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

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.

Download Full-text

Structure based protein engineering to confer selectivity of guanine deaminase

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s205327331409562x ◽

2014 ◽

Vol 70 (a1) ◽

pp. C437-C437

Author(s):

Aruna Bitra ◽

Ruchi Anand

Keyword(s):

Crystal Structures ◽

Active Site ◽

Catalytic Mechanism ◽

Catalytic Efficiency ◽

Structural Basis ◽

Active Site Residues ◽

X Ray ◽

Secondary Activity ◽

Guanine Deaminase ◽

Differential Selectivity

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.

Download Full-text

Faculty Opinions recommendation of Oxygen pressurized X-ray crystallography: probing the dioxygen binding site in cofactorless urate oxidase and implications for its catalytic mechanism.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1164733.626543 ◽

2009 ◽

Author(s):

Andrea Mattevi

Keyword(s):

Binding Site ◽

Catalytic Mechanism ◽

Urate Oxidase ◽

X Ray ◽

X Ray Crystallography

Download Full-text

Oxygen Pressurized X-Ray Crystallography: Probing the Dioxygen Binding Site in Cofactorless Urate Oxidase and Implications for Its Catalytic Mechanism

Biophysical Journal ◽

10.1529/biophysj.107.122184 ◽

2008 ◽

Vol 95 (5) ◽

pp. 2415-2422 ◽

Cited By ~ 41

Author(s):

Nathalie Colloc’h ◽

Laure Gabison ◽

Gérald Monard ◽

Muhannad Altarsha ◽

Mohamed Chiadmi ◽

...

Keyword(s):

Binding Site ◽

Catalytic Mechanism ◽

Urate Oxidase ◽

X Ray ◽

X Ray Crystallography

Download Full-text

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

Philosophical Transactions of the Royal Society of London Series A Physical and Engineering Sciences ◽

10.1098/rsta.1992.0069 ◽

1992 ◽

Vol 340 (1657) ◽

pp. 311-321 ◽

Cited By ~ 5

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.

Download Full-text

Copper–oxygen Dynamics in Tyrosinase

10.26434/chemrxiv.9255251 ◽

2019 ◽

Author(s):

Nobutaka Fujieda ◽

Sachiko Yanagisawa ◽

Minoru Kubo ◽

Genji Kurisu ◽

Shinobu Itoh

Keyword(s):

Catalytic Mechanism ◽

Substrate Binding ◽

Active Form ◽

Copper Ion ◽

Atomic Resolution ◽

Oxygen Species ◽

X Ray ◽

Oxygen Dynamics ◽

Insight Into ◽

Copper Centre

To unveil the activation of dioxygen on the copper centre (Cu2O2core) of tyrosinase, we performed X-ray crystallograpy with active-form tyrosinase at near atomic resolution. This study provided a novel insight into the catalytic mechanism of the tyrosinase, including the rearrangement of copper-oxygen species as well as the intramolecular migration of copper ion induced by substrate-binding.

Download Full-text

Enhancement of Thermostability of Aspergillus flavus Urate Oxidase by Immobilization on the Ni-Based Magnetic Metal–Organic Framework

Nanomaterials ◽

10.3390/nano11071759 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1759

Author(s):

Neda Motamedi ◽

Mahmood Barani ◽

Azadeh Lohrasbi-Nejad ◽

Mojtaba Mortazavi ◽

Ali Riahi-Medvar ◽

...

Keyword(s):

Aspergillus Flavus ◽

Metal Organic Framework ◽

Urate Oxidase ◽

Significant Activity ◽

Original Activity ◽

Long Term Stability ◽

Magnetic Metal ◽

Metal Organic ◽

Almost All ◽

Organic Framework

The improvement in the enzyme activity of Aspergillus flavus urate oxidase (Uox) was attained by immobilizing it on the surface of a Ni-based magnetic metal–organic framework (NimMOF) nanomaterial; physicochemical properties of NimMOF and its application as an enzyme stabilizing support were evaluated, which revealed a significant improvement in its stability upon immobilization on NimMOF (Uox@NimMOF). It was affirmed that while the free Uox enzyme lost almost all of its activity at ~40–45 °C, the immobilized Uox@NimMOF retained around 60% of its original activity, even retaining significant activity at 70 °C. The activation energy (Ea) of the enzyme was calculated to be ~58.81 kJ mol−1 after stabilization, which is approximately half of the naked Uox enzyme. Furthermore, the external spectroscopy showed that the MOF nanomaterials can be coated by hydrophobic areas of the Uox enzyme, and the immobilized enzyme was active over a broad range of pH and temperatures, which bodes well for the thermal and long-term stability of the immobilized Uox on NimMOF.

