bourreau, D., P. Guillon, and M. Chatard-Moulin. "Complex permittivity measurement of optoelectronic substrates." Electronics Letters 22, no. 7 (1986): 399. http://dx.doi.org/10.1049/el:19860271.
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Neal, Sonya, Raymond Mak, Eric J. Bennett, and Randolph Hampton. "A Cdc48 “Retrochaperone” Function Is Required for the Solubility of Retrotranslocated, Integral Membrane Endoplasmic Reticulum-associated Degradation (ERAD-M) Substrates." Journal of Biological Chemistry 292, no. 8 (January 11, 2017): 3112–28. http://dx.doi.org/10.1074/jbc.m116.770610.
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A surprising feature of endoplasmic reticulum (ER)-associated degradation (ERAD) is the movement, or retrotranslocation, of ubiquitinated substrates from the ER lumen or membrane to the cytosol where they are degraded by the 26S proteasome. Multispanning ER membrane proteins, called ERAD-M substrates, are retrotranslocated to the cytosol as full-length intermediates during ERAD, and we have investigated how they maintain substrate solubility. Using an in vivo assay, we show that retrotranslocated ERAD-M substrates are moved to the cytoplasm as part of the normal ERAD pathway, where they are part of a solely proteinaceous complex. Using proteomics and direct biochemical confirmation, we found that Cdc48 serves as a critical “retrochaperone” for these ERAD-M substrates. Cdc48 binding to retrotranslocated, ubiquitinated ERAD-M substrates is required for their solubility; removal of the polyubiquitin chains or competition for binding by addition of free polyubiquitin liberated Cdc48 from retrotranslocated proteins and rendered them insoluble. All components of the canonical Cdc48 complex Cdc48-Npl4-Ufd1 were present in solubilized ERAD-M substrates. This function of the complex was observed for both HRD and DOA pathway substrates. Thus, in addition to the long known ATP-dependent extraction of ERAD substrates during retrotranslocation, the Cdc48 complex is generally and critically needed for the solubility of retrotranslocated ERAD-M intermediates.
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Saunders, Reuben A., Benjamin M. Stinson, Tania A. Baker, and Robert T. Sauer. "Multistep substrate binding and engagement by the AAA+ ClpXP protease." Proceedings of the National Academy of Sciences 117, no. 45 (October 26, 2020): 28005–13. http://dx.doi.org/10.1073/pnas.2010804117.
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Escherichia coliClpXP is one of the most thoroughly studied AAA+ proteases, but relatively little is known about the reactions that allow it to bind and then engage specific protein substrates before the adenosine triphosphate (ATP)-fueled mechanical unfolding and translocation steps that lead to processive degradation. Here, we employ a fluorescence-quenching assay to study the binding of ssrA-tagged substrates to ClpXP. Polyphasic stopped-flow association and dissociation kinetics support the existence of at least three distinct substrate-bound complexes. These kinetic data fit well to a model in which ClpXP and substrate form an initial recognition complex followed by an intermediate complex and then, an engaged complex that is competent for substrate unfolding. The initial association and dissociation steps do not require ATP hydrolysis, but subsequent forward and reverse kinetic steps are accelerated by faster ATP hydrolysis. Our results, together with recent cryo-EM structures of ClpXP bound to substrates, support a model in which the ssrA degron initially binds in the top portion of the axial channel of the ClpX hexamer and then is translocated deeper into the channel in steps that eventually pull the native portion of the substrate against the channel opening. Reversible initial substrate binding allows ClpXP to check potential substrates for degrons, potentially increasing specificity. Subsequent substrate engagement steps allow ClpXP to grip a wide variety of sequences to ensure efficient unfolding and translocation of almost any native substrate.
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Twomey, Edward C., Zhejian Ji, Thomas E. Wales, Nicholas O. Bodnar, Scott B. Ficarro, Jarrod A. Marto, John R. Engen, and Tom A. Rapoport. "Substrate processing by the Cdc48 ATPase complex is initiated by ubiquitin unfolding." Science 365, no. 6452 (June 27, 2019): eaax1033. http://dx.doi.org/10.1126/science.aax1033.
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The Cdc48 adenosine triphosphatase (ATPase) (p97 or valosin-containing protein in mammals) and its cofactor Ufd1/Npl4 extract polyubiquitinated proteins from membranes or macromolecular complexes for subsequent degradation by the proteasome. How Cdc48 processes its diverse and often well-folded substrates is unclear. Here, we report cryo–electron microscopy structures of the Cdc48 ATPase in complex with Ufd1/Npl4 and polyubiquitinated substrate. The structures show that the Cdc48 complex initiates substrate processing by unfolding a ubiquitin molecule. The unfolded ubiquitin molecule binds to Npl4 and projects its N-terminal segment through both hexameric ATPase rings. Pore loops of the second ring form a staircase that acts as a conveyer belt to move the polypeptide through the central pore. Inducing the unfolding of ubiquitin allows the Cdc48 ATPase complex to process a broad range of substrates.
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Ike, Michihiko, Yukihiro Okada, Takaaki Narui, Kosuke Sakai, Masashi Kuroda, Satoshi Soda, and Daisuke Inoue. "Potential of waste activated sludge to accumulate polyhydroxyalkanoates and glycogen using industrial wastewater/liquid wastes as substrates." Water Science and Technology 80, no. 12 (December 15, 2019): 2373–80. http://dx.doi.org/10.2166/wst.2020.059.
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Abstract Recovery of the organics in industrial wastewaters/liquid wastes as polyhydroxyalkanoate (PHA) and/or glycogen (GLG) in waste activated sludge is a useful strategy to not only improve the resource value of waste activated sludge but also reduce the energy and cost of waste disposal and wastewater treatment. This study aimed to evaluate the potential of activated sludge to accumulate PHA and GLG using complex substrates (actual and simulated industrial wastewaters/liquid wastes) in addition to various simple organic substrates (organic acids, saccharides, and glycerol). The 24 h PHA and GLG accumulation experiments resulted in the accumulation of up to 25.5%, 6.0% and 14.1% of polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV) and GLG, respectively, from simple substrates, and up to 9.8%, 0.1% and 14.6%, respectively, from complex substrates. The results indicated that activated sludge can accumulate PHA and GLG even from complex wastewater substrates, although the accumulated PHA and GLG levels were not sufficiently high. The results also indicated that the PHA and GLG accumulation abilities of activated sludge from complex substrates can be drastically enhanced by a short-term acclimation to the corresponding substrate. This study will present the practical implications for value-added resource production through the combined use of waste activated sludge and industrial wastewaters.
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Windsor, Ian W., and Ronald T. Raines. "A substrate selected by phage display exhibits enhanced side-chain hydrogen bonding to HIV-1 protease." Acta Crystallographica Section D Structural Biology 74, no. 7 (June 27, 2018): 690–94. http://dx.doi.org/10.1107/s2059798318006691.
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Crystal structures of inactive variants of HIV-1 protease bound to peptides have revealed how the enzyme recognizes its endogenous substrates. The best of the known substrates is, however, a nonnatural substrate that was identified by directed evolution. The crystal structure of the complex between this substrate and the D25N variant of the protease is reported at a resolution of 1.1 Å. The structure has several unprecedented features, especially the formation of additional hydrogen bonds between the enzyme and the substrate. This work expands the understanding of molecular recognition by HIV-1 protease and informs the design of new substrates and inhibitors.
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Phayungphan, K., N. Rakmak, and A. Promraksa. "Application of monod two-substrate kinetics with an intermediate for anaerobic co-digestion of distillery wastewater and molasses/glycerol waste in batch experiments." Water Practice and Technology 15, no. 4 (August 10, 2020): 1068–82. http://dx.doi.org/10.2166/wpt.2020.081.
