scholarly journals Three-dimensional structures of two heavily N-glycosylatedAspergillussp. family GH3 β-D-glucosidases

2016 ◽  
Vol 72 (2) ◽  
pp. 254-265 ◽  
Author(s):  
Jon Agirre ◽  
Antonio Ariza ◽  
Wendy A. Offen ◽  
Johan P. Turkenburg ◽  
Shirley M. Roberts ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Plant Biomass ◽  
Minimum Energy ◽  
Three Dimensional ◽  
Chair Conformation ◽  
Product Inhibition ◽  
Dimensional Structure ◽  
Glycoside Hydrolase Family ◽  
Major Drawback ◽  
Polysaccharide Monooxygenases

The industrial conversion of cellulosic plant biomass into useful products such as biofuels is a major societal goal. These technologies harness diverse plant degrading enzymes, classical exo- and endo-acting cellulases and, increasingly, cellulose-active lytic polysaccharide monooxygenases, to deconstruct the recalcitrant β-D-linked polysaccharide. A major drawback with this process is that the exo-acting cellobiohydrolases suffer from severe inhibition from their cellobiose product. β-D-Glucosidases are therefore important for liberating glucose from cellobiose and thereby relieving limiting product inhibition. Here, the three-dimensional structures of two industrially important family GH3 β-D-glucosidases fromAspergillus fumigatusandA. oryzae, solved by molecular replacement and refined at 1.95 Å resolution, are reported. Both enzymes, which share 78% sequence identity, display a three-domain structure with the catalytic domain at the interface, as originally shown for barley β-D-glucan exohydrolase, the first three-dimensional structure solved from glycoside hydrolase family GH3. Both enzymes show extensive N-glycosylation, with only a few external sites being truncated to a single GlcNAc molecule. Those glycans N-linked to the core of the structure are identified purely as high-mannose trees, and establish multiple hydrogen bonds between their sugar components and adjacent protein side chains. The extensive glycans pose special problems for crystallographic refinement, and new techniques and protocols were developed especially for this work. These protocols ensured that all of the D-pyranosides in the glycosylation trees were modelled in the preferred minimum-energy4C1chair conformation and should be of general application to refinements of other crystal structures containing O- or N-glycosylation. TheAspergillusGH3 structures, in light of other recent three-dimensional structures, provide insight into fungal β-D-glucosidases and provide a platform on which to inform and inspire new generations of variant enzymes for industrial application.

scholarly journals Crystal structure of inactivated Thermotoga maritima invertase in complex with the trisaccharide substrate raffinose

2006 ◽  
Vol 395 (3) ◽  
pp. 457-462 ◽  
Author(s):  
François Alberto ◽  
Emmanuelle Jordi ◽  
Bernard Henrissat ◽  
Mirjam Czjzek
Keyword(s):  
Crystal Structure ◽  
Catalytic Domain ◽  
Thermotoga Maritima ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Dimensional Structure ◽  
Glycoside Hydrolase Family ◽  
Acid Base ◽  
Hydrolase Family ◽  
Binding Subsites

Thermotoga maritima invertase (β-fructosidase), a member of the glycoside hydrolase family GH-32, readily releases β-D-fructose from sucrose, raffinose and fructan polymers such as inulin. These carbohydrates represent major carbon and energy sources for prokaryotes and eukaryotes. The invertase cleaves β-fructopyranosidic linkages by a double-displacement mechanism, which involves a nucleophilic aspartate and a catalytic glutamic acid acting as a general acid/base. The three-dimensional structure of invertase shows a bimodular enzyme with a five bladed β-propeller catalytic domain linked to a β-sandwich of unknown function. In the present study we report the crystal structure of the inactivated invertase in interaction with the natural substrate molecule α-D-galactopyranosyl-(1,6)-α-D-glucopyranosyl-β-D-fructofuranoside (raffinose) at 1.87 Å (1 Å=0.1 nm) resolution. The structural analysis of the complex reveals the presence of three binding-subsites, which explains why T. maritima invertase exhibits a higher affinity for raffinose than sucrose, but a lower catalytic efficiency with raffinose as substrate than with sucrose.

