scholarly journals X-Ray Crystallographic and Mutational Studies of Fluoroacetate Dehalogenase from Burkholderia sp. Strain FA1

2009 ◽  
Vol 191 (8) ◽  
pp. 2630-2637 ◽  
Author(s):  
Keiji Jitsumori ◽  
Rie Omi ◽  
Tatsuo Kurihara ◽  
Atsushi Kurata ◽  
Hisaaki Mihara ◽  
...  
Keyword(s):  
Active Site ◽  
Three Dimensional ◽  
Chloride Ion ◽  
Catalytic Triad ◽  
Dimensional Structure ◽  
Active Site Residues ◽  
Density Peak ◽  
Core Domain ◽  
X Ray ◽  
The Core

ABSTRACT Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of a carbon-fluorine bond of an aliphatic compound: the bond energy of the carbon-fluorine bond is among the highest found in natural products. The enzyme also acts on chloroacetate, although much less efficiently. We here determined the X-ray crystal structure of the enzyme from Burkholderia sp. strain FA1 as the first experimentally determined three-dimensional structure of fluoroacetate dehalogenase. The enzyme belongs to the α/β hydrolase superfamily and exists as a homodimer. Each subunit consists of core and cap domains. The catalytic triad, Asp104-His271-Asp128, of which Asp104 serves as the catalytic nucleophile, was found in the core domain at the domain interface. The active site was composed of Phe34, Asp104, Arg105, Arg108, Asp128, His271, and Phe272 of the core domain and Tyr147, His149, Trp150, and Tyr212 of the cap domain. An electron density peak corresponding to a chloride ion was found in the vicinity of the Nε1 atom of Trp150 and the Nε2 atom of His149, suggesting that these are the halide ion acceptors. Site-directed replacement of each of the active-site residues, except for Trp150, by Ala caused the total loss of the activity toward fluoroacetate and chloroacetate, whereas the replacement of Trp150 caused the loss of the activity only toward fluoroacetate. An interaction between Trp150 and the fluorine atom is probably an absolute requirement for the reduction of the activation energy for the cleavage of the carbon-fluorine bond.

The three-dimensional structure of interleukin-1β

1988 ◽  
Vol 16 (6) ◽  
pp. 949-953 ◽  
Author(s):  
JOHN P. PRIESTLE ◽  
HANS-PETER SCHÄR ◽  
MARKUS G. GRÜTTER
Keyword(s):  
Polypeptide Chain ◽  
Three Dimensional ◽  
Interleukin 1Β ◽  
Dimensional Structure ◽  
X Ray ◽  
Three Dimensional Structure ◽  
R Factor ◽  
The Core ◽  
Internal Sequence ◽  
Network Of Hydrogen Bonds

Summary The three-dimensional structure of human recombinant interleukin-1β has been determined at 0.24 nm resolution by X-ray crystallographic techniques. The partially refined model has a crystallographic R-factor of just under 19%. The structure is composed of 12 β-strands forming a complex network of hydrogen bonds. The core of the structure can best be described as a tetrahedron whose edges are each formed by two antiparallel β-strands. The interior of this structure is filled with hydrophobic side-chains. There is a 3-fold repeat in the folding of the polypeptide chain. Although this folding pattern suggests gene triplication, no significant internal sequence homology between topologically corresponding residues exists. The folding topology of interleukin-1β is very similar to that described by A. D. McLachlan [(1979) J. Mol. Biol. 133, 557–563] for soybean trypsin inhibitor.

scholarly journals The three-dimensional structure of a class-Pi glutathione S-transferase complexed with glutathione: the active-site hydration provides insights into the reaction mechanism

1998 ◽  
Vol 333 (3) ◽  
pp. 811-816 ◽  
Author(s):  
Antonio PÁRRAGA ◽  
Isabel GARCÍA-SÁEZ ◽  
Sinead B. WALSH ◽  
Timothy J. MANTLE ◽  
Miquel COLL
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Water Molecules ◽  
X Ray Diffraction ◽  
Glutathione S Transferase ◽  
X Ray ◽  
The Right

The structure of mouse liver glutathione S-transferase P1-1 complexed with its substrate glutathione (GSH) has been determined by X-ray diffraction analysis. No conformational changes in the glutathione moiety or in the protein, other than small adjustments of some side chains, are observed when compared with glutathione adduct complexes. Our structure confirms that the role of Tyr-7 is to stabilize the thiolate by hydrogen bonding and to position it in the right orientation. A comparison of the enzyme–GSH structure reported here with previously described structures reveals rearrangements in a well-defined network of water molecules in the active site. One of these water molecules (W0), identified in the unliganded enzyme (carboxymethylated at Cys-47), is displaced by the binding of GSH, and a further water molecule (W4) is displaced following the binding of the electrophilic substrate and the formation of the glutathione conjugate. The possibility that one of these water molecules participates in the proton abstraction from the glutathione thiol is discussed.

