Catalytic Mechanism of Nucleoside Diphosphate Kinase Investigated Using Nucleotide Analogues, Viscosity Effects, and 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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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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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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Crystal structure of glycosyltrehalose synthase fromSulfolobus shibataeDSM5389

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x1801453x ◽

2018 ◽

Vol 74 (11) ◽

pp. 741-746

Author(s):

Nobuo Okazaki ◽

Michael Blaber ◽

Ryota Kuroki ◽

Taro Tamada

Keyword(s):

Crystal Structure ◽

Amino Acids ◽

Catalytic Mechanism ◽

Catalytic Domain ◽

Structural Basis ◽

Hyperthermophilic Archaeon ◽

X Ray ◽

X Ray Crystallography ◽

Sulfolobus Shibatae ◽

Glucosidic Bond

Glycosyltrehalose synthase (GTSase) converts the glucosidic bond between the last two glucose residues of amylose from an α-1,4 bond to an α-1,1 bond, generating a nonreducing glycosyl trehaloside, in the first step of the biosynthesis of trehalose. To better understand the structural basis of the catalytic mechanism, the crystal structure of GTSase from the hyperthermophilic archaeonSulfolobus shibataeDSM5389 (5389-GTSase) has been determined to 2.4 Å resolution by X-ray crystallography. The structure of 5389-GTSase can be divided into five domains. The central domain contains the (β/α)8-barrel fold that is conserved as the catalytic domain in the α-amylase family. Three invariant catalytic carboxylic amino acids in the α-amylase family are also found in GTSase at positions Asp241, Glu269 and Asp460 in the catalytic domain. The shape of the catalytic cavity and the pocket size at the bottom of the cavity correspond to the intramolecular transglycosylation mechanism proposed from previous enzymatic studies.

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Structure, Folding and Stability of Nucleoside Diphosphate Kinases

International Journal of Molecular Sciences ◽

10.3390/ijms21186779 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6779

Author(s):

Florian Georgescauld ◽

Yuyu Song ◽

Alain Dautant

Keyword(s):

Catalytic Mechanism ◽

Catalytic Efficiency ◽

Structural Data ◽

Membrane Remodeling ◽

Oligomeric Proteins ◽

X Ray ◽

X Ray Crystallography ◽

Assembly Structure ◽

Nucleoside Diphosphate Kinases ◽

Cytoskeleton Dynamics

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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High Consistency of Structure-Based Design and X-Ray Crystallography: Design, Synthesis, Kinetic Evaluation and Crystallographic Binding Mode Determination of Biphenyl-N-acyl-β-d-Glucopyranosylamines as Glycogen Phosphorylase Inhibitors

Molecules ◽

10.3390/molecules24071322 ◽

2019 ◽

Vol 24 (7) ◽

pp. 1322 ◽

Cited By ~ 2

Author(s):

Thomas Fischer ◽

Symeon M. Koulas ◽

Anastasia S. Tsagkarakou ◽

Efthimios Kyriakis ◽

George A. Stravodimos ◽

...

Keyword(s):

Glycogen Phosphorylase ◽

Catalytic Mechanism ◽

Liver Glycogen ◽

Binding Mode ◽

Structural Water ◽

Design Synthesis ◽

Kinetic Evaluation ◽

X Ray ◽

X Ray Crystallography ◽

Design And Synthesis

Structure-based design and synthesis of two biphenyl-N-acyl-β-d-glucopyranosylamine derivatives as well as their assessment as inhibitors of human liver glycogen phosphorylase (hlGPa, a pharmaceutical target for type 2 diabetes) is presented. X-ray crystallography revealed the importance of structural water molecules and that the inhibitory efficacy correlates with the degree of disturbance caused by the inhibitor binding to a loop crucial for the catalytic mechanism. The in silico-derived models of the binding mode generated during the design process corresponded very well with the crystallographic data.

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