Structure, Folding and Stability of Nucleoside Diphosphate Kinases

Nucleoside diphosphate kinases (NDPK) are oligomeric proteins involved in the synthesis of nucleoside triphosphates. Their tridimensional structure has been solved by X-ray crystallography and shows that individual subunits present a conserved ferredoxin fold of about 140 residues in prokaryotes, archaea, eukaryotes and viruses. Monomers are functionally independent from each other inside NDPK complexes and the nucleoside kinase catalytic mechanism involves transient phosphorylation of the conserved catalytic histidine. To be active, monomers must assemble into conserved head to tail dimers, which further assemble into hexamers or tetramers. The interfaces between these oligomeric states are very different but, surprisingly, the assembly structure barely affects the catalytic efficiency of the enzyme. While it has been shown that assembly into hexamers induces full formation of the catalytic site and stabilizes the complex, it is unclear why assembly into tetramers is required for function. Several additional activities have been revealed for NDPK, especially in metastasis spreading, cytoskeleton dynamics, DNA binding and membrane remodeling. However, we still lack the high resolution structural data of NDPK in complex with different partners, which is necessary for deciphering the mechanism of these diverse functions. In this review we discuss advances in the structure, folding and stability of NDPKs.

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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.

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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.

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Cu(II) and Fe(III) Complexes Derived from N-Acetylpyrazine-2-Carbohydrazide as Efficient Catalysts Towards Neat Microwave Assisted Oxidation of Alcohols

Catalysts ◽

10.3390/catal9121053 ◽

2019 ◽

Vol 9 (12) ◽

pp. 1053 ◽

Cited By ~ 7

Author(s):

Manas Sutradhar ◽

Tannistha Roy Barman ◽

Armando J. L. Pombeiro ◽

Luísa M. D. R. S. Martins

Keyword(s):

Catalytic Activity ◽

Ir Spectroscopy ◽

Maximum Yield ◽

Catalytic Efficiency ◽

Benzyl Alcohols ◽

X Ray ◽

X Ray Crystallography ◽

Microwave Assisted ◽

Tert Butyl Hydroperoxide ◽

Oxidation Of Alcohols

The mononuclear Cu(II) complex [Cu((kNN′O-HL)(H2O)2] (1) was synthesized using N-acetylpyrazine-2-carbohydrazide (H2L) and characterized by elemental analysis, IR spectroscopy, ESI-MS and single crystal X-ray crystallography. Two Fe(III) complexes derived from the same ligand viz, mononuclear [Fe((kNN′O-HL)Cl2] (2) and the binuclear [Fe(kNN′O-HL)Cl(μ-OMe)]2 (3) (synthesized as reported earlier), were also used in this study. The catalytic activity of these three complexes (1–3) was examined towards the oxidation of alcohols using tert-butyl hydroperoxide (TBHP) as oxidising agent under solvent-free microwave irradiation conditions. Primary and secondary benzyl alcohols (benzyl alcohol and 1-phenylethanol), and secondary aliphatic alcohols (cyclohexanol) were used as model substrates for this study. A comparison of their catalytic efficiency was performed. Complex 1 exhibited the highest activity in the presence of TEMPO as promoter for the oxidation of 1-phenylethanol with a maximum yield of 91.3% of acetophenone.

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Integrative Approaches in Structural Biology: A More Complete Picture from the Combination of Individual Techniques

Biomolecules ◽

10.3390/biom9080370 ◽

2019 ◽

Vol 9 (8) ◽

pp. 370 ◽

Cited By ~ 5

Author(s):

Linda Cerofolini ◽

Marco Fragai ◽

Enrico Ravera ◽

Christoph A. Diebolder ◽

Ludovic Renault ◽

...

