scholarly journals Crystal structure ofEscherichia colipurine nucleoside phosphorylase in complex with 7-deazahypoxanthine

2018 ◽  
Vol 74 (6) ◽  
pp. 355-362
Author(s):  
Vladimir I. Timofeev ◽  
Nadezhda E. Zhukhlistova ◽  
Yuliya A. Abramchik ◽  
Ilya I. Fateev ◽  
Maria A. Kostromina ◽  
...  
Keyword(s):  
Active Site ◽  
Enzyme Inhibitors ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Data Set ◽  
Purine Base ◽  
Cell Parameters ◽  
Purine Bases ◽  
Diffusion Technique ◽  
Enzyme Subunits

Purine nucleoside phosphorylases (EC 2.4.2.1; PNPs) reversibly catalyze the phosphorolytic cleavage of glycosidic bonds in purine nucleosides to generate ribose 1-phosphate and a free purine base, and are key enzymes in the salvage pathway of purine biosynthesis. They also catalyze the transfer of pentosyl groups between purine bases (the transglycosylation reaction) and are widely used for the synthesis of biologically important analogues of natural nucleosides, including a number of anticancer and antiviral drugs. Potent inhibitors of PNPs are used in chemotherapeutic applications. The detailed study of the binding of purine bases and their derivatives in the active site of PNPs is of particular interest in order to understand the mechanism of enzyme action and for the development of new enzyme inhibitors. Here, it is shown that 7-deazahypoxanthine (7DHX) is a noncompetitive inhibitor of the phosphorolysis of inosine by recombinantEscherichia coliPNP (EcPNP) with an inhibition constantKiof 0.13 mM. A crystal ofEcPNP in complex with 7DHX was obtained in microgravity by the counter-diffusion technique and the three-dimensional structure of theEcPNP–7DHX complex was solved by molecular replacement at 2.51 Å resolution using an X-ray data set collected at the SPring-8 synchrotron-radiation facility, Japan. The crystals belonged to space groupP6122, with unit-cell parametersa=b= 120.370,c= 238.971 Å, and contained three subunits of the hexameric enzyme molecule in the asymmetric unit. The 7DHX molecule was located with full occupancy in the active site of each of the three crystallographically independent enzyme subunits. The position of 7DHX overlapped with the positions occupied by purine bases in similar PNP complexes. However, the orientation of the 7DHX molecule differs from those of other bases: it is rotated by ∼180° relative to other bases. The peculiarities of the arrangement of 7DHX in theEcPNP active site are 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 Organophosphorus acid anhydrolase fromAlteromonas macleodii: structural study and functional relationship to prolidases

2013 ◽  
Vol 69 (4) ◽  
pp. 346-354 ◽  
Author(s):  
Andrea Štěpánková ◽  
Jarmila Dušková ◽  
Tereza Skálová ◽  
Jindřich Hašek ◽  
Tomáš Koval' ◽  
...  
Keyword(s):  
Active Site ◽  
Three Dimensional ◽  
Size Exclusion ◽  
Dimensional Structure ◽  
Cell Parameters ◽  
Bacterial Enzyme ◽  
Organophosphorus Acid Anhydrolase ◽  
Full Structure ◽  
Exclusion Chromatography ◽  
Pita Bread

The bacterial enzyme organophosphorus acid anhydrolase (OPAA) is able to catalyze the hydrolysis of both proline dipeptides (Xaa-Pro) and several types of organophosphate (OP) compounds. The full three-dimensional structure of the manganese-dependent OPAA enzyme is presented for the first time. This enzyme, which was originally isolated from the marine bacteriumAlteromonas macleodii, was prepared recombinantly inEscherichia coli. The crystal structure was determined at 1.8 Å resolution in space groupC2, with unit-cell parametersa= 133.8,b= 49.2,c= 97.3 Å, β = 125.0°. The enzyme forms dimers and their existence in solution was confirmed by dynamic light scattering and size-exclusion chromatography. The enzyme shares the pita-bread fold of its C-terminal domain with related prolidases. The binuclear manganese centre is located in the active site within the pita-bread domain. Moreover, an Ni2+ion from purification was localized according to anomalous signal. This study presents the full structure of this enzyme with complete surroundings of the active site and provides a critical analysis of its relationship to prolidases.

scholarly journals Crystallization and X-ray analysis of the transcription-activator protein C1 of bacteriophage P22 in complex with the PREpromoter element

2015 ◽  
Vol 71 (10) ◽  
pp. 1286-1291
Author(s):  
Avisek Mondal ◽  
Rajagopal Chattopadhyaya ◽  
Ajit Bikram Datta ◽  
Pradeep Parrack
Keyword(s):  
Molecular Basis ◽  
Three Dimensional ◽  
Direct Repeat ◽  
Dimensional Structure ◽  
Transcription Activator ◽  
Bacteriophage P22 ◽  
Data Set ◽  
Cell Parameters ◽  
Activator Protein ◽  
Promoter Dna

