scholarly journals The substrate/product-binding modes of a novel GH120 β-xylosidase (XylC) from Thermoanaerobacterium saccharolyticum JW/SL-YS485

2012 ◽  
Vol 448 (3) ◽  
pp. 401-407 ◽  
Author(s):  
Chun-Hsiang Huang ◽  
Yu Sun ◽  
Tzu-Ping Ko ◽  
Chun-Chi Chen ◽  
Yingying Zheng ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Product Inhibition ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Binding Modes ◽  
Glycosyl Hydrolases ◽  
Core Domain ◽  
Thermoanaerobacterium Saccharolyticum ◽  
Cazy Database ◽  
Binding Cleft

Xylan-1,4-β-xylosidase (β-xylosidase) hydrolyses xylo-oligomers at their non-reducing ends into individual xylose units. Recently, XylC, a β-xylosidase from Thermoanaerobacterium saccharolyticum JW/SL-YS485, was found to be structurally different from corresponding glycosyl hydrolases in the CAZy database (http://www.cazy.org/), and was subsequently classified as the first member of a novel family of glycoside hydrolases (GH120). In the present paper, we report three crystal structures of XylC in complex with Tris, xylobiose and xylose at 1.48–2.05 Å (1 Å=0.1 nm) resolution. XylC assembles into a tetramer, and each monomer comprises two distinct domains. The core domain is a right-handed parallel β-helix (residues 1–75 and 201–638) and the flanking region (residues 76–200) folds into a β-sandwich domain. The enzyme contains an open carbohydrate-binding cleft, allowing accommodation of longer xylo-oligosaccharides. On the basis of the crystal structures and in agreement with previous kinetic data, we propose that XylC cleaves the glycosidic bond by the retaining mechanism using two acidic residues Asp382 (nucleophile) and Glu405 (general acid/base). In addition to the active site, nine other xylose-binding sites were consistently observed in each of the four monomers, providing a possible reason for the high tolerance of product inhibition.

Crystal structures ofEscherichia colibranching enzyme in complex with cyclodextrins

2016 ◽  
Vol 72 (5) ◽  
pp. 641-647 ◽  
Author(s):  
Lei Feng ◽  
Remie Fawaz ◽  
Stacy Hovde ◽  
Fang Sheng ◽  
Meisam Nosrati ◽  
...  
Keyword(s):  
Binding Site ◽  
Crystal Structures ◽  
Binding Sites ◽  
Carbohydrate Binding ◽  
Binding Modes ◽  
Branch Points ◽  
Branching Enzymes ◽  
Similarities And Differences ◽  
Amp Activated Protein Kinase ◽  
Shed Light

Branching enzyme (BE) is responsible for the third step in glycogen/starch biosynthesis. It catalyzes the cleavage of α-1,4 glucan linkages and subsequent reattachment to form α-1,6 branch points. These branches are crucial to the final structure of glycogen and starch. The crystal structures ofEscherichia coliBE (EcBE) in complex with α-, β- and γ-cyclodextrin were determined in order to better understand substrate binding. Four cyclodextrin-binding sites were identified inEcBE; they were all located on the surface of the enzyme, with none in the vicinity of the active site. While three of the sites were also identified as linear polysaccharide-binding sites, one of the sites is specific for cyclodextrins. In previous work three additional binding sites were identified as exclusively binding linear malto-oligosaccharides. Comparison of the binding sites shed light on this apparent specificity. Binding site IV is located in the carbohydrate-binding module 48 (CBM48) domain ofEcBE and superimposes with the cyclodextrin-binding site found in the CBM48 domain of 5′-AMP-activated protein kinase (AMPK). Comparison of these sites shows the similarities and differences in the two binding modes. While some of the binding sites were found to be conserved between branching enzymes of different organisms, some are quite divergent, indicating both similarities and differences between oligosaccharide binding in branching enzymes from various sources.

scholarly journals Deamidation and glycation of a Bacillus licheniformis α-amylase during industrial fermentation can improve detergent wash performance