Keyword(s):

Structural Biology ◽

Catalytic Mechanism ◽

X Ray ◽

X Ray Crystallography ◽

X Ray Scattering ◽

Protein Model ◽

Pros And Cons ◽

Transient Interactions ◽

Single Technique ◽

Nmr Restraints

With the recent technological and computational advancements, structural biology has begun to tackle more and more difficult questions, including complex biochemical pathways and transient interactions among macromolecules. This has demonstrated that, to approach the complexity of biology, one single technique is largely insufficient and unable to yield thorough answers, whereas integrated approaches have been more and more adopted with successful results. Traditional structural techniques (X-ray crystallography and Nuclear Magnetic Resonance (NMR)) and the emerging ones (cryo-electron microscopy (cryo-EM), Small Angle X-ray Scattering (SAXS)), together with molecular modeling, have pros and cons which very nicely complement one another. In this review, three examples of synergistic approaches chosen from our previous research will be revisited. The first shows how the joint use of both solution and solid-state NMR (SSNMR), X-ray crystallography, and cryo-EM is crucial to elucidate the structure of polyethylene glycol (PEG)ylated asparaginase, which would not be obtainable through any of the techniques taken alone. The second deals with the integrated use of NMR, X-ray crystallography, and SAXS in order to elucidate the catalytic mechanism of an enzyme that is based on the flexibility of the enzyme itself. The third one shows how it is possible to put together experimental data from X-ray crystallography and NMR restraints in order to refine a protein model in order to obtain a structure which simultaneously satisfies both experimental datasets and is therefore closer to the ‘real structure’.

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Assignment of the Ferriheme Resonances of the Low-Spin Complexes of Nitrophorins 1 and 4 by1H and13C NMR Spectroscopy: Comparison to Structural Data Obtained from X-ray Crystallography

Inorganic Chemistry ◽

10.1021/ic061408l ◽

2007 ◽

Vol 46 (6) ◽

pp. 2041-2056 ◽

Cited By ~ 29

Author(s):

Tatiana Kh. Shokhireva ◽

Andrzej Weichsel ◽

Kevin M. Smith ◽

Robert E. Berry ◽

Nikolai V. Shokhirev ◽

...

Keyword(s):

Nmr Spectroscopy ◽

Structural Data ◽

X Ray ◽

X Ray Crystallography

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Catalytic Mechanism of Nucleoside Diphosphate Kinase Investigated Using Nucleotide Analogues, Viscosity Effects, and X-ray Crystallography†,‡

Biochemistry ◽

10.1021/bi982990v ◽

1999 ◽

Vol 38 (22) ◽

pp. 7265-7272 ◽

Cited By ~ 45

Author(s):

Philippe Gonin ◽

Yingwu Xu ◽

Laurence Milon ◽

Sandrine Dabernat ◽

Michael Morr ◽

...

Keyword(s):

Catalytic Mechanism ◽

Nucleoside Diphosphate Kinase ◽

Nucleotide Analogues ◽

X Ray ◽

X Ray Crystallography ◽

Viscosity Effects

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New insights into the catalytic active-site structure of multicopper oxidases

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004713033051 ◽

2014 ◽

Vol 70 (3) ◽

pp. 772-779 ◽

Cited By ~ 16

Author(s):

Hirofumi Komori ◽

Ryosuke Sugiyama ◽

Kunishige Kataoka ◽

Kentaro Miyazaki ◽

Yoshiki Higuchi ◽

...

Keyword(s):

Electron Density ◽

Active Sites ◽

Catalytic Mechanism ◽

Reduction Process ◽

Multicopper Oxidases ◽

Site Structure ◽

X Ray ◽

X Ray Crystallography ◽

Catalytic Active Site ◽

Hydrated Electrons

Structural models determined by X-ray crystallography play a central role in understanding the catalytic mechanism of enzymes. However, X-ray radiation generates hydrated electrons that can cause significant damage to the active sites of metalloenzymes. In the present study, crystal structures of the multicopper oxidases (MCOs) CueO fromEscherichia coliand laccase from a metagenome were determined. Diffraction data were obtained from a single crystal under low to high X-ray dose conditions. At low levels of X-ray exposure, unambiguous electron density for an O atom was observed inside the trinuclear copper centre (TNC) in both MCOs. The gradual reduction of copper by hydrated electrons monitored by measurement of the Cu K-edge X-ray absorption spectra led to the disappearance of the electron density for the O atom. In addition, the size of the copper triangle was enlarged by a two-step shift in the location of the type III coppers owing to reduction. Further, binding of O2to the TNC after its full reduction was observed in the case of the laccase. Based on these novel structural findings, the diverse resting structures of the MCOs and their four-electron O2-reduction process are discussed.

