Complex structures of MoeN5 with substrate analogues suggest sequential catalytic mechanism

2019 ◽  
Vol 511 (4) ◽  
pp. 800-805 ◽  
Author(s):  
Lilan Zhang ◽  
Tzu-Ping Ko ◽  
Satish R. Malwal ◽  
Weidong Liu ◽  
Shuyu Zhou ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Complex Structures ◽  
Substrate Analogues

scholarly journals Large crystal growth by thermal control allows combined X-ray and neutron crystallographic studies to elucidate the protonation states in Aspergillus flavus urate oxidase

2009 ◽  
Vol 6 (suppl_5) ◽  
Author(s):  
E. Oksanen ◽  
M. P. Blakeley ◽  
F. Bonneté ◽  
M. T. Dauvergne ◽  
F. Dauvergne ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Catalytic Mechanism ◽  
Direct Determination ◽  
Thermal Control ◽  
European Synchrotron Radiation Facility ◽  
Urate Oxidase ◽  
Nuclear Scattering ◽  
Active Site Residues ◽  
X Ray ◽  
Substrate Analogues

Urate oxidase (Uox) catalyses the oxidation of urate to allantoin and is used to reduce toxic urate accumulation during chemotherapy. X-ray structures of Uox with various inhibitors have been determined and yet the detailed catalytic mechanism remains unclear. Neutron crystallography can provide complementary information to that from X-ray studies and allows direct determination of the protonation states of the active-site residues and substrate analogues, provided that large, well-ordered deuterated crystals can be grown. Here, we describe a method and apparatus used to grow large crystals of Uox ( Aspergillus flavus ) with its substrate analogues 8-azaxanthine and 9-methyl urate, and with the natural substrate urate, in the presence and absence of cyanide. High-resolution X-ray (1.05–1.20 Å) and neutron diffraction data (1.9–2.5 Å) have been collected for the Uox complexes at the European Synchrotron Radiation Facility and the Institut Laue-Langevin, respectively. In addition, room temperature X-ray data were also collected in preparation for joint X-ray and neutron refinement. Preliminary results indicate no major structural differences between crystals grown in H 2 O and D 2 O even though the crystallization process is affected. Moreover, initial nuclear scattering density maps reveal the proton positions clearly, eventually providing important information towards unravelling the mechanism of catalysis.

scholarly journals The structure of a complex of the lactonohydrolase zearalenone hydrolase with the hydrolysis product of zearalenone at 1.60 Å resolution

2017 ◽  
Vol 73 (7) ◽  
pp. 376-381 ◽  
Author(s):  
Qi Qi ◽  
Wen-Jing Yang ◽  
Hu-Jian Zhou ◽  
Deng-Ming Ming ◽  
Kai-Lei Sun ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Bond Cleavage ◽  
Hydrolysis Product ◽  
Complex Structures ◽  
Substrate Complex ◽  
Translational Movement ◽  
Enzyme Substrate ◽  
Naturally Occurring ◽  
Phenol Ring ◽  
Resolution Structure

Zearalenone hydrolase (ZHD) is an α/β-hydrolase that detoxifies and degrades the lactone zearalenone (ZEN), a naturally occurring oestrogenic mycotoxin that contaminates crops. Several apoenzyme and enzyme–substrate complex structures have been reported in the resolution range 2.4–2.6 Å. However, the properties and mechanism of this enzyme are not yet fully understood. Here, a 1.60 Å resolution structure of a ZHD–product complex is reported which was determined from a C-terminally His6-tagged ZHD crystal soaked with 2 mMZEN for 30 min. It shows that after the lactone-bond cleavage, the phenol-ring region moves closer to residues Leu132, Tyr187 and Pro188, while the lactone-ring region barely moves. Comparisons of the ZHD–substrate and ZHD–product structures show that the hydrophilic interactions change, especially Trp183 N∊1, which shifts from contacting O2 to O12′, suggesting that Trp183 is responsible for the unidirectional translational movement of the phenol ring. This structure provides information on the final stage of the catalytic mechanism of zearalenone hydrolysis.

scholarly journals Probing the Catalytic Mechanism ofS-Ribosylhomocysteinase (LuxS) with Catalytic Intermediates and Substrate Analogues

2009 ◽  
Vol 131 (3) ◽  
pp. 1243-1250 ◽  
Author(s):  
Bhaskar Gopishetty ◽  
Jinge Zhu ◽  
Rakhi Rajan ◽  
Adam J. Sobczak ◽  
Stanislaw F. Wnuk ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Substrate Analogues ◽  
Catalytic Intermediates

scholarly journals Substrate analogues as probes of the catalytic mechanism of l-mandelate dehydrogenase from Rhodotorula graminis

1994 ◽  
Vol 297 (3) ◽  
pp. 647-652 ◽  
Author(s):  
O Smékal ◽  
G A Reid ◽  
S K Chapman
Keyword(s):  
Transition State ◽  
Catalytic Mechanism ◽  
Activation Parameters ◽  
Linear Free Energy Relationship ◽  
Free Energies ◽  
Linear Free Energy ◽  
Kinetic Isotope ◽  
Substrate Analogues ◽  
Reaction Constant ◽  
Delta G

A detailed kinetic analysis of the oxidation of mono-substituted mandelates catalysed by L-(+)-mandelate dehydrogenase (L-MDH) from Rhodotorula graminis has been carried out to elucidate the role of the substrate in the catalytic mechanism. Values of Km and kcat. (25 degrees C, pH 7.5) were determined for mandelate and eight substrate analogues. Values of the activation parameters, delta H++ and delta S++ (determined over the range 5-37 degrees C), for mandelate and all substrate analogues were compensatory resulting in similar low values for free energies of activation delta G++ (approx. 60 kJ.mol-1 at 298.15 K) in all cases. A kinetic-isotope-effect value of 1.1 +/- 0.1 was observed using D,L-[2-2H]mandelate as substrate and was invariant over the temperature range studied. The logarithm of kcat. values for the enzymic oxidation of mandelate and all substrate analogues (except 4-hydroxymandelate) showed good correlation with Taft's dual substituent constant omega (where omega = omega I + 0.64 omega +R) and gave a positive reaction constant value, rho, of 0.36 +/- 0.07. This linear free-energy relationship was verified by analysing the data using isokinetic methods. These findings support the hypothesis that the enzyme-catalysed reaction proceeds via the same transition state for each substrate and indicates that this transition state is relatively nonpolar but has an electron-rich centre at the alpha-carbon position.