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Abstract Anaerobic digestion is a highly complex process, particularly in co-digestion between poorly-defined, complex co-substrates like distillery wastewater, molasses, and crude glycerine. Thus, in this article, the authors tackled the problems by using Monod two-substrate with an intermediate (M2SI) model to represent accumulated biomethane evolution (ABE) obtained from the co-substrates, including easily degradable, slowly degradable substrates and intermediate. The M2SI model predictions were compared with the traditional Monod model's simulation results to clarify an outstanding of the present model in the aspect of modeling and control. Different behaviors of ABE curves from batch experiments were used to calibrate the M2SI model prediction with sensitivity analysis of the model parameters. It was found that the M2SI model gives a correct trend to describe the co-digestion process with multiple substrates and complex microbial activities with satisfactory fitting accuracy. At the same time, simple Monod kinetics have a good fit for dilute pure distillery wastewater, but the estimated microbial growth kinetics were counterintuitive. Therefore, the M2SI Model has a broader range of applications for co-digestion dealing with the complexity of multiple microbial activities to consume inherently complex or artificial co-substrates.
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Gardner, Richard G., Alexander G. Shearer, and Randolph Y. Hampton. "In Vivo Action of the HRD Ubiquitin Ligase Complex: Mechanisms of Endoplasmic Reticulum Quality Control and Sterol Regulation." Molecular and Cellular Biology 21, no. 13 (July 1, 2001): 4276–91. http://dx.doi.org/10.1128/mcb.21.13.4276-4291.2001.
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ABSTRACT Ubiquitination is used to target both normal proteins for specific regulated degradation and misfolded proteins for purposes of quality control destruction. Ubiquitin ligases, or E3 proteins, promote ubiquitination by effecting the specific transfer of ubiquitin from the correct ubiquitin-conjugating enzyme, or E2 protein, to the target substrate. Substrate specificity is usually determined by specific sequence determinants, or degrons, in the target substrate that are recognized by the ubiquitin ligase. In quality control, however, a potentially vast collection of proteins with characteristic hallmarks of misfolding or misassembly are targeted with high specificity despite the lack of any sequence similarity between substrates. In order to understand the mechanisms of quality control ubiquitination, we have focused our attention on the first characterized quality control ubiquitin ligase, the HRD complex, which is responsible for the endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous ER-resident proteins. Using an in vivo cross-linking assay, we directly examined the association of the separate HRDcomplex components with various ERAD substrates. We have discovered that the HRD ubiquitin ligase complex associates with both ERAD substrates and stable proteins, but only mediates ubiquitin-conjugating enzyme association with ERAD substrates. Our studies with the sterol pathway-regulated ERAD substrate Hmg2p, an isozyme of the yeast cholesterol biosynthetic enzyme HMG-coenzyme A reductase (HMGR), indicated that the HRD complex discerns between a degradation-competent “misfolded” state and a stable, tightly folded state. Thus, it appears that the physiologically regulated, HRD-dependent degradation of HMGR is effected by a programmed structural transition from a stable protein to a quality control substrate.
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Makarov, V., L. Kucheryavykh, Y. Kucheryavykh, A. Rivera, M. J. Eaton, S. N. Skatchkov, and M. Inyushin. "Transport Reversal during Heteroexchange: A Kinetic Study." Journal of Biophysics 2013 (October 26, 2013): 1–14. http://dx.doi.org/10.1155/2013/683256.
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It is known that secondary transporters, which utilize transmembrane ionic gradients to drive their substrates up a concentration gradient, can reverse the uptake and instead release their substrates. Unfortunately, the Michaelis-Menten kinetic scheme, which is popular in transporter studies, does not include transporter reversal, and it completely neglects the possibility of equilibrium between the substrate concentrations on both sides of the membrane. We have developed a complex two-substrate kinetic model that includes transport reversal. This model allows us to construct analytical formulas allowing the calculation of a “heteroexchange” and “transacceleration” using standard Michaelis coefficients for respective substrates. This approach can help to understand how glial and other cells accumulate substrates without synthesis and are able to release such substrates and gliotransmitters.
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Nakatsukasa, Kunio, Jeffrey L. Brodsky, and Takumi Kamura. "A stalled retrotranslocation complex reveals physical linkage between substrate recognition and proteasomal degradation during ER-associated degradation." Molecular Biology of the Cell 24, no. 11 (June 2013): 1765–75. http://dx.doi.org/10.1091/mbc.e12-12-0907.
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During endoplasmic reticulum–associated degradation (ERAD), misfolded lumenal and membrane proteins in the ER are recognized by the transmembrane Hrd1 ubiquitin ligase complex and retrotranslocated to the cytosol for ubiquitination and degradation. Although substrates are believed to be delivered to the proteasome only after the ATPase Cdc48p/p97 acts, there is limited knowledge about how the Hrd1 complex coordinates with Cdc48p/p97 and the proteasome to orchestrate substrate recognition and degradation. Here we provide evidence that inactivation of Cdc48p/p97 stalls retrotranslocation and triggers formation of a complex that contains the 26S proteasome, Cdc48p/p97, ubiquitinated substrates, select components of the Hrd1 complex, and the lumenal recognition factor, Yos9p. We propose that the actions of Cdc48p/p97 and the proteasome are tightly coupled during ERAD. Our data also support a model in which the Hrd1 complex links substrate recognition and degradation on opposite sides of the ER membrane.
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S. B., Dimova, Derkach S. M., and Volkohon V. V. "ACTIVITY OF ENZYMATIC CELLULOLYTIC COMPLEX AND ANTAGONISTIC PROPERTIES OF TRICHODERMA HARZIANUM 128." Agriciltural microbiology 33 (June 18, 2021): 13–24. http://dx.doi.org/10.35868/1997-3004.33.13-24.
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Objective. To study the activity of enzymes of the cellulolytic complex of the association of micromycetes Trichoderma harzianum 128 and their antagonistic activity; to study the possibility of the influence of fungal introduction to the composted substrate on the composting rate and quality of composts. Methods. Microbiological (for growing micromycetes in digest media, accounting the number of T. harzianum 128 and determining the antagonistic activity of the association), agrochemical (to determine the characteristics of composting chicken manure-based substrates and content of carbon and nitrogen in these substrates), biochemical (study of activity of cellulolytic enzymes), production inspection (on the basis of Agrofirma KOLOS LLC, Region of Kyiv). Results. The association of T. harzianum 128 micromycetes is characterized by high activity of exoglucanase, endoglucanase and β-glucosidase, which contributes to reduction in the composting time of organic matter upon the introduction of fungi to the chicken manure-based substrate. T. harzianum 128 has pronounced antifungal properties against pathogens of certain diseases of crops (Fusarium oxysporum, F. sulfurum, Nigrospora oryzae). Enrichment of composted substrates with the studied micromycetes provides their active development (up to 8,150 thous CFU/g of dry compost), accumulation of carbon and nitrogen in compost, which improves the quality of the final product. Conclusion. The association of T. harzianum 128 micromycetes is characterized by high activity of enzymes of the cellulolytic complex, has a pronounced antagonistic activity against pathogens of certain diseases of crops. The introduction of T. harzianum 128 to composted chicken manure-based substrates reduces the composting time and improves the quality of the final product, which can increase the productivity of agrocenoses by including biofertilizers in crop fertilization systems.
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Sartori, Tanara, Heloisa Tibolla, Elenizi Prigol, Luciane Maria Colla, Jorge Alberto Vieira Costa, and Telma Elita Bertolin. "Enzymatic Saccharification of Lignocellulosic Residues by Cellulases Obtained from Solid State Fermentation UsingTrichoderma viride." BioMed Research International 2015 (2015): 1–9. http://dx.doi.org/10.1155/2015/342716.