The first crystal structure of the peptidase domain of the U32 peptidase family

2015 ◽  
Vol 71 (12) ◽  
pp. 2505-2512 ◽  
Author(s):  
Magdalena Schacherl ◽  
Angelika A. M. Montada ◽  
Elena Brunstein ◽  
Ulrich Baumann
Keyword(s):  
Crystal Structure ◽  
Catalytic Mechanism ◽  
Quaternary Structure ◽  
Catalytic Domain ◽  
Three Dimensional ◽  
Zinc Ion ◽  
Crystal Structure Analysis ◽  
Dimensional Structure ◽  
Sequence Motifs ◽  
Conserved Sequence

The U32 family is a collection of over 2500 annotated peptidases in the MEROPS database with unknown catalytic mechanism. They mainly occur in bacteria and archaea, but a few representatives have also been identified in eukarya. Many of the U32 members have been linked to pathogenicity, such as proteins fromHelicobacterandSalmonella. The first crystal structure analysis of a U32 catalytic domain fromMethanopyrus kandleri(genemk0906) reveals a modified (βα)8TIM-barrel fold with some unique features. The connecting segment between strands β7 and β8 is extended and helix α7 is located on top of the C-terminal end of the barrel body. The protein exhibits a dimeric quaternary structure in which a zinc ion is symmetrically bound by histidine and cysteine side chains from both monomers. These residues reside in conserved sequence motifs. No typical proteolytic motifs are discernible in the three-dimensional structure, and biochemical assays failed to demonstrate proteolytic activity. A tunnel in which an acetate ion is bound is located in the C-terminal part of the β-barrel. Two hydrophobic grooves lead to a tunnel at the C-terminal end of the barrel in which an acetate ion is bound. One of the grooves binds to aStrep-Tag II of another dimer in the crystal lattice. Thus, these grooves may be binding sites for hydrophobic peptides or other ligands.

scholarly journals Crystal structure of 2-benzylamino-4-(4-methoxyphenyl)-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-3-carbonitrile

2014 ◽  
Vol 70 (12) ◽  
pp. 525-527 ◽  
Author(s):  
R. A. Nagalakshmi ◽  
J. Suresh ◽  
S. Maharani ◽  
R. Ranjith Kumar ◽  
P. L. Nilantha Lakshman
Keyword(s):  
Crystal Structure ◽  
Pyridine Ring ◽  
Three Dimensional ◽  
Chair Conformation ◽  
Dimensional Structure ◽  
Compound C ◽  
Centroid Distance ◽  
Steric Crowding ◽  
Benzene Rings ◽  
Group A

The title compound, C25H25N3O, comprises a 2-aminopyridine ring fused with a cycloheptane ring, which adopts a chair conformation. The central pyridine ring (r.m.s. deviation = 0.013 Å) carries three substituents,viz.a benzylamino group, a methoxyphenyl ring and a carbonitrile group. The N atom of the carbonitrile group is significantly displaced [by 0.2247 (1) Å] from the plane of the pyridine ring, probably due to steric crowding involving the adjacent substituents. The phenyl and benzene rings are inclined to one another by 58.91 (7)° and to the pyridine ring by 76.68 (7) and 49.80 (6)°, respectively. In the crystal, inversion dimers linked by pairs of N—H...Nnitrilehydrogen bonds generateR22(14) loops. The dimers are linked by C—H...π and slipped parallel π–π interactions [centroid–centroid distance = 3.6532 (3) Å] into a three-dimensional structure.

scholarly journals A processive endoglucanase with multi-substrate specificity is characterized from porcine gut microbiota

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Weijun Wang ◽  
Tania Archbold ◽  
Joseph S. Lam ◽  
Matthew S. Kimber ◽  
Ming Z. Fan
Keyword(s):  
Gut Microbiome ◽  
Catalytic Domain ◽  
Hydrolysis Product ◽  
Three Dimensional ◽  
Homology Model ◽  
Industrial Applications ◽  
Glycoside Hydrolase Family ◽  
Expression Library ◽  
Plant Polysaccharides ◽  
Processive Endoglucanase

Abstract Cellulases play important roles in the dietary fibre digestion in pigs, and have multiple industrial applications. The porcine intestinal microbiota display a unique feature in rapid cellulose digestion. Herein, we have expressed a cellulase gene, p4818Cel5_2A, which singly encoded a catalytic domain belonging to glycoside hydrolase family 5 subfamily 2, and was previously identified from a metagenomic expression library constructed from porcine gut microbiome after feeding grower pigs with a cellulose-supplemented diet. The activity of purified p4818Cel5_2A was maximal at pH 6.0 and 50 °C and displayed resistance to trypsin digestion. This enzyme exhibited activities towards a wide variety of plant polysaccharides, including cellulosic substrates of avicel and solka-Floc®, and the hemicelluloses of β-(1 → 4)/(1 → 3)-glucans, xyloglucan, glucomannan and galactomannan. Viscosity, reducing sugar distribution and hydrolysis product analyses further revealed that this enzyme was a processive endo-β-(1 → 4)-glucanase capable of hydrolyzing cellulose into cellobiose and cellotriose as the primary end products. These catalytic features of p4818Cel5_2A were further explored in the context of a three-dimensional homology model. Altogether, results of this study report a microbial processive endoglucanase identified from the porcine gut microbiome, and it may be tailored as an efficient biocatalyst candidate for potential industrial applications.

