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 Ribokinase fromLeishmania donovani: purification, characterization and X-ray crystallographic analysis

2018 ◽  
Vol 74 (2) ◽  
pp. 99-104 ◽  
Author(s):  
Santhosh Gatreddi ◽  
Sayanna Are ◽  
Insaf Ahmed Qureshi
Keyword(s):  
Functional Characterization ◽  
Three Dimensional ◽  
Protozoan Parasite ◽  
Crystallographic Analysis ◽  
Size Exclusion ◽  
Dimensional Structure ◽  
Diffusion Method ◽  
Gene Encoding ◽  
Cell Parameters ◽  
Α Helix

Leishmaniais an auxotrophic protozoan parasite which acquires D-ribose by transporting it from the host cell and also by the hydrolysis of nucleosides. The enzyme ribokinase (RK) catalyzes the first step of ribose metabolism by phosphorylating D-ribose using ATP to produce D-ribose-5-phosphate. To understand its structure and function, the gene encoding RK fromL. donovaniwas cloned, expressed and purified using affinity and size-exclusion chromatography. Circular-dichroism spectroscopy of the purified protein showed comparatively more α-helix in the secondary-structure content, and thermal unfolding revealed theTmto be 317.2 K. Kinetic parameters were obtained by functional characterization ofL. donovaniRK, and theKmvalues for ribose and ATP were found to be 296 ± 36 and 116 ± 9.0 µM, respectively. Crystals obtained by the hanging-drop vapour-diffusion method diffracted to 1.95 Å resolution and belonged to the hexagonal space groupP61, with unit-cell parametersa=b= 100.25,c= 126.77 Å. Analysis of the crystal content indicated the presence of two protomers in the asymmetric unit, with a Matthews coefficient (VM) of 2.45 Å3 Da−1and 49.8% solvent content. Further study revealed that human counterpart of this protein could be used as a template to determine the first three-dimensional structure of the RK from trypanosomatid parasites.

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.

scholarly journals Structure of Human Cyclophilin A in Complex with the Novel Immunosuppressant Sanglifehrin A at 1.6 Å Resolution

2005 ◽  
Vol 280 (23) ◽  
pp. 21965-21971 ◽  
Author(s):  
Joerg Kallen ◽  
Richard Sedrani ◽  
Gerhard Zenke ◽  
Juergen Wagner
Keyword(s):  
Crystal Structure ◽  
Three Dimensional ◽  
Cyclophilin A ◽  
The Other ◽  
Size Exclusion ◽  
Dimensional Structure ◽  
The Novel ◽  
Immunosuppressive Activity ◽  
Exclusion Chromatography ◽  
Sanglifehrin A

Sanglifehrin A (SFA) is a novel immunosuppressant isolated from Streptomyces sp. that binds strongly to the human immunophilin cyclophilin A (CypA). SFA exerts its immunosuppressive activity through a mode of action different from that of all other known immunophilin-binding substances, namely cyclosporine A (CsA), FK506, and rapamycin. We have determined the crystal structure of human CypA in complex with SFA at 1.6 Å resolution. The high resolution of the structure revealed the absolute configuration at all 17 chiral centers of SFA as well as the details of the CypA/SFA interactions. In particular, it was shown that the 22-membered macrocycle of SFA is deeply embedded in the same binding site as CsA and forms six direct hydrogen bonds with CypA. The effector domain of SFA, on the other hand, has a chemical and three-dimensional structure very different from CsA, already strongly suggesting different immunosuppressive mechanisms. Furthermore, two CypA·SFA complexes form a dimer in the crystal as well as in solution as shown by light scattering and size exclusion chromatography experiments. This observation raises the possibility that the dimer of CypA·SFA complexes is the molecular species mediating the immunosuppressive effect.

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 Cloning, expression, purification, crystallization and preliminary X-ray diffraction of a lysine-specific permease fromPseudomonas aeruginosa

2014 ◽  
Vol 70 (10) ◽  
pp. 1362-1367 ◽  
Author(s):  
Emmanuel Nji ◽  
Dianfan Li ◽  
Declan A. Doyle ◽  
Martin Caffrey
Keyword(s):  
Metal Ion ◽  
Cell Volume Regulation ◽  
Ion Concentration ◽  
Size Exclusion ◽  
Substrate Selectivity ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Exclusion Chromatography

The prokaryotic lysine-specific permease (LysP) belongs to the amino acid–polyamine–organocation (APC) transporter superfamily. In the cell, members of this family are responsible for the uptake and recycling of nutrients, for the maintenance of a constant internal ion concentration and for cell volume regulation. The detailed mechanism of substrate selectivity and transport of L-lysine by LysP is not understood. A high-resolution crystal structure would enormously facilitate such an understanding. To this end, LysP fromPseudomonas aeruginosawas recombinantly expressed inEscherichia coliand purified to near homogeneity by immobilized metal ion-affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Hexagonal- and rod-shaped crystals were obtained in the presence of L-lysine and the L-lysine analogue L-4-thialysine by vapour diffusion and diffracted to 7.5 Å resolution. The diffraction data were indexed in space groupP21, with unit-cell parametersa= 169.53,b= 169.53,c= 290.13 Å, γ = 120°.

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 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.

scholarly journals Characterization of a monoclonal antibody directed against the biologically active site of human interleukin 1.

1986 ◽  
Vol 163 (2) ◽  
pp. 463-468 ◽  
Author(s):  
A Köck ◽  
M Danner ◽  
B M Stadler ◽  
T A Luger
Keyword(s):  
Monoclonal Antibody ◽  
Active Site ◽  
Biological Effects ◽  
Interleukin 1 ◽  
Biologically Active ◽  
Size Exclusion ◽  
Fibroblast Proliferation ◽  
Initial Activity ◽  
Exclusion Chromatography

Human IL-1 was successfully used to produce an anti-IL-1 mAb. Anti-IL-1 (IgG2a) blocked IL-1-mediated thymocyte and fibroblast proliferation, but did not interfere with the biological effects of other lymphokines, such as IL-2 or IL-3. The antibody immunoprecipitated biosynthetically radiolabeled 33, 17, and 4 kD IL-1. An immunoadsorbent column yielded 20% of initial activity, and upon HPLC size-exclusion chromatography, affinity-purified IL-1 had a molecular mass of approximately 4 kD. These results provide first evidence of a monoclonal anti-IL-1 that reacts with different species of IL-1 and apparently binds to an epitope close to the active site of IL-1. Thus, anti-IL-1 IgG may be very helpful for further investigations of the molecular as well as biological characteristics of IL-1 and related mediators.

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