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The aim of this study was to verify the viability of lignocellulosic substrates to obtain renewable energy source, through characterization of the cellulolytic complex, which was obtained by solid state fermentation usingTrichoderma viride. Enzymatic activity of the cellulosic complex was measured during saccharification of substrates filter paper, eucalyptus sawdust, and corncob, and compared with the activity of commercial cellulase. The characterization of the enzymes was performed by a 22Full Factorial Design, where the pH and temperature were the variables of study. Enzymatic saccharification of different substrates appearedviable until 12 to be viable until 12 h; after this period the activity decreased for both enzymatic forms (cellulolytic complex and commercial cellulase). The enzymatic activity of the commercial cellulase was favored with the use of corncob as substrate, while the cellulolytic complex does not show any difference in its specificity by the substrates studied. The largest activities of both enzymes were obtained in the temperature and pH range between 40°C and 50°C and 4.8 and 5.2, respectively. The cellulolytic complex obtained appeared to be viable for the saccharification of lignocellulosic residues compared with the commercial cellulase.
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Bono, Mario de, and Andres Villu Maricq. "NEURONAL SUBSTRATES OF COMPLEX BEHAVIORS INC. ELEGANS." Annual Review of Neuroscience 28, no. 1 (July 21, 2005): 451–501. http://dx.doi.org/10.1146/annurev.neuro.27.070203.144259.
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Bennett, J. W., A. Henderberg, and K. Grossman. "Sterigmatocystin production on complex and defined substrates." Mycopathologia 105, no. 1 (January 1989): 35–38. http://dx.doi.org/10.1007/bf00443827.
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Sun, G. S., J. M. Li, M. C. Luo, S. R. Zhu, L. Wang, F. F. Zhang, and L. Y. Lin. "Epitaxial growth of SiC on complex substrates." Journal of Crystal Growth 227-228 (July 2001): 811–15. http://dx.doi.org/10.1016/s0022-0248(01)00889-2.
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Lu, Yong-Feng. "Laser-induced temperature profiles in complex substrates." Applied Surface Science 79-80 (May 1994): 481–90. http://dx.doi.org/10.1016/0169-4332(94)90459-6.
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Tramonte, Rafael Prandini, Nicolli Cristina Osório, Flávio Henrique Ragonha, Gisele Daiane Pinha, Liliana Rodrigues, and Roger Paulo Mormul. "Periphyton consumption by an invasive snail species is greater in simplified than in complex habitats." Canadian Journal of Zoology 97, no. 1 (January 2019): 13–21. http://dx.doi.org/10.1139/cjz-2017-0359.
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Habitat complexity may stabilize consumer–resource interactions and reduce the probability of invasion in aquatic habitats. We tested the hypotheses that (i) higher habitat complexity reduces resource consumption independently of grazer species, but that (ii) invasive grazers have a greater influence on decreasing resources independently of habitat complexity. We performed an experiment using artificial substrates to simulate different complexity levels. We evaluated Melanoides tuberculata (O.F. Müller, 1774) and Aylacostoma chloroticum Hylton Scott, 1954 consumption of specific algal groups and the interaction between habitat complexity and grazer species. Moreover, we evaluated grazer activity on the different substrates during the experiment. The results support only the first hypothesis and indicate lower resource consumption on complex substrates compared with simpler substrates. Additionally, the effect of the grazing of the invasive species on taxon richness was greater in simplified than in complex habitats. The grazing activity on the substrate suggests a relationship between resource exploitation and habitat complexity in which the invasive grazing species visited the simple habitat less frequently. However, the effects of invasive grazers on food resources were higher on the simple substrate. The effects of grazing activity on food resources depend on the interaction between habitat complexity and grazer species. In this way, the introduction of an invasive species may have negative impacts on the structure and function of periphytic communities, mainly in simplified aquatic ecosystems.
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Brouwer, Charlotte, Mark G. Hazekamp, and Katja Zeppenfeld. "Anatomical Substrates and Ablation of Reentrant Atrial and Ventricular Tachycardias in Repaired Congenital Heart Disease." Arrhythmia & Electrophysiology Review 5, no. 2 (2016): 150. http://dx.doi.org/10.15420/aer.2016.19.2.
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Advances in surgical repair techniques for various types of congenital heart disease have improved survival into adulthood over the past decades, thus exposing these patients to a high risk of atrial and ventricular arrhythmias later in life. These arrhythmias arise from complex arrhythmogenic substrates. Substrate formation may depend on both pathological myocardial remodelling and variable anatomical boundaries, determined by the type and timing of prior corrective surgery. Accordingly, arrhythmogenic substrates after repair have changed as a result of evolving surgical techniques. Radiofrequency catheter ablation offers an important therapeutic option but remains challenging due to the variable anatomy, surgically created obstacles and the complex arrhythmogenic substrates. Recent technical developments including electroanatomical mapping and image integration for delineating the anatomy facilitate complex catheter ablation procedures. The purpose of this review is to provide an update on the changing anatomical arrhythmogenic substrates and their potential impact on catheter ablation in patients with repaired congenital heart disease and tachyarrhythmias.
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Jagadamma, S., M. A. Mayes, J. M. Steinweg, and S. M. Schaeffer. "Substrate quality alters the microbial mineralization of added substrate and soil organic carbon." Biogeosciences 11, no. 17 (September 3, 2014): 4665–78. http://dx.doi.org/10.5194/bg-11-4665-2014.
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Abstract. The rate and extent of decomposition of soil organic carbon (SOC) is dependent, among other factors, on substrate chemistry and microbial dynamics. Our objectives were to understand the influence of substrate chemistry on microbial decomposition of carbon (C), and to use model fitting to quantify differences in pool sizes and mineralization rates. We conducted an incubation experiment for 270 days using four uniformly labeled 14C substrates (glucose, starch, cinnamic acid and stearic acid) on four different soils (a temperate Mollisol, a tropical Ultisol, a sub-arctic Andisol, and an arctic Gelisol). The 14C labeling enabled us to separate CO2 respired from added substrates and from native SOC. Microbial gene copy numbers were quantified at days 4, 30 and 270 using quantitative polymerase chain reaction (qPCR). Substrate C respiration was always higher for glucose than other substrates. Soils with cinnamic and stearic acid lost more native SOC than glucose- and starch-amended soils. Cinnamic and stearic acid amendments also exhibited higher fungal gene copy numbers at the end of incubation compared to unamended soils. We found that 270 days were sufficient to model the decomposition of simple substrates (glucose and starch) with three pools, but were insufficient for more complex substrates (cinnamic and stearic acid) and native SOC. This study reveals that substrate quality exerts considerable control on the microbial decomposition of newly added and native SOC, and demonstrates the need for multi-year incubation experiments to constrain decomposition parameters for the most recalcitrant fractions of SOC and complex substrates.
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Brown, Nicholas G., Ryan VanderLinden, Edmond R. Watson, Renping Qiao, Christy R. R. Grace, Masaya Yamaguchi, Florian Weissmann, et al. "RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex." Proceedings of the National Academy of Sciences 112, no. 17 (March 30, 2015): 5272–79. http://dx.doi.org/10.1073/pnas.1504161112.