scholarly journals Preliminary crystallographic analysis of Xyn52B2, a GH52 β-D-xylosidase fromGeobacillus stearothermophilusT6

2014 ◽  
Vol 70 (12) ◽  
pp. 1675-1682 ◽  
Author(s):  
Roie Dann ◽  
Shifra Lansky ◽  
Noa Lavid ◽  
Arie Zehavi ◽  
Valery Belakhov ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Crystal Form ◽  
Thermophilic Bacterium ◽  
Crystallographic Analysis ◽  
Dimensional Structure ◽  
Glycoside Hydrolase Family ◽  
Data Sets ◽  
X Ray Diffraction ◽  
Cell Parameters ◽  
Average Unit

Geobacillus stearothermophilusT6 is a thermophilic bacterium that possesses an extensive hemicellulolytic system, including over 40 specific genes that are dedicated to this purpose. For the utilization of xylan, the bacterium uses an extracellular xylanase which degrades xylan to decorated xylo-oligomers that are imported into the cell. These oligomers are hydrolyzed by side-chain-cleaving enzymes such as arabinofuranosidases, acetylesterases and a glucuronidase, and finally by an intracellular xylanase and a number of β-xylosidases. One of these β-xylosidases is Xyn52B2, a GH52 enzyme that has already proved to be useful for various glycosynthesis applications. In addition to its demonstrated glycosynthase properties, interest in the structural aspects of Xyn52B2 stems from its special glycoside hydrolase family, GH52, the structures and mechanisms of which are only starting to be resolved. Here, the cloning, overexpression, purification and crystallization of Xyn52B2 are reported. The most suitable crystal form that has been obtained belonged to the orthorhombicP212121space group, with average unit-cell parametersa = 97.7,b= 119.1,c = 242.3 Å. Several X-ray diffraction data sets have been collected from flash-cooled crystals of this form, including the wild-type enzyme (3.70 Å resolution), the E335G catalytic mutant (2.95 Å resolution), a potential mercury derivative (2.15 Å resolution) and a selenomethionine derivative (3.90 Å resolution). These data are currently being used for detailed three-dimensional structure determination of the Xyn52B2 protein.

scholarly journals Crystal structure of 13-(2-methoxyphenyl)-3,4-dihydro-2H-indazolo[1,2-b]phthalazine-1,6,11(13H)-trione

2015 ◽  
Vol 71 (8) ◽  
pp. o604-o605 ◽  
Author(s):  
Abdelmalek Bouraiou ◽  
Sofiane Bouacida ◽  
Hocine Merazig ◽  
Aissa Chibani ◽  
Zouhair Bouaziz
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonds ◽  
Three Dimensional ◽  
Chair Conformation ◽  
Dimensional Structure ◽  
Π Interactions ◽  
Compound C ◽  
Cyclohexene Ring ◽  
Centroid Distance ◽  
The Mean

In the title compound, C22H18N2O4, the three fused rings of the pyrazolophthalazine moiety are coplanar (r.m.s. deviation = 0.027 Å). The cyclohexene ring fused to the pyrazolidine ring, so forming the indazolophthalazine unit, has a half-chair conformation. The benzene ring is almost normal to the mean plane of the pyrazolophthalazine moiety, with a dihedral angle of 87.21 (6)° between their planes. In the crystal, molecules are linked by pairs of C—H...O hydrogen bonds forming inversion dimers. The dimers are linkedviaC—H...π interactions, forming slabs parallel to (100). Between the slabs there are weak π–π interactions [shortest inter-centroid distance = 3.6664 (9) Å], leading to the formation of a three-dimensional structure.

scholarly journals Crystal structure of 1-[(2S*,4R*)-6-fluoro-2-methyl-1,2,3,4-tetrahydroquinolin-4-yl]pyrrolidin-2-one