The transcription-activator protein C1 of the temperate phage P22 ofSalmonella typhimuriumplays a key role in the lyticversuslysogenic switch of the phage. A homotetramer of 92-residue polypeptides, C1 binds to an approximate direct repeat similar to the transcription activator CII of coliphage λ. Despite this and several other similarities, including 57% sequence identity to coliphage CII, many biochemical observations on P22 C1 cannot be explained based on the structure of CII. To understand the molecular basis of these differences, C1 was overexpressed and purified and subjected to crystallization trials. Although no successful hits were obtained for the apoprotein, crystals could be obtained when the protein was subjected to crystallization trials in complex with a 23-mer promoter DNA fragment (PRE). These crystals diffracted very well at the home source, allowing the collection of a 2.2 Å resolution data set. The C1–DNA crystals belonged to space groupP21, with unit-cell parametersa= 87.27,b= 93.58,c= 111.16 Å, β = 94.51°. Solvent-content analysis suggests that the asymmetric unit contains three tetramer–DNA complexes. The three-dimensional structure is expected to shed light on the mechanism of activation by C1 and the molecular basis of its specificity.

Three Dimensional Structure of UMo8O26: Ordering of U-Vacancies

2002 ◽  
Vol 718 ◽  
Author(s):  
N.D. Zakharov ◽  
P. Werner
Keyword(s):  
Low Temperatures ◽  
Driving Force ◽  
Three Dimensional ◽  
Solid State Reaction Method ◽  
Dimensional Structure ◽  
Unit Cell Parameters ◽  
Cell Parameters ◽  
Selected Area Electron Diffraction ◽  
Transmission Electron ◽  
Edx Microanalysis

AbstractThe structure and composition of UMo8O26 synthesized by solid state reaction method have been investigated by High Resolution Transmission Electron Microscopy (HRTEM), Selected Area Electron Diffraction, and EDX microanalysis. The ordering of U vacancies results in considerable enlargement of unit cell parameters: an=6.44 nm, bn=1.45 nm, cn=1.6 nm. It is build up of four layers piled up in c direction. Each following layer is shifted relative to previous one by vector bn/4. Eight hexagonal tunnels in each layer are filled by U atoms, while the eight others are vacant (V). Interaction between U cations and vacancies is driving force for ordering. The variation of stoichiometry can be a reason for appearance of incommensurate modulations in these crystals. It seems plausible that this structure might also exhibit superconductivity at low temperatures.

scholarly journals Crystallization and preliminary X-ray analysis of the major peanut allergen Ara h 1 core region

2010 ◽  
Vol 66 (9) ◽  
pp. 1071-1073 ◽  
Author(s):  
Cerrone Cabanos ◽  
Hiroyuki Urabe ◽  
Taro Masuda ◽  
Mary Rose Tandang-Silvas ◽  
Shigeru Utsumi ◽  
...  
Keyword(s):  
Sodium Citrate ◽  
Three Dimensional ◽  
Core Region ◽  
Dimensional Structure ◽  
Monoclinic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Peg 400 ◽  
Ara H 1 ◽  
Peanut Allergens

Peanuts contain some of the most potent food allergens known to date. Ara h 1 is one of the three major peanut allergens. As a first step towards three-dimensional structure elucidation, recombinant Ara h 1 core region was cloned, expressed inEscherichia coliand purified to homogeneity. Crystals were obtained using 0.1 Msodium citrate pH 5.6, 0.1 MNaCl, 15% PEG 400 as precipitant. The crystals diffracted to 2.25 Å resolution using synchrotron radiation and belonged to the monoclinic space groupC2, with unit-cell parametersa= 156.521,b= 88.991,c= 158.971 Å, β = 107.144°. Data were collected at the BL-38B1 station of SPring-8 (Hyogo, Japan).

scholarly journals Solvent Accessibility of Residues Undergoing Pathogenic Variations in Humans: From Protein Structures to Protein Sequences

2021 ◽  
Vol 7 ◽  
Author(s):  
Castrense Savojardo ◽  
Matteo Manfredi ◽  
Pier Luigi Martelli ◽  
Rita Casadio
Keyword(s):  
Solvent Accessibility ◽  
Protein Structures ◽  
Three Dimensional ◽  
Protein Sequences ◽  
Large Data ◽  
Human Protein ◽  
Dimensional Structure ◽  
Wild Type ◽  
Solvent Exposure ◽  
Data Set