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For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2∼Ub intermediate to a lysine on a remotely bound disordered substrate. One such E3 is the gigantic, multisubunit 1.2-MDa anaphase-promoting complex/cyclosome (APC), which controls cell division by ubiquitinating cell cycle regulators to drive their timely degradation. Intrinsically disordered substrates are typically recruited via their KEN-box, D-box, and/or other motifs binding to APC and a coactivator such as CDH1. On the opposite side of the APC, the dynamic catalytic core contains the cullin-like subunit APC2 and its RING partner APC11, which collaborates with the E2 UBCH10 (UBE2C) to ubiquitinate substrates. However, how dynamic RING–E2∼Ub catalytic modules such as APC11–UBCH10∼Ub collide with distally tethered disordered substrates remains poorly understood. We report structural mechanisms of UBCH10 recruitment to APCCDH1 and substrate ubiquitination. Unexpectedly, in addition to binding APC11’s RING, UBCH10 is corecruited via interactions with APC2, which we visualized in a trapped complex representing an APCCDH1–UBCH10∼Ub–substrate intermediate by cryo-electron microscopy, and in isolation by X-ray crystallography. To our knowledge, this is the first structural view of APC, or any cullin–RING E3, with E2 and substrate juxtaposed, and it reveals how tripartite cullin–RING–E2 interactions establish APC’s specificity for UBCH10 and harness a flexible catalytic module to drive ubiquitination of lysines within an accessible zone. We propose that multisite interactions reduce the degrees of freedom available to dynamic RING E3–E2∼Ub catalytic modules, condense the search radius for target lysines, increase the chance of active-site collision with conformationally fluctuating substrates, and enable regulation.
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Oh, Jang-Hyun, Ju-Yeon Hyun, Shun-Jia Chen, and Alexander Varshavsky. "Five enzymes of the Arg/N-degron pathway form a targeting complex: The concept of superchanneling." Proceedings of the National Academy of Sciences 117, no. 20 (May 4, 2020): 10778–88. http://dx.doi.org/10.1073/pnas.2003043117.
Full textAbstract:
The Arg/N-degron pathway targets proteins for degradation by recognizing their N-terminal (Nt) residues. If a substrate bears, for example, Nt-Asn, its targeting involves deamidation of Nt-Asn, arginylation of resulting Nt-Asp, binding of resulting (conjugated) Nt-Arg to the UBR1-RAD6 E3-E2 ubiquitin ligase, ligase-mediated synthesis of a substrate-linked polyubiquitin chain, its capture by the proteasome, and substrate’s degradation. We discovered that the human Nt-Asn–specific Nt-amidase NTAN1, Nt-Gln–specific Nt-amidase NTAQ1, arginyltransferase ATE1, and the ubiquitin ligase UBR1-UBE2A/B (or UBR2-UBE2A/B) form a complex in which NTAN1 Nt-amidase binds to NTAQ1, ATE1, and UBR1/UBR2. In addition, NTAQ1 Nt-amidase and ATE1 arginyltransferase also bind to UBR1/UBR2. In the yeast Saccharomyces cerevisiae, the Nt-amidase, arginyltransferase, and the double-E3 ubiquitin ligase UBR1-RAD6/UFD4-UBC4/5 are shown to form an analogous targeting complex. These complexes may enable substrate channeling, in which a substrate bearing, for example, Nt-Asn, would be captured by a complex-bound Nt-amidase, followed by sequential Nt modifications of the substrate and its polyubiquitylation at an internal Lys residue without substrate’s dissociation into the bulk solution. At least in yeast, the UBR1/UFD4 ubiquitin ligase interacts with the 26S proteasome, suggesting an even larger Arg/N-degron–targeting complex that contains the proteasome as well. In addition, specific features of protein-sized Arg/N-degron substrates, including their partly sequential and partly nonsequential enzymatic modifications, led us to a verifiable concept termed “superchanneling.” In superchanneling, the synthesis of a substrate-linked poly-Ub chain can occur not only after a substrate’s sequential Nt modifications, but also before them, through a skipping of either some or all of these modifications within a targeting complex.
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Melzer, Björn, Tina Steinbrecher, Robin Seidel, Oliver Kraft, Ruth Schwaiger, and Thomas Speck. "The attachment strategy of English ivy: a complex mechanism acting on several hierarchical levels." Journal of The Royal Society Interface 7, no. 50 (May 12, 2010): 1383–89. http://dx.doi.org/10.1098/rsif.2010.0140.
Full textAbstract:
English ivy ( Hedera helix L.) is able to grow on vertical substrates such as trees, rocks and house plaster, thereby attaching so firmly to the surface that when removed by force typically whole pieces of the climbing substrate are torn off. The structural details of the attachment process are not yet entirely understood. We studied the attachment process of English ivy in detail and suggest a four-phase process to describe the attachment strategy: (i) initial physical contact, (ii) form closure of the root with the substrate, (iii) chemical adhesion, and (iv) shape changes of the root hairs and form-closure with the substrate. These four phases and their variations play an important role in the attachment to differently structured surfaces. We demonstrate that, in English ivy, different mechanisms work together to allow the plant's attachment to various climbing substrates and reveal the importance of micro-fibril orientation in the root hairs for the attachment based on structural changes at the subcellular level.
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Langosch, Dieter, and Harald Steiner. "Substrate processing in intramembrane proteolysis by γ-secretase – the role of protein dynamics." Biological Chemistry 398, no. 4 (April 1, 2017): 441–53. http://dx.doi.org/10.1515/hsz-2016-0269.
Full textAbstract:
Abstract Intramembrane proteases comprise a number of different membrane proteins with different types of catalytic sites. Their common denominator is cleavage within the plane of the membrane, which usually results in peptide bond scission within the transmembrane helices of their substrates. Despite recent progress in the determination of high-resolution structures, as illustrated here for the γ-secretase complex and its substrate C99, it is still unknown how these enzymes function and how they distinguish between substrates and non-substrates. In principle, substrate/non-substrate discrimination could occur at the level of substrate binding and/or cleavage. Focusing on the γ-secretase/C99 pair, we will discuss recent observations suggesting that global motions within a substrate transmembrane helix may be much more important for defining a substrate than local unraveling at cleavage sites.
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Kim, Tai Young, Priscila F. Siesser, Kent L. Rossman, Dennis Goldfarb, Kathryn Mackinnon, Feng Yan, XianHua Yi, et al. "Substrate Trapping Proteomics Reveals Targets of the βTrCP2/FBXW11 Ubiquitin Ligase." Molecular and Cellular Biology 35, no. 1 (October 20, 2014): 167–81. http://dx.doi.org/10.1128/mcb.00857-14.
Full textAbstract:
Defining the full complement of substrates for each ubiquitin ligase remains an important challenge. Improvements in mass spectrometry instrumentation and computation and in protein biochemistry methods have resulted in several new methods for ubiquitin ligase substrate identification. Here we used the parallel adapter capture (PAC) proteomics approach to study βTrCP2/FBXW11, a substrate adaptor for the SKP1–CUL1–F-box (SCF) E3 ubiquitin ligase complex. The processivity of the ubiquitylation reaction necessitates transient physical interactions between FBXW11 and its substrates, thus making biochemical purification of FBXW11-bound substrates difficult. Using the PAC-based approach, we inhibited the proteasome to “trap” ubiquitylated substrates on the SCFFBXW11E3 complex. Comparative mass spectrometry analysis of immunopurified FBXW11 protein complexes before and after proteasome inhibition revealed 21 known and 23 putatively novel substrates. In focused studies, we found that SCFFBXW11bound, polyubiquitylated, and destabilized RAPGEF2, a guanine nucleotide exchange factor that activates the small GTPase RAP1. High RAPGEF2 protein levels promoted cell-cell fusion and, consequently, multinucleation. Surprisingly, this occurred independently of the guanine nucleotide exchange factor (GEF) catalytic activity and of the presence of RAP1. Our data establish new functions for RAPGEF2 that may contribute to aneuploidy in cancer. More broadly, this report supports the continued use of substrate trapping proteomics to comprehensively define targets for E3 ubiquitin ligases. All proteomic data are available via ProteomeXchange with identifier PXD001062.
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Dallaire, Frédéric, Paola Blanchette, and Philip E. Branton. "A Proteomic Approach To Identify Candidate Substrates of Human Adenovirus E4orf6-E1B55K and Other Viral Cullin-Based E3 Ubiquitin Ligases." Journal of Virology 83, no. 23 (September 16, 2009): 12172–84. http://dx.doi.org/10.1128/jvi.01169-09.
