scholarly journals An unprecedented insight into the catalytic mechanism of copper nitrite reductase from atomic-resolution and damage-free structures

2021 ◽  
Vol 7 (1) ◽  
pp. eabd8523
Author(s):  
Samuel L. Rose ◽  
Svetlana V. Antonyuk ◽  
Daisuke Sasaki ◽  
Keitaro Yamashita ◽  
Kunio Hirata ◽  
...  
Keyword(s):  
Bound States ◽  
Catalytic Mechanism ◽  
Catalytic Efficiency ◽  
Atomic Resolution ◽  
Reaction Intermediates ◽  
X Ray ◽  
Nitrite Reductases ◽  
Radiation Induced ◽  
Insight Into ◽  
Shed Light

Copper-containing nitrite reductases (CuNiRs), encoded by nirK gene, are found in all kingdoms of life with only 5% of CuNiR denitrifiers having two or more copies of nirK. Recently, we have identified two copies of nirK genes in several α-proteobacteria of the order Rhizobiales including Bradyrhizobium sp. ORS 375, encoding a four-domain heme-CuNiR and the usual two-domain CuNiR (Br2DNiR). Compared with two of the best-studied two-domain CuNiRs represented by the blue (AxNiR) and green (AcNiR) subclasses, Br2DNiR, a blue CuNiR, shows a substantially lower catalytic efficiency despite a sequence identity of ~70%. Advanced synchrotron radiation and x-ray free-electron laser are used to obtain the most accurate (atomic resolution with unrestrained SHELX refinement) and damage-free (free from radiation-induced chemistry) structures, in as-isolated, substrate-bound, and product-bound states. This combination has shed light on the protonation states of essential catalytic residues, additional reaction intermediates, and how catalytic efficiency is modulated.

Copper–oxygen Dynamics in Tyrosinase

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 (Cu<sub>2</sub>O<sub>2</sub>core) 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.<br>

scholarly journals Copper–oxygen Dynamics in Tyrosinase

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 (Cu<sub>2</sub>O<sub>2</sub>core) 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.<br>

scholarly journals Structure based protein engineering to confer selectivity of guanine deaminase

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.

scholarly journals Structure, Folding and Stability of Nucleoside Diphosphate Kinases

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.

Structural study of fluoro-tetraphenylporphyrins relating to large variability in solubilities of the para-F-TPP, ortho-F-TPP and meta-F-TPP

2018 ◽  
Vol 22 (04) ◽  
pp. 355-358 ◽  
Author(s):  
Ruxian Han ◽  
Scott Kim ◽  
Kenneth J. Janda ◽  
Everly B. Fleischer
Keyword(s):  
Structural Study ◽  
Large Variability ◽  
X Ray ◽  
Temperature Factors ◽  
Differential Solubility ◽  
Insight Into ◽  
Shed Light

We present the X-ray structures of the crystals of the ortho-, meta-, and para-F-tetraphenylporphyrins crystallized from different solvents to shed light on the differential solubility of these three isomers. The meta-F-TPP is 4300 times more soluble than the para-F-TPP isomer in chloroform. It is shown that the packing of the porphyrins in the crystal is not a key issue in this solubility difference. However, we discovered a large difference in the temperature factors of the phenyls and Fs in the various isomers and this gives us insight into the solubility differences.

scholarly journals Structural studies on dehydroshikimate dehydratase

2014 ◽  
Vol 70 (a1) ◽  
pp. C475-C475
Author(s):  
James Peek ◽  
Dinesh Christendat
Keyword(s):  
Sole Carbon Source ◽  
Catalytic Mechanism ◽  
Substrate Recognition ◽  
Structural Studies ◽  
Sugar Phosphate ◽  
X Ray ◽  
Structural Insight ◽  
Functional Relevance ◽  
Insight Into