Solvent accessibility (SASA) is a key feature of proteins for determining their folding and stability. SASA is computed from protein structures with different algorithms, and from protein sequences with machine-learning based approaches trained on solved structures. Here we ask the question as to which extent solvent exposure of residues can be associated to the pathogenicity of the variation. By this, SASA of the wild-type residue acquires a role in the context of functional annotation of protein single-residue variations (SRVs). By mapping variations on a curated database of human protein structures, we found that residues targeted by disease related SRVs are less accessible to solvent than residues involved in polymorphisms. The disease association is not evenly distributed among the different residue types: SRVs targeting glycine, tryptophan, tyrosine, and cysteine are more frequently disease associated than others. For all residues, the proportion of disease related SRVs largely increases when the wild-type residue is buried and decreases when it is exposed. The extent of the increase depends on the residue type. With the aid of an in house developed predictor, based on a deep learning procedure and performing at the state-of-the-art, we are able to confirm the above tendency by analyzing a large data set of residues subjected to variations and occurring in some 12,494 human protein sequences still lacking three-dimensional structure (derived from HUMSAVAR). Our data support the notion that surface accessible area is a distinguished property of residues that undergo variation and that pathogenicity is more frequently associated to the buried property than to the exposed one.

scholarly journals Structural Features of a Bacteroidetes-Affiliated Cellulase Linked with a Polysaccharide Utilization Locus

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
A.E. Naas ◽  
A.K. MacKenzie ◽  
B. Dalhus ◽  
V.G.H. Eijsink ◽  
P.B. Pope
Keyword(s):  
Active Site ◽  
Three Dimensional ◽  
Structural Features ◽  
Structure And Function ◽  
Dimensional Structure ◽  
Active Site Cleft ◽  
Polysaccharide Utilization Locus ◽  
And Function ◽  
Rumen Metagenome

Abstract Previous gene-centric analysis of a cow rumen metagenome revealed the first potentially cellulolytic polysaccharide utilization locus, of which the main catalytic enzyme (AC2aCel5A) was identified as a glycoside hydrolase (GH) family 5 endo-cellulase. Here we present the 1.8 Å three-dimensional structure of AC2aCel5A and characterization of its enzymatic activities. The enzyme possesses the archetypical (β/α)8-barrel found throughout the GH5 family and contains the two strictly conserved catalytic glutamates located at the C-terminal ends of β-strands 4 and 7. The enzyme is active on insoluble cellulose and acts exclusively on linear β-(1,4)-linked glucans. Co-crystallization of a catalytically inactive mutant with substrate yielded a 2.4 Å structure showing cellotriose bound in the −3 to −1 subsites. Additional electron density was observed between Trp178 and Trp254, two residues that form a hydrophobic “clamp”, potentially interacting with sugars at the +1 and +2 subsites. The enzyme’s active-site cleft was narrower compared to the closest structural relatives, which in contrast to AC2aCel5A, are also active on xylans, mannans and/or xyloglucans. Interestingly, the structure and function of this enzyme seem adapted to less-substituted substrates such as cellulose, presumably due to the insufficient space to accommodate the side-chains of branched glucans in the active-site cleft.

PREDICTION OF THE THREE-DIMENSIONAL STRUCTURE OF THE ENZYMATIC PART OF T-PA

1987 ◽  
Author(s):  
A Heckel ◽  
K M Hasselbach
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Serine Proteases ◽  
Three Dimensional ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
Salt Bridges ◽  
Three Dimensional Structure ◽  
Insertions And Deletions

Up to now the three-dimensional structure of t-PA or parts of this enzyme is unknown. Using computer graphical methods the spatial structure of the enzymatic part of t-PA is predicted on the hypothesis, the three-dimensional backbone structure of t-PA being similar to that of other serine proteases. The t-PA model was built up in three steps:1) Alignment of the t-PA sequence with other serine proteases. Comparison of enzyme structures available from Brookhaven Protein Data Bank proved elastase as a basis for modeling.2) Exchange of amino acids of elastase differing from the t-PA sequence. The replacement of amino acids was performed such that backbone atoms overlapp completely and side chains superpose as far as possible.3) Modeling of insertions and deletions. To determine the spatial arrangement of insertions and deletions parts of related enzymes such as chymotrypsin or trypsin were used whenever possible. Otherwise additional amino acid sequences were folded to a B-turn at the surface of the proteine, where all insertions or deletions are located. Finally the side chain torsion angles of amino acids were optimised to prevent close contacts of neigh bouring atoms and to improve hydrogen bonds and salt bridges.The resulting model was used to explain binding of arginine 560 of plasminogen to the active site of t-PA. Arginine 560 interacts with Asp 189, Gly 19 3, Ser 19 5 and Ser 214 of t-PA (chymotrypsin numbering). Furthermore interaction of chromo-genic substrate S 2288 with the active site of t-PA was studied. The need for D-configuration of the hydrophobic amino acid at the N-terminus of this tripeptide derivative could be easily explained.

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.

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