Full textAbstract:
ABSTRACT It has been known for some time that the human adenovirus serotype 5 (Ad5) E4orf6 and E1B55K proteins work in concert to degrade p53 and to regulate selective export of late viral mRNAs during productive infection. Both of these functions rely on the formation by the Ad5 E4orf6 protein of a cullin 5-based E3 ubiquitin ligase complex containing elongins B and C. E1B55K is believed to function as the substrate recognition module for the complex and, in addition to p53, Mre11 and DNA ligase IV have also been identified as substrates. To discover additional substrates we have taken a proteomic approach by using two-dimensional difference gel electrophoresis to detect cellular proteins that decrease significantly in amount in p53-null H1299 human lung carcinoma cells after expression of E1B55K and E4orf6 using adenovirus vectors. Several species were detected and identified by mass spectroscopy, and for one of these, integrin α3, we went on in a parallel study to confirm it as a bone fide substrate of the complex (F. Dallaire et al., J. Virol. 83:5329-5338, 2009). Although the system has some limitations, it may still be of some general use in identifying candidate substrates of any viral cullin-based E3 ubiquitin ligase complex, and we suggest a series of criteria for substrate validation.
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Fierobe, Henri-Pierre, Florence Mingardon, Adva Mechaly, Anne Bélaïch, Marco T. Rincon, Sandrine Pagès, Raphael Lamed, Chantal Tardif, Jean-Pierre Bélaïch, and Edward A. Bayer. "Action of Designer Cellulosomes on HomogeneousVersusComplex Substrates." Journal of Biological Chemistry 280, no. 16 (February 10, 2005): 16325–34. http://dx.doi.org/10.1074/jbc.m414449200.
Full textAbstract:
In recent work (Fierobe, H.-P., Bayer, E. A., Tardif, C., Czjzek, M., Mechaly, A., Belaïch, A., Lamed, R., Shoham, Y., and Belaich, J.-P. (2002)J. Biol. Chem. 277, 49621–49630), we reported the self-assembly of a comprehensive set of defined “bifunctional” chimeric cellulosomes. Each complex contained the following: (i) a chimeric scaffoldin possessing a cellulose-binding module and two cohesins of divergent specificity and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. This approach allowed the controlled integration of desired enzymes into a multiprotein complex of predetermined stoichiometry and topology. The observed enhanced synergy on recalcitrant substrates by the bifunctional designer cellulosomes was ascribed to two major factors: substrate targeting and proximity of the two catalytic components. In the present work, the capacity of the previously described chimeric cellulosomes was amplified by developing a third divergent cohesin-dockerin device. The resultant trifunctional designer cellulosomes were assayed on homogeneous and complex substrates (microcrystalline cellulose and straw, respectively) and found to be considerably more active than the corresponding free enzyme or bifunctional systems. The results indicate that the synergy between two prominent cellulosomal enzymes (from the family-48 and -9 glycoside hydrolases) plays a crucial role during the degradation of cellulose by cellulosomes and that one dominant family-48 processive endoglucanase per complex is sufficient to achieve optimal levels of synergistic activity. Furthermore cooperation within a cellulosome chimera between cellulases and a hemicellulase from different microorganisms was achieved, leading to a trifunctional complex with enhanced activity on a complex substrate.
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Freeman, C., and J. J. Hopwood. "Human liver N-acetylglucosamine-6-sulphate sulphatase. Catalytic properties." Biochemical Journal 246, no. 2 (September 1, 1987): 355–65. http://dx.doi.org/10.1042/bj2460355.
Full textAbstract:
Kinetic parameters (Km and kcat.) of the two major forms (A and B) and a minor form (C) of human liver N-acetylglucosamine-6-sulphate sulphatase [Freeman, Clements & Hopwood (1987) Biochem. J. 246, 347-354] were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparin, heparan sulphate and keratan sulphate. Enzyme activity is highly specific towards glucosamine 6-sulphate or glucose 6-sulphate residues. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, are hydrolysed with catalytic efficiencies up to 3900 times above that observed for the monosaccharide substrate N-acetylglucosamine 6-sulphate. Forms A and B both desulphate substrates derived from keratan sulphate and heparin. Aglycone structures that influence substrate binding and/or enzyme activity were penultimate-residue 6-carboxy and 2-sulphate ester groups for heparin-derived substrates and penultimate-residue 6-sulphate ester groups for keratan sulphate-derived substrates. The 4-hydroxy group of the N-acetylglucosamine 6-sulphate or the 2-sulphaminoglucosamine 6-sulphate under enzymic attack is involved in the catalytic mechanism. The presence of a 2-amino group in place of a 2-acetamido or a 2-sulphoamino group considerably decreases the catalytic efficiency of the sulphatase, particularly in the absence of a penultimate-aglycone-residue 6-carboxy group. Both forms A and B are exo-enzymes, since activity towards internal sulphate ester bonds was not observed. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure. The presence of aglycone 2-sulphate ester, 6-carboxy group and 6-sulphate ester groups on the glucosamine 6-sulphate residue under attack considerably affects the pH response. Sulphate and phosphate ions are potent inhibitors of enzyme activity.
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Cadet, F., and J. C. Meunier. "pH and kinetic studies of chloroplast sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea)." Biochemical Journal 253, no. 1 (July 1, 1988): 249–54. http://dx.doi.org/10.1042/bj2530249.
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The aim of this paper is to study some steady-state kinetic properties of sedoheptulose-1,7-bisphosphatase, its pH-dependence and the effect of a substrate analogue, fructose 2,6-bisphosphate. Studies were carried out with sedoheptulose 1,7-bisphosphate and with fructose 1,6-bisphosphate, an alternative substrate. The pK values are identical for both substrates, and fructose 2,6-bisphosphate behaves like a competitive inhibitor. These results suggest that there exists a unique active site for either sedoheptulose 1,7-bisphosphate or fructose 1,6-bisphosphate on the enzyme molecule. Increasing Mg2+ concentrations shifted the optimum pH. As for fructose-1,6-bisphosphatase, we believe that this shift is due to the neutralization of negative charges near the active centre [Cadet, Meunier & Ferté (1987) Eur. J. Biochem. 162, 393-398]. The free species of sedoheptulose 1,7-bisphosphate and fructose 1,6-bisphosphate are not the usual substrates of enzyme, nor is Mg2+. But the kinetics relative to the (Mg2+-substrate4-)2- complex is not consistent with this complex being the substrate. An explanation of this discrepancy is proposed, involving both the negative charges near the active centre and the positive charges of Mg2+. The observed Vmax. of the reduced enzyme is 65% of the theoretical Vmax. for both substrates, but the observed Vmax. relative to sedoheptulose 1,7-bisphosphate is 3 times the one relative to fructose 1,6-bisphosphate. The specificity constant (kcat./Km), 1.62 × 10(6) M-1.s-1 with respect to sedoheptulose 1,7-bisphosphate compared with 5.5 × 10(4) M-1.s-1 with respect to fructose 1,6-bisphosphate, indicates that the enzyme specificity towards sedoheptulose 1,7-bisphosphate is high but not absolute.
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Cooke, Charles, and James C. Alwine. "Characterization of Specific Protein-RNA Complexes Associated with the Coupling of Polyadenylation and Last-Intron Removal." Molecular and Cellular Biology 22, no. 13 (July 1, 2002): 4579–86. http://dx.doi.org/10.1128/mcb.22.13.4579-4586.2002.