Static Laue Diffraction Studies on Acetylcholinesterase

1998 ◽  
Vol 54 (6) ◽  
pp. 1359-1366 ◽  
Author(s):  
Raimond B. G. Ravelli ◽  
Mia L. Raves ◽  
Zhong Ren ◽  
Dominique Bourgeois ◽  
Michel Roth ◽  
...  
Keyword(s):  
Structural Change ◽  
Exposure Time ◽  
Active Site ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Reversible Inhibitor ◽  
Data Set ◽  
Time Resolved ◽  
X Ray ◽  
Density Maps

Acetylcholinesterase (AChE) is one of nature's fastest enzymes, despite the fact that its three-dimensional structure reveals its active site to be deeply sequestered within the molecule. This raises questions with respect to traffic of substrate to, and products from, the active site, which may be investigated by time-resolved crystallography. In order to address one aspect of the feasibility of performing time-resolved studies on AChE, a data set has been collected using the Laue technique on a trigonal crystal of Torpedo californica AChE soaked with the reversible inhibitor edrophonium, using a total X-ray exposure time of 24 ms. Electron-density maps obtained from the Laue data, which are of surprisingly good quality compared with similar maps from monochromatic data, show essentially the same features. They clearly reveal the bound ligand, as well as a structural change in the conformation of the active-site Ser200 induced upon binding.

scholarly journals Urkinase: Structure of Acetate Kinase, a Member of the ASKHA Superfamily of Phosphotransferases

2001 ◽  
Vol 183 (2) ◽  
pp. 680-686 ◽  
Author(s):  
Kathryn A. Buss ◽  
David R. Cooper ◽  
Cheryl Ingram-Smith ◽  
James G. Ferry ◽  
David Avram Sanders ◽  
...  
Keyword(s):  
Common Ancestor ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Acetate Kinase ◽  
Active Site Residues ◽  
Methanosarcina Thermophila ◽  
The Core ◽  
Binding Domains ◽  
The Common ◽  
Actin Superfamily

ABSTRACT Acetate kinase, an enzyme widely distributed in theBacteria and Archaea domains, catalyzes the phosphorylation of acetate. We have determined the three-dimensional structure of Methanosarcina thermophila acetate kinase bound to ADP through crystallography. As we previously predicted, acetate kinase contains a core fold that is topologically identical to that of the ADP-binding domains of glycerol kinase, hexokinase, the 70-kDa heat shock cognate (Hsc70), and actin. Numerous charged active-site residues are conserved within acetate kinases, but few are conserved within the phosphotransferase superfamily. The identity of the points of insertion of polypeptide segments into the core fold of the superfamily members indicates that the insertions existed in the common ancestor of the phosphotransferases. Another remarkable shared feature is the unusual, epsilon conformation of the residue that directly precedes a conserved glycine residue (Gly-331 in acetate kinase) that binds the α-phosphate of ADP. Structural, biochemical, and geochemical considerations indicate that an acetate kinase may be the ancestral enzyme of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases.

GTPases: a family of molecular switches and clocks

1995 ◽  
Vol 349 (1329) ◽  
pp. 283-289 ◽  
Keyword(s):  
Single Gene ◽  
Three Dimensional ◽  
Molecular Switches ◽  
Eukaryotic Cells ◽  
Dimensional Structure ◽  
Gtpase Domain ◽  
Core Domain ◽  
The Core ◽  
Amino Acid Side Chains ◽  
Gtpase Cycle

Members of the GTPase superfamily share a core domain with a conserved three-dimensional structure and a common GTPase cycle, but perform a wide variety of regulatory tasks in eukaryotic cells. Evolution has created functional diversity from the conserved GTPase structure in two principal ways: (i) by combining in the product of a single gene the core GTPase domain attached to one or more additional folded domains; (ii) by building around a core GTPase an assembly of proteins encoded by different genes. Analysis of the patterns of conserved amino acid side chains on surfaces of Ga proteins reveals interfaces with other proteins in the G-protein signal linking device.

SdsA polymorph isolation and improvement of their crystal quality using nonconventional crystallization techniques

2015 ◽  
Vol 48 (5) ◽  
pp. 1551-1559 ◽  
Author(s):  
Eugenio De la Mora ◽  
Edith Flores-Hernández ◽  
Jean Jakoncic ◽  
Vivian Stojanoff ◽  
Dritan Siliqi ◽  
...  
Keyword(s):  
Magnetic Field ◽  
Active Site ◽  
Crystal Packing ◽  
Sodium Dodecyl ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
X Ray Diffraction ◽  
Oligomeric State ◽  
X Ray ◽  
Internal Order