Download Full-text

Discovery of fungal surface NADases predominantly present in pathogenic species

Nature Communications ◽

10.1038/s41467-021-21307-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Øyvind Strømland ◽

Juha P. Kallio ◽

Annica Pschibul ◽

Renate H. Skoge ◽

Hulda M. Harðardóttir ◽

...

Keyword(s):

Cell Death ◽

Aspergillus Fumigatus ◽

Neurospora Crassa ◽

Binding Site ◽

Host Cell ◽

Nicotinamide Adenine Dinucleotide ◽

Catalytic Mechanism ◽

Pathogenic Species ◽

X Ray ◽

Cellular Bioenergetics

AbstractNicotinamide adenine dinucleotide (NAD) is a key molecule in cellular bioenergetics and signalling. Various bacterial pathogens release NADase enzymes into the host cell that deplete the host’s NAD+ pool, thereby causing rapid cell death. Here, we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa. The enzymes harbour a tuberculosis necrotizing toxin (TNT) domain and are predominately present in pathogenic species. The 1.6 Å X-ray structure of the homodimeric A. fumigatus protein reveals unique properties including N-linked glycosylation and a Ca2+-binding site whose occupancy regulates activity. The structure in complex with a substrate analogue suggests a catalytic mechanism that is distinct from those of known NADases, ADP-ribosyl cyclases and transferases. We propose that fungal NADases may convey advantages during interaction with the host or competing microorganisms.

Download Full-text

Design of a novel Peltier-based cooling device and its use in neutron diffraction data collection of perdeuterated yeast pyrophosphatase

Journal of Applied Crystallography ◽

10.1107/s0021889810027111 ◽

2010 ◽

Vol 43 (5) ◽

pp. 1113-1120 ◽

Cited By ~ 1

Author(s):

Esko Oksanen ◽

François Dauvergne ◽

Adrian Goldman ◽

Monika Budayova-Spano

Keyword(s):

Data Collection ◽

Neutron Diffraction ◽

Diffraction Data ◽

Catalytic Mechanism ◽

Inorganic Pyrophosphatase ◽

Neutron Diffraction Data ◽

Cooling Device ◽

Data Set ◽

X Ray ◽

X Ray Crystallography

H atoms play a central role in enzymatic mechanisms, but H-atom positions cannot generally be determined by X-ray crystallography. Neutron crystallography, on the other hand, can be used to determine H-atom positions but it is experimentally very challenging. Yeast inorganic pyrophosphatase (PPase) is an essential enzyme that has been studied extensively by X-ray crystallography, yet the details of the catalytic mechanism remain incompletely understood. The temperature instability of PPase crystals has in the past prevented the collection of a neutron diffraction data set. This paper reports how the crystal growth has been optimized in temperature-controlled conditions. To stabilize the crystals during neutron data collection a Peltier cooling device that minimizes the temperature gradient along the capillary has been developed. This device allowed the collection of a full neutron diffraction data set.

Download Full-text

A large-solid-angle X-ray Raman scattering spectrometer at ID20 of the European Synchrotron Radiation Facility

Journal of Synchrotron Radiation ◽

10.1107/s1600577516020579 ◽

2017 ◽

Vol 24 (2) ◽

pp. 521-530 ◽

Cited By ~ 46

Author(s):

S. Huotari ◽

Ch. J. Sahle ◽

Ch. Henriquet ◽

A. Al-Zein ◽

K. Martel ◽

...

Keyword(s):

Synchrotron Radiation ◽

Raman Scattering ◽

Solid Angle ◽

European Synchrotron Radiation Facility ◽

Electronic Excitations ◽

Raman Scattering Spectroscopy ◽

X Ray ◽

Area Detector ◽

Spectroscopic Tool

An end-station for X-ray Raman scattering spectroscopy at beamline ID20 of the European Synchrotron Radiation Facility is described. This end-station is dedicated to the study of shallow core electronic excitations using non-resonant inelastic X-ray scattering. The spectrometer has 72 spherically bent analyzer crystals arranged in six modular groups of 12 analyzer crystals each for a combined maximum flexibility and large solid angle of detection. Each of the six analyzer modules houses one pixelated area detector allowing for X-ray Raman scattering based imaging and efficient separation of the desired signal from the sample and spurious scattering from the often used complicated sample environments. This new end-station provides an unprecedented instrument for X-ray Raman scattering, which is a spectroscopic tool of great interest for the study of low-energy X-ray absorption spectra in materials under in situ conditions, such as in operando batteries and fuel cells, in situ catalytic reactions, and extreme pressure and temperature conditions.

Download Full-text