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ABSTRACT Polyadenylation and splicing are highly coordinated on substrate RNAs capable of coupled polyadenylation and splicing. Individual elements of both splicing and polyadenylation signals are required for the in vitro coupling of the processing reactions. In order to understand more about the coupling mechanism, we examined specific protein-RNA complexes formed on RNA substrates, which undergo coupled splicing and polyadenylation. We hypothesized that formation of a coupling complex would be adversely affected by mutations of either splicing or polyadenylation elements known to be required for coupling. We defined three specific complexes (AC′, AC, and BC) that form rapidly on a coupled splicing and polyadenylation substrate, well before the appearance of spliced and/or polyadenylated products. The AC′ complex is formed by 30 s after mixing, the AC complex is formed between 1 and 2 min after mixing, and the BC complex is formed by 2 to 3 min after mixing. AC′ is a precursor of AC, and the AC′ and/or AC complex is a precursor of BC. Of the three complexes, BC appears to be a true coupling complex in that its formation was consistently diminished by mutations or experimental conditions known to disrupt coupling. The characteristics of the AC′ complex suggest that it is analogous to the spliceosomal A complex, which forms on splicing-only substrates. Formation of the AC′ complex is dependent on the polypyrimidine tract. The transition from AC′ to AC appears to require an intact 3′-splice site. Formation of the BC complex requires both splicing elements and the polyadenylation signal. A unique polyadenylation-specific complex formed rapidly on substrates containing only the polyadenylation signal. This complex, like the AC′ complex, formed very transiently on the coupled splicing and polyadenylation substrate; we suggest that these two complexes coordinate, resulting in the BC complex. We also suggest a model in which the coupling mechanism may act as a dominant checkpoint in which aberrant definition of one exon overrides the normal processing at surrounding wild-type sites.
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Turner, John, Dave Snowden, and Nigel Thurlow. "The Substrate-Independence Theory: Advancing Constructor Theory to Scaffold Substrate Attributes for the Recursive Interaction between Knowledge and Information." Systems 10, no. 1 (January 5, 2022): 7. http://dx.doi.org/10.3390/systems10010007.
Full textAbstract:
The substrate-independence theory utilizes sensemaking techniques to provide cognitively based scaffolds that guide and structure learning. Scaffolds are cognitive abstractions of constraints that relate to information within a system. The substrate-independence theory concentrates on the flow of information as the underlying property of the host system. The substrate-independence theory views social systems as complex adaptive systems capable of repurposing their structure to combat external threats by utilizing constructors and substrates. Constructor theory is used to identify potential construction tasks, the legitimate input and output states that are possible, to map the desired change in the substrate’s attributes. Construction tasks can be mapped in advance for ordered and known environments. Construction tasks may also be mapped in either real-time or post hoc for unordered and complex environments using current sensemaking techniques. Mapping of the construction tasks in real-time becomes part of the landscape, and scaffolds are implemented to aid in achieving the desired state or move to a more manageable environment (e.g., from complex to complicated).
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Liu, Decheng, Wen Yue, Jiajie Kang, and Chengbiao Wang. "Effects of Different Substrates on the Formability and Densification Behaviors of Cemented Carbide Processed by Laser Powder Bed Fusion." Materials 14, no. 17 (September 2, 2021): 5027. http://dx.doi.org/10.3390/ma14175027.
Full textAbstract:
Cemented carbide materials are widely applied in cutting tools, drill tools, and mold fabrication due to their superior hardness and wear resistance. Producing cemented carbide parts via the laser powder bed fusion (L-PBF) method has the advantage of fabricating complex structures with a rapid manufacturing speed; however, they were underdeveloped due to their low density and crack formation on the blocks. This work studied the effect of different substrates including 316L substrates, Ni200 substrates, and YG15 substrates on the forming quality of WC-17Co parts fabricated by L-PBF, with the aim of finding the optimal substrate for fabrication. The results revealed that the Ni200 substrates had a better wettability for the single tracks formation than other substrates, and bonding between the built block and the Ni200 substrate was firm without separation during processing with a large range of laser energy inputs. This guaranteed the fabrication of a relatively dense block with fewer cracks. Although the high laser energy input that led to fine crack formation on the blocks formed on the Ni200 substrate, it was found to be better suited to restricting cracks than other substrates.
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Böhmer, Christoph, Angelika Bröer, Michael Munzinger, Sonja Kowalczuk, John E. J. Rasko, Florian Lang, and Stefan Bröer. "Characterization of mouse amino acid transporter B0AT1 (slc6a19)." Biochemical Journal 389, no. 3 (July 26, 2005): 745–51. http://dx.doi.org/10.1042/bj20050083.
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The mechanism of the mouse (m)B0AT1 (slc6a19) transporter was studied in detail using two electrode voltage-clamp techniques and tracer studies in the Xenopus oocyte expression system. All neutral amino acids induced inward currents at physiological potentials, but large neutral non-aromatic amino acids were the preferred substrates of mB0AT1. Substrates were transported with K0.5 values ranging from approx. 1 mM to approx. 10 mM. The transporter mediates Na+–amino acid co-transport with a stoichiometry of 1:1. No other ions were involved in the transport mechanism. An increase in the extracellular Na+ concentration reduced the K0.5 for leucine, and vice versa. Moreover, the K0.5 values and Vmax values of both substrates varied with the membrane potential. As a result, K0.5 and Vmax values are a complex function of the concentration of substrate and co-substrate and the membrane potential. A model is presented assuming random binding order and a positive charge associated with the ternary [Na+–substrate–transporter] complex, which is consistent with the experimental data.
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Vomastek, Tomáš, Marcin P. Iwanicki, W. Richard Burack, Divya Tiwari, Devanand Kumar, J. Thomas Parsons, Michael J. Weber, and Vinay Kumar Nandicoori. "Extracellular Signal-Regulated Kinase 2 (ERK2) Phosphorylation Sites and Docking Domain on the Nuclear Pore Complex Protein Tpr Cooperatively Regulate ERK2-Tpr Interaction." Molecular and Cellular Biology 28, no. 22 (September 15, 2008): 6954–66. http://dx.doi.org/10.1128/mcb.00925-08.
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ABSTRACT Identifying direct substrates of mitogen-activated protein kinases (MAPKs) and understanding how those substrates are selected is central to understanding how these ubiquitously activated enzymes generate diverse biological responses. In previous work, we identified several new candidate substrates for the MAPK ERK2 (extracellular signal-regulated kinase 2), including the nuclear pore complex protein Tpr (translocated promoter region). In this report, we identify sites on Tpr for ERK2 phosphorylation and binding and demonstrate their functional interaction. ERK2 phosphorylation and dimerization are necessary for ERK2-Tpr binding, and this occurs through a DEF (docking site for ERK2, FXF) domain on Tpr. Surprisingly, the DEF domain and the phosphorylation sites displayed positive cooperativity to promote ERK2 binding to Tpr, in contrast to substrates where phosphorylation reduces binding. Ectopic expression or depletion of Tpr resulted in decreased movement of activated ERK2 from the cytoplasm to the nucleus, implying a role for Tpr in ERK2 translocation. Collectively, the data provide direct evidence that a component of the nuclear pore complex is a bona fide substrate of ERK2 in vivo and that activated ERK2 stably associates with this substrate after phosphorylation, where it could play a continuing role in nuclear pore function. We propose that Tpr is both a substrate and a scaffold for activated ERKs.
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Sulaiman, Noorul Aini. "Combining Docking and Molecular Dynamic of Protease from Bacillus lehensis G1." Journal of Engineering and Science Research 2, no. 1 (February 28, 2018): 1–5. http://dx.doi.org/10.26666/rmp.jesr.2018.1.1.
