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941.
John Raedts Magnus Lundgren Servé W. M. Kengen Jin-Ping Li John van der Oost 《The Journal of biological chemistry》2013,288(34):24332-24339
Glycosaminoglycans are biologically active polysaccharides that are found ubiquitously in the animal kingdom. The biosynthesis of these complex polysaccharides involves complicated reactions that turn the simple glycosaminoglycan backbone into highly heterogeneous structures. One of the modification reactions is the epimerization of d-glucuronic acid to its C5-epimer l-iduronic acid, which is essential for the function of heparan sulfate. Although l-iduronic acid residues have been shown to exist in polysaccharides of some prokaryotes, there has been no experimental evidence for the existence of a prokaryotic d-glucuronyl C5-epimerase. This work for the first time reports on the identification of a bacterial enzyme with d-glucuronyl C5-epimerase activity. A gene of the marine bacterium Bermanella marisrubri sp. RED65 encodes a protein (RED65_08024) of 448 amino acids that has an overall 37% homology to the human d-glucuronic acid C5-epimerase. Alignment of this peptide with the human and mouse sequences revealed a 60% similarity at the carboxyl terminus. The recombinant protein expressed in Escherichia coli showed epimerization activity toward substrates generated from heparin and the E. coli K5 capsular polysaccharide, thereby providing the first evidence for bacterial d-glucuronyl C5-epimerase activity. These findings may eventually be used for modification of mammalian glycosaminoglycans. 相似文献
942.
943.
Ana S. Luís Immacolata Venditto Max J. Temple Artur Rogowski Arnaud Baslé Jie Xue J. Paul Knox José A.M. Prates Luís M. A. Ferreira Carlos M. G. A. Fontes Shabir Najmudin Harry J. Gilbert 《The Journal of biological chemistry》2013,288(7):4799-4809
Plant biomass is central to the carbon cycle and to environmentally sustainable industries exemplified by the biofuel sector. Plant cell wall degrading enzymes generally contain noncatalytic carbohydrate binding modules (CBMs) that fulfil a targeting function, which enhances catalysis. CBMs that bind β-glucan chains often display broad specificity recognizing β1,4-glucans (cellulose), β1,3-β1,4-mixed linked glucans and xyloglucan, a β1,4-glucan decorated with α1,6-xylose residues, by targeting structures common to the three polysaccharides. Thus, CBMs that recognize xyloglucan target the β1,4-glucan backbone and only accommodate the xylose decorations. Here we show that two closely related CBMs, CBM65A and CBM65B, derived from EcCel5A, a Eubacterium cellulosolvens endoglucanase, bind to a range of β-glucans but, uniquely, display significant preference for xyloglucan. The structures of the two CBMs reveal a β-sandwich fold. The ligand binding site comprises the β-sheet that forms the concave surface of the proteins. Binding to the backbone chains of β-glucans is mediated primarily by five aromatic residues that also make hydrophobic interactions with the xylose side chains of xyloglucan, conferring the distinctive specificity of the CBMs for the decorated polysaccharide. Significantly, and in contrast to other CBMs that recognize β-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln106 is central to cellulose recognition, but is not required for binding to mixed linked glucans. This report reveals the mechanism by which β-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated β-glucans. 相似文献
944.
945.