Amylase ◽  
2021 ◽  
Vol 5 (1) ◽  
pp. 38-49
Author(s):  
Connie Pontoppidan ◽  
Svend G. Kaasgaard ◽  
Carsten P. Sønksen ◽  
Carsten Andersen ◽  
Birte Svensson
Keyword(s):  
Bacillus Licheniformis ◽  
Mutational Analysis ◽  
Peptide Mapping ◽  
Substrate Binding ◽  
Carbohydrate Binding ◽  
Surface Binding ◽  
Lysine Residues ◽  
Tandem Mass Spectrometric ◽  
Binding Cleft ◽  
Household Detergents

Abstract The industrial thermostable Bacillus licheniformis α-amylase (BLA) has wide applications, including in household detergents, and efforts to improve its performance are continuously ongoing. BLA during the industrial production is deamidated and glycated resulting in multiple forms with different isoelectric points. Forty modified positions were identified by tandem mass spectrometric peptide mapping of BLA forms separated by isoelectric focusing. These modified 12 asparagine, 9 glutamine, 8 arginine and 11 lysine residues are mostly situated on the enzyme surface and several belong to regions involved in stability, activity and carbohydrate binding. Eight residues presumed to interact with starch at the active site and surface binding sites (SBSs) were subjected to mutational analysis. Five mutants mimicking deamidation (N→D, Q→E) at the substrate binding cleft showed moderate to no effect on thermostability and k cat and K M for maltoheptaose and amylose. Notably, the mutations improved laundry wash efficiency in detergents at pH 8.5 and 10.0. Replacing three reducing sugar reactive side chains (K→M, R→L) at a distant substrate binding region and two SBSs enhanced wash performance especially in liquid detergent at pH 8.5, slightly improved enzymatic activity and maintained thermostability. Wash performance was most improved (5-fold) for the N265D mutant near substrate binding subsite +3.

scholarly journals Genome and Secretome Analysis of Staphylotrichum longicolleum DSM105789 Cultured on Agro-Residual and Chitinous Biomass

2021 ◽  
Vol 9 (8) ◽  
pp. 1581
Author(s):  
Arslan Ali ◽  
Bernhard Ellinger ◽  
Sophie C. Brandt ◽  
Christian Betzel ◽  
Martin Rühl ◽  
...  
Keyword(s):  
Sugarcane Bagasse ◽  
Chitinase Activity ◽  
Growth Conditions ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Waste Products ◽  
Enzyme Mixture ◽  
Secretome Analysis ◽  
Polysaccharide Lyases ◽  
Carbohydrate Esterases

Staphylotrichum longicolleum FW57 (DSM105789) is a prolific chitinolytic fungus isolated from wood, with a chitinase activity of 0.11 ± 0.01 U/mg. We selected this strain for genome sequencing and annotation, and compiled its growth characteristics on four different chitinous substrates as well as two agro-industrial waste products. We found that the enzymatic mixture secreted by FW57 was not only able to digest pre-treated sugarcane bagasse, but also untreated sugarcane bagasse and maize leaves. The efficiency was comparable to a commercial enzymatic cocktail, highlighting the potential of the S. longicolleum enzyme mixture as an alternative pretreatment method. To further characterize the enzymes, which efficiently digested polymers such as cellulose, hemicellulose, pectin, starch, and lignin, we performed in-depth mass spectrometry-based secretome analysis using tryptic peptides from in-gel and in-solution digestions. Depending on the growth conditions, we were able to detect from 442 to 1092 proteins, which were annotated to identify from 134 to 224 putative carbohydrate-active enzymes (CAZymes) in five different families: glycoside hydrolases, auxiliary activities, carbohydrate esterases, polysaccharide lyases, glycosyl transferases, and proteins containing a carbohydrate-binding module, as well as combinations thereof. The FW57 enzyme mixture could be used to replace commercial enzyme cocktails for the digestion of agro-residual substrates.

scholarly journals Factor G Utilizes a Carbohydrate-Binding Cleft That Is Conserved between Horseshoe Crab and Bacteria for the Recognition of β-1,3-d-Glucans