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Protease is an enzyme that catalysed the hydrolysis of protein into peptide. Application of protease in industry has been linked with cost effective substrates and complex of enzyme-substrate stability. Molecular docking approach has identified casein as a preference substrates. However, lack of data on casein mode of binding to protease and enzyme stability represents a limitation for its production and structural optimization. In this study, we have used a molecular dynamic (MD) to examine the stability of complex enzyme-substrate of protease from Bacillus lehensis G1. The 3D structure of protease (BleG1_1979) was docked with substrate casein using AutoDock Vina. Structural analysis of the substrate-binding cleft revealed a binding site of casein was predominantly at the hydrophobic region of BleG1_1979. The MD of complex BleG1_1979-casein was tested with two temperatures; 298 K and 310 K using GROMACS v5.1.4. MD simulation showed a stable behaviour of BleG1_1979 over the 20 ns simulation period. The molecular docking and MD simulation suggested that the production of protease from B. lehensis G1 by utilization of casein and the stability of complex protease-casein could be a potential application to generate a cost effective enzyme to be develop for industrial use.
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TAKAHASHI, N., T. ZHANG, M. SPANGENBERG, D. GREIG, T. H. SHEN, S. CORNELIUS, E. A. SEDDON, and J. A. D. MATTHEW. "SPIN-RESOLVED PHOTOELECTRON SPECTROSCOPY OF ULTRATHIN Fe FILMS ON GaAs(001)." Surface Review and Letters 09, no. 02 (April 2002): 693–98. http://dx.doi.org/10.1142/s0218625x02002816.
Full textAbstract:
Thin epitaxial Fe films were grown on singular and vicinal GaAs(001) substrates, and their magnetic and electronic structures were investigated by synchrotron-based spin-resolved and spin-integrated photoelectron spectroscopy with different Fe thickness. There were two types of substrates: one was a Si-doped n-type GaAs(001) surface with doping concentration of 2 × 1018 cm -3 (singular substrate), and the other was orientated by 3° toward the (111)A direction (vicinal substrate). Spin polarization of the secondary electron peak at different growth stages of Fe coverage for the singular substrate sample and the vicinal one were measured. In the case of singular substrates, there was a dependence of their initial surface reconstruction, which is associated with complex domain structure, while no such the dependence was observed in the case of vicinal substrates. The result from the vicinal sample suggests the geometrical influence of the initial surface stoichiometry of the substrate.
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Chan, Nickie C., and Trevor Lithgow. "The Peripheral Membrane Subunits of the SAM Complex Function Codependently in Mitochondrial Outer Membrane Biogenesis." Molecular Biology of the Cell 19, no. 1 (January 2008): 126–36. http://dx.doi.org/10.1091/mbc.e07-08-0796.
Full textAbstract:
The sorting and assembly machinery (SAM) complex functions in the assembly of β-barrel proteins into the mitochondrial outer membrane. It is related to the Omp85/YaeT machinery in bacterial outer membranes, but the eukaryotic SAM complex is distinguished by two peripheral subunits, Sam37 and Sam35, that sit on the cytosolic face of the complex. The function of these subunits in β-barrel protein assembly is currently unclear. By screening a library of sam35 mutants, we show that 13 distinct alleles were each specifically suppressed by overexpression of SAM37. Two of these mutants, sam35-409 and sam35-424, show distinct phenotypes that enable us to distinguish the function of Sam35 from that of Sam37. Sam35 is required for the SAM complex to bind outer membrane substrate proteins: destabilization of Sam35 inhibits substrate binding by Sam50. Sam37 acts later than Sam35, apparently to assist release of substrates from the SAM complex. Very different environments surround bacteria and mitochondria, and we discuss the role of Sam35 and Sam37 in terms of the problems peculiar to mitochondrial protein substrates.
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Baker, Michael J., Chaille T. Webb, David A. Stroud, Catherine S. Palmer, Ann E. Frazier, Bernard Guiard, Agnieszka Chacinska, Jacqueline M. Gulbis, and Michael T. Ryan. "Structural and Functional Requirements for Activity of the Tim9–Tim10 Complex in Mitochondrial Protein Import." Molecular Biology of the Cell 20, no. 3 (February 2009): 769–79. http://dx.doi.org/10.1091/mbc.e08-09-0903.
Full textAbstract:
The Tim9–Tim10 complex plays an essential role in mitochondrial protein import by chaperoning select hydrophobic precursor proteins across the intermembrane space. How the complex interacts with precursors is not clear, although it has been proposed that Tim10 acts in substrate recognition, whereas Tim9 acts in complex stabilization. In this study, we report the structure of the yeast Tim9–Tim10 hexameric assembly determined to 2.5 Å and have performed mutational analysis in yeast to evaluate the specific roles of Tim9 and Tim10. Like the human counterparts, each Tim9 and Tim10 subunit contains a central loop flanked by disulfide bonds that separate two extended N- and C-terminal tentacle-like helices. Buried salt-bridges between highly conserved lysine and glutamate residues connect alternating subunits. Mutation of these residues destabilizes the complex, causes defective import of precursor substrates, and results in yeast growth defects. Truncation analysis revealed that in the absence of the N-terminal region of Tim9, the hexameric complex is no longer able to efficiently trap incoming substrates even though contacts with Tim10 are still made. We conclude that Tim9 plays an important functional role that includes facilitating the initial steps in translocating precursor substrates into the intermembrane space.
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Freeman, C., and J. J. Hopwood. "Human liver sulphamate sulphohydrolase. Determinations of native protein and subunit Mr values and influence of substrate agylcone structure on catalytic properties." Biochemical Journal 234, no. 1 (February 15, 1986): 83–92. http://dx.doi.org/10.1042/bj2340083.
Full textAbstract:
Human sulphamate sulphohydrolase was purified at least 20,000-fold to homogeneity from liver with a three-step four-column procedure, which consisted of a concanavalin A-Sepharose/Blue A agarose coupled step, and Bio-Gel HT step and then a CM-Sepharose step. The procedure was also used to purify enzyme from kidney and placenta. The subunit Mr of liver, kidney and placenta sulphamate sulphohydrolase was assessed to be 56,000 by using SDS/polacrylamide-gel electrophoresis. The native protein Mr of enzyme from all three tissue sources was assessed by gel-permeation chromatography to be approx. 120,000 on Sephacryl S-300 and 100,000 on Fractogel TSK. It is probable that the native enzyme results from dimerization of subunits. Kinetic parameters (km and kcat.) of human liver sulphamate sulphohydrolase were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparin and heparan sulphate. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, are turned over up to 372000 times faster than the monosaccharide substrate 2-sulphaminoglucosamine. Aglycone structures that influence substrate binding and/or enzyme activity were penultimate-residue C-6 carboxy and C-2 sulphate ester groups and a post-penultimate 2-sulphaminoglucosamine residue. The C-4 hydroxy group of the 2-sulphaminoglucosamine under enzymic attack is involved in binding of substrate to enzyme. The presence of C-6 sulphate ester on the non-reducing end 2-sulphaminoglucosamine stimulates sulphamate bond hydrolysis and substrate affinity if the adjacent monosaccharide residue is idose or 2-sulphoidose, but strongly inhibits hydrolysis if the adjacent monosaccharide residue is iduronic acid. Sulphamate sulphohydrolase is an exoenzyme, since activity toward internal sulphamate bonds was not detected. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure. The presence of aglycone C-2 sulphate ester and aglycone C-6 carboxy groups and C-6 sulphate ester groups on the 2-sulphaminoglucosamine residue under attack considerably affect the pH response. Structurally complex substrates had two pH optima. Incubation temperature and buffer ionic strength markedly influenced pH optima and enzyme activity. Cu2+ and SO4(2-)ions are potent inhibitors of enzyme activity.
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Huber, Sylvia, Sabine Minnebusch, Stefan Wuertz, Peter A. Wilderer, and Brigitte Helmreich. "Impact of different substrates on biomass protein composition during wastewater treatment investigated by two-dimensional electrophoresis." Water Science and Technology 37, no. 4-5 (February 1, 1998): 363–66. http://dx.doi.org/10.2166/wst.1998.0667.