Shoko Shinya Takayuki Ohnuma Reina Yamashiro Hisashi Kimoto Hideo Kusaoke Padmanabhan Anbazhagan André H. Juffer Tamo Fukamizo 《The Journal of biological chemistry》2013,288(42):30042-30053
Two carbohydrate binding modules (DD1 and DD2) belonging to CBM32 are located at the C terminus of a chitosanase from Paenibacillus sp. IK-5. We produced three proteins, DD1, DD2, and tandem DD1/DD2 (DD1+DD2), and characterized their binding ability. Transition temperature of thermal unfolding (Tm) of each protein was elevated by the addition of cello-, laminari-, chitin-, or chitosan-hexamer (GlcN)6. The Tm elevation (ΔTm) in DD1 was the highest (10.3 °C) upon the addition of (GlcN)6 and was markedly higher than that in DD2 (1.0 °C). A synergistic effect was observed (ΔTm = 13.6 °C), when (GlcN)6 was added to DD1+DD2. From isothermal titration calorimetry experiments, affinities to DD1 were not clearly dependent upon chain length of (GlcN)n; ΔGr° values were −7.8 (n = 6), −7.6 (n = 5), −7.6 (n = 4), −7.6 (n = 3), and −7.1 (n = 2) kcal/mol, and the value was not obtained for GlcN due to the lowest affinity. DD2 bound (GlcN)n with the lower affinities (ΔGr° = −5.0 (n = 3) ∼ −5.2 (n = 6) kcal/mol). Isothermal titration calorimetry profiles obtained for DD1+DD2 exhibited a better fit when the two-site model was used for analysis and provided greater affinities to (GlcN)6 for individual DD1 and DD2 sites (ΔGr° = −8.6 and −6.4 kcal/mol, respectively). From NMR titration experiments, (GlcN)n (n = 2∼6) were found to bind to loops extruded from the core β-sandwich of individual DD1 and DD2, and the interaction sites were similar to each other. Taken together, DD1+DD2 is specific to chitosan, and individual modules synergistically interact with at least two GlcN units, facilitating chitosan hydrolysis. 相似文献
946.
Joakim Lundqvist Ilka Braumann Marzena Kurowska André H. Müller Mats Hansson 《The Journal of biological chemistry》2013,288(33):24012-24019
The ATP-dependent insertion of Mg2+ into protoporphyrin IX is the first committed step in the chlorophyll biosynthetic pathway. The reaction is catalyzed by magnesium chelatase, which consists of three gene products: BchI, BchD, and BchH. The BchI and BchD subunits belong to the family of AAA+ proteins (ATPases associated with various cellular activities) and form a two-ring complex with six BchI subunits in one layer and six BchD subunits in the other layer. This BchID complex is a two-layered trimer of dimers with the ATP binding site located at the interface between two neighboring BchI subunits. ATP hydrolysis by the BchID motor unit fuels the insertion of Mg2+ into the porphyrin by the BchH subunit. In the present study, we explored mutations that were originally identified in semidominant barley (Hordeum vulgare L.) mutants. The resulting recombinant BchI proteins have marginal ATPase activity and cannot contribute to magnesium chelatase activity although they apparently form structurally correct complexes with BchD. Mixing experiments with modified and wild-type BchI in various combinations showed that an exchange of BchI subunits in magnesium chelatase occurs during the catalytic cycle, which indicates that dissociation of the complex may be part of the reaction mechanism related to product release. Mixing experiments also showed that more than three functional interfaces in the BchI ring structure are required for magnesium chelatase activity. 相似文献
947.
Hugh Kim Hervé Falet Karin M. Hoffmeister John H. Hartwig 《The Journal of biological chemistry》2013,288(48):34352-34363
Platelets are immunologically competent cells containing cytokines such as TGF-β1 that regulate cell-mediated immunity. However, the mechanisms underlying cytokine secretion from platelets are undefined. The Wiskott-Aldrich syndrome protein (WASp) regulates actin polymerization in nucleated hematopoietic cells but has other role(s) in platelets. WASp-null (WASp−/−) platelets stimulated with a PAR-4 receptor agonist had increased TGF-β1 release compared with WT platelets; inhibiting WASp function with wiskostatin augmented TRAP-induced TGF-β1 release in human platelets. TGF-β1 release is dissociated from α-granule secretion (P-selectin up-regulation) and occurs more gradually, with ∼10–15% released after 30–60 min. Blockade of Src family kinase-mediated WASp Tyr-291/Tyr-293 phosphorylation increased TGF-β1 release, with no additive effect in WASp−/− platelets, signifying that phosphorylation is critical for WASp-limited TGF-β1 secretion. Inhibiting F-actin assembly with cytochalasin D enhanced secretion in WT platelets and further increased TGF-β1 release in WASp−/− platelets, indicating that WASp and actin assembly independently regulate TGF-β1 release. A permeabilized platelet model was used to test the role of upstream small GTPases in TGF-β1 release. N17Cdc42, but not Rac1 mutants, increased TGF-β1 secretion and abrogated WASp phosphorylation. We conclude that WASp function restricts TGF-β1 secretion in a Cdc42- and Src family kinase-dependent manner and independently of actin assembly. 相似文献
948.