2014 ◽  
Vol 70 (9) ◽  
pp. 153-156
Author(s):  
P. S. Pradeep ◽  
S. Naveen ◽  
M. N. Kumara ◽  
K. M. Mahadevan ◽  
N. K. Lokanath
Keyword(s):  
Hydrogen Bonds ◽  
Three Dimensional ◽  
Bond Distance ◽  
Chair Conformation ◽  
Dimensional Structure ◽  
Pyrrolidine Ring ◽  
Charge Delocalization ◽  
Compound C ◽  
The Mean ◽  
Bond Character

In the title compound, C14H17FN2O, the 1,2,3,4-tetrahydropyridine ring of the quinoline moiety adopts a half-chair conformation, while the pyrrolidine ring has an envelope conformation with the central methylene C atom as the flap. The pyrrolidine ring lies in the equatorial plane and its mean plane is normal to the mean plane of the quinoline ring system, with a dihedral angle value of 88.37 (9)°. The bridging N—C bond distance [1.349 (3) Å] is substantially shorter than the sum of the covalent radii (dcov: C—N = 1.47 Å and C=N = 1.27 Å), which indicates partial double-bond character for this bond, resulting in a certain degree of charge delocalization. In the crystal, molecules are linked by N—H...O and C—H...O hydrogen bonds, forming sheets lying parallel to (10-1). These two-dimensional networks are linkedviaC—H...F hydrogen bonds and C—H...π interactions, forming a three-dimensional structure.

scholarly journals 4-(Pyrimidin-2-yl)piperazin-1-ium (E)-3-carboxyprop-2-enoate

2014 ◽  
Vol 70 (6) ◽  
pp. o702-o703 ◽  
Author(s):  
Thammarse S. Yamuna ◽  
Manpreet Kaur ◽  
Jerry P. Jasinski ◽  
H. S. Yathirajan
Keyword(s):  
Hydrogen Bond ◽  
Hydrogen Bonds ◽  
Intermolecular Hydrogen Bond ◽  
Three Dimensional ◽  
Chair Conformation ◽  
Dimensional Structure ◽  
Three Dimensional Structure ◽  
Axis Direction ◽  
Π Interactions ◽  
Centroid Distance

In the cation of the title salt, C8H13N4+·C4H3O4−, the piperazinium ring adopts a slightly distorteded chair conformation. In the crystal, a single strong O—H...O intermolecular hydrogen bond links the anions, forming chains along thec-axis direction. The chains of anions are linked by the cations,viaN—H...O hydrogen bonds, forming sheets parallel to (100). These layers are linked by weak C—H...O hydrogen bonds, forming a three-dimensional structure. In addition, there are weak π–π interactions [centroid–centroid distance = 3.820 (9) Å] present involving inversion-related pyrimidine rings.

scholarly journals Three-dimensional Structure of Acanthamoeba castellanii Myosin-IB (MIB) Determined by Cryoelectron Microscopy of Decorated Actin Filaments

1998 ◽  
Vol 141 (1) ◽  
pp. 155-162 ◽  
Author(s):  
James D. Jontes ◽  
E. Michael Ostap ◽  
Thomas D. Pollard ◽  
Ronald A. Milligan
Keyword(s):  
Actin Filaments ◽  
Catalytic Domain ◽  
Three Dimensional ◽  
Acanthamoeba Castellanii ◽  
Dimensional Structure ◽  
Cryoelectron Microscopy ◽  
Tail Domain ◽  
Three Dimensional Structure ◽  
Myosin I ◽  
Carboxy Terminal

The Acanthamoeba castellanii myosin-Is were the first unconventional myosins to be discovered, and the myosin-I class has since been found to be one of the more diverse and abundant classes of the myosin superfamily. We used two-dimensional (2D) crystallization on phospholipid monolayers and negative stain electron microscopy to calculate a projection map of a “classical” myosin-I, Acanthamoeba myosin-IB (MIB), at ∼18 Å resolution. Interpretation of the projection map suggests that the MIB molecules sit upright on the membrane. We also used cryoelectron microscopy and helical image analysis to determine the three-dimensional structure of actin filaments decorated with unphosphorylated (inactive) MIB. The catalytic domain is similar to that of other myosins, whereas the large carboxy-terminal tail domain differs greatly from brush border myosin-I (BBM-I), another member of the myosin-I class. These differences may be relevant to the distinct cellular functions of these two types of myosin-I. The catalytic domain of MIB also attaches to F-actin at a significantly different angle, ∼10°, than BBM-I. Finally, there is evidence that the tails of adjacent MIB molecules interact in both the 2D crystal and in the decorated actin filaments.

Sign in / Sign up

Export Citation Format

Share Document