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PLP undergoes conformational changes during the course of an enzymatic reaction

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004713031283 ◽

2014 ◽

Vol 70 (2) ◽

pp. 596-606 ◽

Cited By ~ 14

Author(s):

Ho-Phuong-Thuy Ngo ◽

Nuno M. F. S. A. Cerqueira ◽

Jin-Kwang Kim ◽

Myoung-Ki Hong ◽

Pedro Alexandrino Fernandes ◽

...

Keyword(s):

Conformational Change ◽

Conformational Changes ◽

Catalytic Mechanism ◽

Enzymatic Reaction ◽

Nucleophilic Attack ◽

The Novel ◽

X Ray ◽

X Ray Crystallography ◽

Starting Point ◽

High Level

Numerous enzymes, such as the pyridoxal 5′-phosphate (PLP)-dependent enzymes, require cofactors for their activities. Using X-ray crystallography, structural snapshots of the L-serine dehydratase catalytic reaction of a bacterial PLP-dependent enzyme were determined. In the structures, the dihedral angle between the pyridine ring and the Schiff-base linkage of PLP varied from 18° to 52°. It is proposed that the organic cofactor PLP directly catalyzes reactions by active conformational changes, and the novel catalytic mechanism involving the PLP cofactor was confirmed by high-level quantum-mechanical calculations. The conformational change was essential for nucleophilic attack of the substrate on PLP, for concerted proton transfer from the substrate to the protein and for directing carbanion formation of the substrate. Over the whole catalytic cycle, the organic cofactor catalyzes a series of reactions, like the enzyme. The conformational change of the PLP cofactor in catalysis serves as a starting point for identifying the previously unknown catalytic roles of organic cofactors.

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Expansion of Substrate Specificity and Catalytic Mechanism of Azoreductase by X-ray Crystallography and Site-directed Mutagenesis

Journal of Biological Chemistry ◽

10.1074/jbc.m710070200 ◽

2008 ◽

Vol 283 (20) ◽

pp. 13889-13896 ◽

Cited By ~ 42

Author(s):

Kosuke Ito ◽

Masayuki Nakanishi ◽

Woo-Cheol Lee ◽

Yuehua Zhi ◽

Hiroshi Sasaki ◽

...

Keyword(s):

Substrate Specificity ◽

Catalytic Mechanism ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

X Ray ◽

X Ray Crystallography

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Cocktailed fragment screening by X-ray crystallography of the antibacterial target undecaprenyl pyrophosphate synthase from Acinetobacter baumannii

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x19017199 ◽

2020 ◽

Vol 76 (1) ◽

pp. 40-46

Author(s):

James H. Thorpe ◽

Ian D. Wall ◽

Robert H. Sinnamon ◽

Amy N. Taylor ◽

Robert A. Stavenger

Keyword(s):

Drug Discovery ◽

Acinetobacter Baumannii ◽

Structural Data ◽

X Ray ◽

Traditional Medicines ◽

X Ray Crystallography ◽

Fragment Screening ◽

Binding Pockets ◽

Antibacterial Target ◽

Robustness Check

Direct soaking of protein crystals with small-molecule fragments grouped into complementary clusters is a useful technique when assessing the potential of a new crystal system to support structure-guided drug discovery. It provides a robustness check prior to any extensive crystal screening, a double check for assay binding cutoffs and structural data for binding pockets that may or may not be picked out in assay measurements. The structural output from this technique for three novel fragment molecules identified to bind to the antibacterial target Acinetobacter baumannii undecaprenyl pyrophosphate synthase are reported, and the different physicochemical requirements of a successful antibiotic are compared with traditional medicines.

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