SdsA, a sodium dodecyl sulfate hydrolase, fromPseudomonas aeruginosawas crystallized in three different crystal polymorphs and their three-dimensional structure was determined. The different polymorphs present different crystal packing habits. One of the polymorphs suggests the existence of a tetramer, an oligomeric state not observed previously, while the crystal packing of the remaining two polymorphs obstructs the active site entrance but stabilizes flexible regions of the protein. Nonconventional crystallization methods that minimize convection, such as counterdiffusion in polyvinyl alcohol gel coupled with the influence of a 500 MHz (10.2 T) magnetic field, were necessary to isolate the poorest diffracting polymorph and increase its internal order to determine its structure by X-ray diffraction. The results obtained show the effectiveness of nonconventional crystallographic methods to isolate different crystal polymorphs.

scholarly journals Crystal structure of the catalytic core domain of the family 6 cellobiohydrolase II, Cel6A, from Humicola insolens, at 1.92 Å resolution

1999 ◽  
Vol 337 (2) ◽  
pp. 297-304 ◽  
Author(s):  
Annabelle VARROT ◽  
Sven HASTRUP ◽  
Martin SCHÜLEIN ◽  
Gideon J. DAVIES
Keyword(s):  
Active Site ◽  
Three Dimensional ◽  
Structural Similarity ◽  
Data Bank ◽  
Dimensional Structure ◽  
Active Site Residues ◽  
Catalytic Core ◽  
Humicola Insolens ◽  
The Family ◽  
Cellobiohydrolase Ii

The three-dimensional structure of the catalytic core of the family 6 cellobiohydrolase II, Cel6A (CBH II), from Humicola insolens has been determined by X-ray crystallography at a resolution of 1.92 Å. The structure was solved by molecular replacement using the homologous Trichoderma reesei CBH II as a search model. The H. insolens enzyme displays a high degree of structural similarity with its T. reeseiequivalent. The structure features both O- (α-linked mannose) and N-linked glycosylation and a hexa-co-ordinate Mg2+ ion. The active-site residues are located within the enclosed tunnel that is typical for cellobiohydrolase enzymes and which may permit a processive hydrolysis of the cellulose substrate. The close structural similarity between the two enzymes implies that kinetics and chain-end specificity experiments performed on the H. insolens enzyme are likely to be applicable to the homologous T. reesei enzyme. These cast doubt on the description of cellobiohydrolases as exo-enzymes since they demonstrated that Cel6A (CBH II) shows no requirement for non-reducing chain-ends, as had been presumed. There is no crystallographic evidence in the present structure to support a mechanism involving loop opening, yet preliminary modelling experiments suggest that the active-site tunnel of Cel6A (CBH II) is too narrow to permit entry of a fluorescenyl-derivatized substrate, known to be a viable substrate for this enzyme. Co-ordinates for the structure described in this paper have been deposited with the Brookhaven Protein Data Bank with accession code 1BVW.PDB.

scholarly journals Structure of the S.coelicolor A3(2) N-acetylhexosaminidase provides insight into GH20 catalysis

2014 ◽  
Vol 70 (a1) ◽  
pp. C1677-C1677
Author(s):  
Nhung Thi Nguyen ◽  
Nicolas Doucet
Keyword(s):  
Active Site ◽  
Streptomyces Coelicolor ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Active Site Residues ◽  
Structural Variations ◽  
Neighboring Group ◽  
Glycosidic Bonds ◽  
Ligand Free ◽  
Neighboring Group Participation

β-N-acetylhexosaminidases (HEX - EC 3.2.1.52) are glycosidases that catalyze the glycosidic linkage hydrolysis of gluco- and galacto-configurations of N-acetyl-β-D-hexosaminides. These enzymes have shown considerable interest due to their importance in human physiology and their potential use for the enzymatic synthesis of carbohydrates and glycomymetics. HEX can cleave the β-1,4-glycosidic bonds of polymers with long saccharide chains, and utilize a double-displacement retaining mechanism with neighboring group participation to yield an oxazolinium intermediate. In this study, the three-dimensional structure of the wild-type and catalytically impaired E302Q HEX variant from the soil bacterium Streptomyces coelicolor A3(2) (ScHEX-family GH20) were solved in ligand-free forms and in the presence of 6-acetamido-6-deoxy-castanospermine (6-Ac-Cas). The E302Q variant was also trapped as an intermediate with oxazoline bound to the active center. The complexed structures reveal an active pocket with multiple subsites packed with four Trp, providing a hydrophobic environment that forms a small active-site architecture suitable for holding polysaccharide chains and protecting the formed oxazolinium intermediate during catalysis. Crystallographic evidence highlights structural variations in the loop 3 environment, suggesting conformational heterogeneity for important active-site residues of this GH20 family member.

scholarly journals The Three-dimensional Structure of the Core Domain of Naf Y fromAzotobacter vinelandiidetermined at 1.8-Å Resolution

2003 ◽  
Vol 278 (34) ◽  
pp. 32150-32156 ◽  
Author(s):  
David H. Dyer ◽  
Luis M. Rubio ◽  
James B. Thoden ◽  
Hazel M. Holden ◽  
Paul W. Ludden ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Core Domain ◽  
Three Dimensional Structure ◽  
The Core
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