Conventional X-ray diffraction approaches to the study of enzyme mechanism: serine proteinases, aminoacyl-tRNA synthetases and xylose isomerase

1992 ◽  
Vol 340 (1657) ◽  
pp. 311-321 ◽  
Keyword(s):  
Catalytic Mechanism ◽  
Xylose Isomerase ◽  
Enzyme Mechanism ◽  
X Ray Diffraction ◽  
Molecular Mechanics Calculations ◽  
Time Resolved ◽  
X Ray ◽  
Trna Synthetases ◽  
Substrate Analogues ◽  
Aminoacyl Trna Synthetases

Techniques that have been used to study enzyme mechanism by conventional steady-state crystallographic techniques are reviewed. Substrates and substrate analogues can often be diffused into crystals, but occasionally co-crystallization is necessary. The poor solubility of substrates and inhibitors may pose a problem. Even if a substrate is present at adequate concentration, it may not be observed by X -ray diffraction. To observe a substrate, special measures may be needed to stop enzyme action, but sometimes this is not necessary because an equilibrium is established. Inhibitors may usefully model a particular reaction state, but one must always question whether the inhibitor provides a correct model. Stabilization of a transition state is often discussed, but rarely achieved. Where practicable, protein engineering can provide a powerful tool to test proposals about the catalytic mechanism. Molecular mechanics calculations can also be useful. These themes are developed in relation to enzymes studied in the authors’ laboratory. Many of the same problems are encountered in the application of time-resolved techniques to the study of enzyme mechanism.

Cell morphology, volume, and surface area determinations from multilevel contouring within HVEM micrographs of serial sections and whole-cell mounts

1987 ◽  
Vol 45 ◽  
pp. 588-589
Author(s):  
M. Marko ◽  
A. Leith ◽  
D. Parsons
Keyword(s):  
Surface Area ◽  
Electron Micrographs ◽  
Cell Morphology ◽  
Individual Variation ◽  
Serial Section ◽  
Stereo Pair ◽  
Complex Structures ◽  
Serial Sections ◽  
Surface Areas ◽  
Computer Based

The use of serial sections and computer-based 3-D reconstruction techniques affords an opportunity not only to visualize the shape and distribution of the structures being studied, but also to determine their volumes and surface areas. Up until now, this has been done using serial ultrathin sections.The serial-section approach differs from the stereo logical methods of Weibel in that it is based on the Information from a set of single, complete cells (or organelles) rather than on a random 2-dimensional sampling of a population of cells. Because of this, it can more easily provide absolute values of volume and surface area, especially for highly-complex structures. It also allows study of individual variation among the cells, and study of structures which occur only infrequently.We have developed a system for 3-D reconstruction of objects from stereo-pair electron micrographs of thick specimens.

Stereo modeling of HVEM specimens by serial tilting and computer graphics

1986 ◽  
Vol 44 ◽  
pp. 34-35
Author(s):  
J.R. McIntosh ◽  
D.L. Stemple ◽  
William Bishop ◽  
G.W. Hannaway
Keyword(s):  
Computer Graphics ◽  
Analytical Methods ◽  
Photographic Film ◽  
Complex Structures ◽  
Multiple Objects ◽  
Multiple Images ◽  
3 Dimensional

EM specimens often contain 3-dimensional information that is lost during micrography on a single photographic film. Two images of one specimen at appropriate orientations give a stereo view, but complex structures composed of multiple objects of graded density that superimpose in each projection are often difficult to decipher in stereo. Several analytical methods for 3-D reconstruction from multiple images of a serially tilted specimen are available, but they are all time-consuming and computationally intense.

EXELFS Studies of Carbon Fibers

1990 ◽  
Vol 48 (2) ◽  
pp. 72-73
Author(s):  
V. Serin ◽  
K. Hssein ◽  
G. Zanchi ◽  
J. Sévely
Keyword(s):  
Carbon Fibers ◽  
Energy Analysis ◽  
Electron Excitation ◽  
Complex Structures ◽  
High Modulus ◽  
Promising Technique ◽  
X Ray ◽  
Fine Structures ◽  
X Ray Absorption

The present developments of electron energy analysis in the microscopes by E.E.L.S. allow an accurate recording of the spectra and of their different complex structures associated with the inner shell electron excitation by the incident electrons (1). Among these structures, the Extended Energy Loss Fine Structures (EXELFS) are of particular interest. They are equivalent to the well known EXAFS oscillations in X-ray absorption spectroscopy. Due to the EELS characteristic, the Fourier analysis of EXELFS oscillations appears as a promising technique for the characterization of composite materials, the major constituents of which are low Z elements. Using EXELFS, we have developed a microstructural study of carbon fibers. This analysis concerns the carbon K edge, which appears in the spectra at 285 eV. The purpose of the paper is to compare the local short range order, determined by this way in the case of Courtauld HTS and P100 ex-polyacrylonitrile carbon fibers, which are high tensile strength (HTS) and high modulus (HM) fibers respectively.

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