2009 ◽  
Vol 183 (6) ◽  
pp. 3810-3818 ◽  
Author(s):  
Yuki Ueda ◽  
Shuhei Ohwada ◽  
Yoshito Abe ◽  
Toshio Shibata ◽  
Manabu Iijima ◽  
...  
Keyword(s):  
Horseshoe Crab ◽  
Carbohydrate Binding ◽  
Binding Cleft ◽  
Factor G

scholarly journals Plasmodium falciparum DHODH inhibitor complexes reveal flexible binding site

2014 ◽  
Vol 70 (a1) ◽  
pp. C1793-C1793
Author(s):  
Paul Rowland ◽  
Onkar SINGH ◽  
Leila Ross ◽  
Francisco Gamo ◽  
Maria Lafuente-Monasterio ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Binding Site ◽  
Crystal Structures ◽  
Point Mutations ◽  
Crystal Form ◽  
Drug Binding ◽  
Binding Modes ◽  
Resistance Mutations ◽  
Drug Binding Site

Malaria is a preventable and treatable disease, yet annually there are still hundreds of thousands of malaria-related deaths. The disease is caused by infection with mosquito-borne Plasmodium parasites. With hundreds of millions of cases each year there is a very high potential for drug resistance and this has compromised many existing therapies. One target under investigation is the enzyme dihydroorotate dehydrogenase (DHODH) which catalyses the rate-limiting step of pyrimidine biosynthesis and is an essential enzyme in the malaria parasite. There are currently several Plasmodium-selective DHODH inhibitors under development. To investigate the potential for drug resistance against DHODH inhibitors in vitro resistance selections were carried out using known inhibitors from different structural classes [1]. These studies identified point mutations in the drug binding site which lead to reduced sensitivity to the inhibitors, and in some cases increased sensitivity to a different inhibitor, suggesting a novel combination therapy approach to combat resistance. To help understand the significance of the inhibitor binding site mutations we determined the crystal structures of P. falciparum DHODH in complex with the inhibitors Genz-669178, IDI-6253 and IDI-6273. Co-crystallisation experiments led to a new crystal form in each case. Here we describe the crystal structures, the binding modes of the inhibitors and the great flexibility of the binding site, which is able to adjust to accommodate different inhibitor series. The structural role of the resistance mutations is also discussed.

scholarly journals Use of Crystallography and Molecular Modeling for the Inhibition of the Botulinum Neurotoxin A Protease

2021 ◽  
Author(s):  
Lewis Turner ◽  
Alexander Lund Nielsen ◽  
Lucy Lin ◽  
Antonio J. Campedelli ◽  
Nicholas Silvaggi ◽  
...  
Keyword(s):  
Molecular Modeling ◽  
Crystal Structures ◽  
Botulinum Neurotoxin ◽  
Enzyme Inhibitor ◽  
Binding Modes ◽  
Botulinum Neurotoxin A ◽  
Inhibitor Binding

We have used crystal structures and molecular modeling to evaluate inhibitor binding modes and design a series of compounds to take advantage of a new, cryptic, hydrophobic sub-pocket. This is a classical SBDD approach to improving enzyme/inhibitor interactions.

scholarly journals Structural basis of AdoMet-dependent aminocarboxypropyl transfer reaction catalyzed by tRNA-wybutosine synthesizing enzyme, TYW2

2009 ◽  
Vol 106 (37) ◽  
pp. 15616-15621 ◽  
Author(s):  
Masataka Umitsu ◽  
Hiroshi Nishimasu ◽  
Akiko Noma ◽  
Tsutomu Suzuki ◽  
Ryuichiro Ishitani ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Binding Pocket ◽  
Structural Basis ◽  
Binding Modes ◽  
Substrate Binding Pocket ◽  
Methyl Group ◽  
Group Transfer ◽  
Canonical Class ◽  
Functional Analyses