Full textAbstract:
The influence of different influent substrates on biomass protein composition was examined by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Activated sludge from six sequencing batch reactors (SBRs) was investigated; four reactors were fed with model substrates and two received effluents from a wood milling process and a paper production process, respectively. Our investigations showed that in 2D-PAGE complex substrates caused a less diverse protein pattern than model wastewater composed of simple and low molecular weight compounds. This may be caused by complex formation by high molecular weight compounds of substrate with proteins. A more likely explanation is the presence of a more diversified microbial population resulting in a lower concentration of individual proteins, so that detection limits after staining were too high to observe discrete spots.
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Lan, Pengfei, Bin Zhou, Ming Tan, Shaobai Li, Mi Cao, Jian Wu, and Ming Lei. "Structural insight into precursor ribosomal RNA processing by ribonuclease MRP." Science 369, no. 6504 (June 25, 2020): 656–63. http://dx.doi.org/10.1126/science.abc0149.
Full textAbstract:
Ribonuclease (RNase) MRP is a conserved eukaryotic ribonucleoprotein complex that plays essential roles in precursor ribosomal RNA (pre-rRNA) processing and cell cycle regulation. In contrast to RNase P, which selectively cleaves transfer RNA–like substrates, it has remained a mystery how RNase MRP recognizes its diverse substrates. To address this question, we determined cryo–electron microscopy structures of Saccharomyces cerevisiae RNase MRP alone and in complex with a fragment of pre-rRNA. These structures and the results of biochemical studies reveal that coevolution of both protein and RNA subunits has transformed RNase MRP into a distinct ribonuclease that processes single-stranded RNAs by recognizing a short, loosely defined consensus sequence. This broad substrate specificity suggests that RNase MRP may have myriad yet unrecognized substrates that could play important roles in various cellular contexts.
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Marcil, Mariannick, Karine Bourduas, Alexis Ascah, and Yan Burelle. "Exercise training induces respiratory substrate-specific decrease in Ca2+-induced permeability transition pore opening in heart mitochondria." American Journal of Physiology-Heart and Circulatory Physiology 290, no. 4 (April 2006): H1549—H1557. http://dx.doi.org/10.1152/ajpheart.00913.2005.
Full textAbstract:
The purpose of this study was to determine whether regular exercise (treadmill running, 10 wk) alters the susceptibility of rat isolated heart mitochondria to Ca2+-induced permeability transition pore (PTP) opening and whether this could be associated with changes in the modulation of PTP opening by selected physiological effectors. Basal leak-driven and ADP-stimulated respiration in the presence of substrates for complex I, II, and IV were not affected by training. Fluorimetric studies revealed that in the control and exercise-trained groups, the amount of Ca2+ required to trigger PTP opening was greater in the presence of complex II vs. I substrates (230 ± 12 vs. 134 ± 7 nmol Ca2+/mg protein, P < 0.01; pooled average of control and trained groups). In addition, with a substrate feeding the complex II, training increased by 45% ( P < 0.01) the amount of Ca2+ required to trigger PTP opening both in the presence and absence of the PTP inhibitor cyclosporin A. However, membrane potential, reactive oxygen species production, NAD(P)H ratio, and Ca2+ uptake kinetics were not different in mitochondria from both groups. Together, these results suggest the existence of a substrate-specific regulation of the PTP in heart mitochondria and suggest that regular exercise results in a reduced sensitivity to Ca2+-induced PTP opening in presence of complex II substrates.
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Amano, Mutsuki, Tomonari Hamaguchi, Md Hasanuzzaman Shohag, Kei Kozawa, Katsuhiro Kato, Xinjian Zhang, Yoshimitsu Yura, et al. "Kinase-interacting substrate screening is a novel method to identify kinase substrates." Journal of Cell Biology 209, no. 6 (June 22, 2015): 895–912. http://dx.doi.org/10.1083/jcb.201412008.
Full textAbstract:
Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 proteins were identified as candidate substrates for Rho-associated kinase (Rho-kinase/ROCK2), including known substrates. The KISS method was also applied to additional kinases, including PKA, MAPK1, CDK5, CaMK1, PAK7, PKN, LYN, and FYN, and a lot of candidate substrates and their phosphorylation sites were determined, most of which have not been reported previously. Among the candidate substrates for Rho-kinase, several functional clusters were identified, including the polarity-associated proteins, such as Scrib. We found that Scrib plays a crucial role in the regulation of subcellular contractility by assembling into a ternary complex with Rho-kinase and Shroom2 in a phosphorylation-dependent manner. We propose that the KISS method is a comprehensive and useful substrate screen for various kinases.
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Schwartz, Rachel A., Seema S. Lakdawala, Heather D. Eshleman, Matthew R. Russell, Christian T. Carson, and Matthew D. Weitzman. "Distinct Requirements of Adenovirus E1b55K Protein for Degradation of Cellular Substrates." Journal of Virology 82, no. 18 (July 9, 2008): 9043–55. http://dx.doi.org/10.1128/jvi.00925-08.
Full textAbstract:
ABSTRACT The E1b55K and E4orf6 proteins of adenovirus type 5 (Ad5) assemble into a complex together with cellular proteins including cullin 5, elongins B and C, and Rbx1. This complex possesses E3 ubiquitin ligase activity and targets cellular proteins for proteasome-mediated degradation. The ligase activity has been suggested to be responsible for all functions of E1b55K/E4orf6, including promoting efficient viral DNA replication, preventing a cellular DNA damage response, and stimulating late viral mRNA nuclear export and late protein synthesis. The known cellular substrates for degradation by E1b55K/E4orf6 are the Mre11/Rad50/Nbs1 DNA repair complex, the tumor suppressor p53, and DNA ligase IV. Here we show that the degradation of individual targets can occur independently of other substrates. Furthermore, we identify separation-of-function mutant forms of E1b55K that can distinguish substrates for binding and degradation. Our results identify distinct regions of E1b55K that are involved in substrate recognition but also imply that there are additional requirements beyond protein association. These mutant proteins will facilitate the determination of the relevance of specific substrates to the functions of E1b55K in promoting infection and inactivating host defenses.
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Fierobe, Henri-Pierre, Edward A. Bayer, Chantal Tardif, Mirjam Czjzek, Adva Mechaly, Anne Bélaı̈ch, Raphael Lamed, Yuval Shoham, and Jean-Pierre Bélaı̈ch. "Degradation of Cellulose Substrates by Cellulosome Chimeras." Journal of Biological Chemistry 277, no. 51 (October 22, 2002): 49621–30. http://dx.doi.org/10.1074/jbc.m207672200.
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A library of 75 different chimeric cellulosomes was constructed as an extension of our previously described approach for the production of model functional complexes (Fierobe, H.-P., Mechaly, A., Tardif, C., Bélaı̈ch, A., Lamed, R., Shoham, Y., Bélaı̈ch, J.-P., and Bayer, E. A. (2001)J. Biol. Chem.276, 21257–21261), based on the high affinity species-specific cohesin-dockerin interaction. Each complex contained three protein components: (i) a chimeric scaffoldin possessing an optional cellulose-binding module and two cohesins of divergent specificity, and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. The activities of the resultant ternary complexes were assayed using different types of cellulose substrates. Organization of cellulolytic enzymes into cellulosome chimeras resulted in characteristically high activities on recalcitrant substrates, whereas the cellulosome chimeras showed little or no advantage over free enzyme systems on tractable substrates. On recalcitrant cellulose, the presence of a cellulose-binding domain on the scaffoldin and enzyme proximity on the resultant complex contributed almost equally to their elevated action on the substrate. For certain enzyme pairs, however, one effect appeared to predominate over the other. The results also indicate that substrate recalcitrance is not necessarily a function of its crystallinity but reflects the overall accessibility of reactive sites.