Ana Casa?al Ulrich Zander Cristina Mu?oz Florine Dupeux Irene Luque Miguel Angel Botella Wilfried Schwab Victoriano Valpuesta José A. Marquez 《The Journal of biological chemistry》2013,288(49):35322-35332
Pathogenesis-related 10 (PR-10) proteins are involved in many aspects of plant biology but their molecular function is still unclear. They are related by sequence and structural homology to mammalian lipid transport and plant abscisic acid receptor proteins and are predicted to have cavities for ligand binding. Recently, three new members of the PR-10 family, the Fra a proteins, have been identified in strawberry, where they are required for the activity of the flavonoid biosynthesis pathway, which is essential for the development of color and flavor in fruits. Here, we show that Fra a proteins bind natural flavonoids with different selectivity and affinities in the low μm range. The structural analysis of Fra a 1 E and a Fra a 3-catechin complex indicates that loops L3, L5, and L7 surrounding the ligand-binding cavity show significant flexibility in the apo forms but close over the ligand in the Fra a 3-catechin complex. Our findings provide mechanistic insight on the function of Fra a proteins and suggest that PR-10 proteins, which are widespread in plants, may play a role in the control of secondary metabolic pathways by binding to metabolic intermediates. 相似文献
949.
Michela Campagna Laura Marcos-Villar Francesca Arnoldi Carlos F. de la Cruz-Herrera Pedro Gallego José González-Santamaría Dolores González Fernando Lopitz-Otsoa Manuel S. Rodriguez Oscar R. Burrone Carmen Rivas 《Journal of virology》2013,87(2):807-817
Posttranslational modification by SUMO provides functional flexibility to target proteins. Viruses interact extensively with the cellular SUMO modification system in order to improve their replication, and there are numerous examples of viral proteins that are SUMOylated. However, thus far the relevance of SUMOylation for rotavirus replication remains unexplored. In this study, we report that SUMOylation positively regulates rotavirus replication and viral protein production. We show that SUMO can be covalently conjugated to the viroplasm proteins VP1, VP2, NSP2, VP6, and NSP5. In addition, VP1, VP2, and NSP2 can also interact with SUMO in a noncovalent manner. We observed that an NSP5 SUMOylation mutant protein retains most of its activities, such as its interaction with VP1 and NSP2, the formation of viroplasm-like structures after the coexpression with NSP2, and the ability to complement in trans the lack of NSP5 in infected cells. However, this mutant is characterized by a high degree of phosphorylation and is impaired in the formation of viroplasm-like structures when coexpressed with VP2. These results reveal for the first time a positive role for SUMO modification in rotavirus replication, describe the SUMOylation of several viroplasm resident rotavirus proteins, and demonstrate a requirement for NSP5 SUMOylation in the production of viroplasm-like structures. 相似文献
950.
Adriano Cavalleri André R. de Souza Fábio Prezoto Laurence A. Mound 《Biological journal of the Linnean Society. Linnean Society of London》2013,109(2):332-341
The insects known as thrips are commonly thought of as flower‐living and pestiferous organisms, but we report here a novel interaction between a phlaeothripine thrips species, Mirothrips arbiter gen. et sp. nov. and three species of social paper wasps in Brazil. This thrips species breeds inside the wasp colonies, and larval and adult thrips feed on wasp eggs, which become severely damaged. Infested nests can contain up to 300 M. arbiter gen. et sp. nov. individuals. The closest relatives of M. arbiter are two presumably predaceous species: Mirothrips bicolor sp. nov. , which inhabits abandoned Cecidomyiidae galls, and Mirothrips analis comb. nov. , described from individuals collected in the silken bags of the caterpillars of Psychidae moths. The behaviour exhibited by M. arbiter represents one of the most evolutionarily advanced lifestyles known among Thysanoptera, and we predict that other polistine species serve as hosts for this thrips in Brazil. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 332–341. 相似文献