S-adenosylmethionine (AdoMet) is a methyl donor used by a wide variety of methyltransferases, and it is also used as the source of an α-amino-α-carboxypropyl (“acp”) group by several enzymes. tRNA-yW synthesizing enzyme-2 (TYW2) is involved in the biogenesis of a hypermodified nucleotide, wybutosine (yW), and it catalyzes the transfer of the “acp” group from AdoMet to the C7 position of the imG-14 base, a yW precursor. This modified nucleoside yW is exclusively located at position 37 of eukaryotic tRNAPhe, and it ensures the anticodon-codon pairing on the ribosomal decoding site. Although this “acp” group has a significant role in preventing decoding frame shifts, the mechanism of the “acp” group transfer by TYW2 remains unresolved. Here we report the crystal structures and functional analyses of two archaeal homologs of TYW2 from Pyrococcus horikoshii and Methanococcus jannaschii. The in vitro mass spectrometric and radioisotope-labeling analyses confirmed that these archaeal TYW2 homologues have the same activity as yeast TYW2. The crystal structures verified that the archaeal TYW2 contains a canonical class-I methyltransferase (MTase) fold. However, their AdoMet-bound structures revealed distinctive AdoMet-binding modes, in which the “acp” group, instead of the methyl group, of AdoMet is directed to the substrate binding pocket. Our findings, which were confirmed by extensive mutagenesis studies, explain why TYW2 transfers the “acp” group, and not the methyl group, from AdoMet to the nucleobase.

scholarly journals On the distinct binding modes of expansin and carbohydrate-binding module proteins on crystalline and nanofibrous cellulose: implications for cellulose degradation by designer cellulosomes

2018 ◽  
Vol 20 (12) ◽  
pp. 8278-8293 ◽  
Author(s):  
Adam Orłowski ◽  
Lior Artzi ◽  
Pierre-Andre Cazade ◽  
Melissabye Gunnoo ◽  
Edward A. Bayer ◽  
...  
Keyword(s):  
Cellulose Degradation ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Binding Modes ◽  
Special Group ◽  
P Transformation ◽  
Hydrolysis Of

Transformation of cellulose into monosaccharides can be achieved by hydrolysis of the cellulose chains, carried out by a special group of enzymes known as cellulases.

scholarly journals A unique combination of glycoside hydrolases in Streptococcus suis specifically and sequentially acts on host-derived αGal-epitope glycans

2020 ◽  
Vol 295 (31) ◽  
pp. 10638-10652
Author(s):  
Ping Chen ◽  
Ran Liu ◽  
Mengmeng Huang ◽  
Jinlu Zhu ◽  
Dong Wei ◽  
...  
Keyword(s):  
Wide Spectrum ◽  
Streptococcus Suis ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Unique Combination ◽  
Enzymatic Assays ◽  
Tracheal Epithelial Cells ◽  
Carbohydrate Binding Modules ◽  
Host Signaling ◽  
Important Host

Infections by many bacterial pathogens rely on their ability to degrade host glycans by producing glycoside hydrolases (GHs). Here, we discovered a conserved multifunctional GH, SsGalNagA, containing a unique combination of two family 32 carbohydrate-binding modules (CBM), a GH16 domain and a GH20 domain, in the zoonotic pathogen Streptococcus suis 05ZYH33. Enzymatic assays revealed that the SsCBM-GH16 domain displays endo-(β1,4)-galactosidase activity specifically toward the host-derived αGal epitope Gal(α1,3)Gal(β1,4)Glc(NAc)-R, whereas the SsGH20 domain has a wide spectrum of exo-β-N-acetylhexosaminidase activities, including exo-(β1,3)-N-acetylglucosaminidase activity, and employs this activity to act in tandem with SsCBM-GH16 on the αGal-epitope glycan. Further, we found that the CBM32 domain adjacent to the SsGH16 domain is indispensable for SsGH16 catalytic activity. Surface plasmon resonance experiments uncovered that both CBM32 domains specifically bind to αGal-epitope glycan, and together they had a KD of 3.5 mm toward a pentasaccharide αGal-epitope glycan. Cell-binding and αGal epitope removal assays revealed that SsGalNagA efficiently binds to both swine erythrocytes and tracheal epithelial cells and removes the αGal epitope from these cells, suggesting that SsGalNagA functions in nutrient acquisition or alters host signaling in S. suis. Both binding and removal activities were blocked by an αGal-epitope glycan. SsGalNagA is the first enzyme reported to sequentially act on a glycan containing the αGal epitope. These findings shed detailed light on the evolution of GHs and an important host-pathogen interaction.

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