The soil bacterium, Pseudomonas putida, is capable of using the alicyclic compound quinate as a sole carbon source. During this process, quinate is converted to 3-dehydroshikimate, which subsequently undergoes a dehydration to form protocatechuate. The latter transformation is performed by the enzyme dehydroshikimate dehydratase (DSD). We have recombinantly produced DSD from P. putida and are currently performing x-ray crystallographic studies on the enzyme to gain structural insight into its catalytic mechanism and mode of substrate recognition. Initial crystals of DSD diffracted to 2.7 Ä resolution, but exhibited strong twinning. A redesigned construct has recently yielded crystals that diffract to similar resolution, but with a significantly reduced tendency toward twinning. Interestingly, sequence analysis of P. putida DSD reveals that the protein is in fact a fusion of two distinct domains: an N-terminal sugar phosphate isomerase-like domain associated with DSD activity, and a C-terminal hydroxyphenylpyruvate dioxygenase (HPPD)-like domain with unknown functional significance. Structural characterization of the protein may provide novel insight into the functional relevance of the unusual HPPD-like domain.

scholarly journals Crystallization and Preliminary X-Ray Analysis of Rotavirus Protein VP6

1998 ◽  
Vol 72 (9) ◽  
pp. 7615-7619 ◽  
Author(s):  
Isabelle Petitpas ◽  
Jean Lepault ◽  
Patrice Vachette ◽  
Annie Charpilienne ◽  
Magali Mathieu ◽  
...  
Keyword(s):  
Atomic Resolution ◽  
Asymmetric Unit ◽  
Tubular Structures ◽  
Solvent Content ◽  
X Ray ◽  
X Ray Scattering ◽  
A Cell ◽  
Crystallization Conditions ◽  
Insight Into ◽  
Better Than

ABSTRACT As a first step to gain insight into the structure of the rotavirus virion at atomic resolution, we report here the expression, purification, and crystallization of recombinant rotavirus protein VP6. This protein has the property of polymerizing in the form of tubular structures in solution which have hindered crystallization thus far. Using a combination of electron microscopy and small-angle X-ray scattering, we found that addition of Ca2+ at concentrations higher than 100 mM results in depolymerization of the tubes, leading to an essentially monodisperse solution of trimeric VP6 even at high protein concentrations (higher than 10 mg/ml), thereby enabling us to search for crystallization conditions. We have thus obtained crystals of VP6 which diffract to better than 2.4 Å resolution and belong to the cubic space group P4132 with a cell dimension a of 160 Å. The crystals contain a trimer of VP6 lying along the diagonal of the cubic unit cell, resulting in one VP6 monomer per asymmetric unit and a solvent content of roughly 70%.

Theoretical insight into the disordered structure of (Z)-2-[(E)-(4-methoxybenzylidene)hydrazinylidene]-1,2-diphenylethanone: the role of noncovalent interactions

2018 ◽  
Vol 74 (9) ◽  
pp. 1058-1067
Author(s):  
Xue-Jie Tan ◽  
Di Wang ◽  
Xu-Gang Lei ◽  
Jun-Peng Chen
Keyword(s):  
Room Temperature ◽  
Noncovalent Interactions ◽  
Poor Quality ◽  
X Ray ◽  
Disordered Structure ◽  
Theoretical Insight ◽  
Insight Into ◽  
Shed Light

A global glide disorder has been discovered during an X-ray investigation of the crystal structure of (Z)-2-[(E)-(4-methoxybenzylidene)hydrazinylidene]-1,2-diphenylethanone (MHDE, C22H18N2O2) at room temperature. In another crystal, however, such disorder disappears (still at room temperature). Even though the disorder may be partly due to the poor quality of the harvested crystal, the structure can shed light on the nature of disorder. With the help of quantum chemical calculations, it is found that the global disorder seems to be connected with the need for stabilization of the somewhat rigid but mobile and unstable molecular structure. The most relevant feature driving the packing of the disordered structure concerns the slight perturbations (such as glide) of two or more disorder components (fractional occupancies) distributed throughout the crystal.

The catalytic mechanism of Pseudomonas aeruginosa cd1 nitrite reductase

2011 ◽  
Vol 39 (1) ◽  
pp. 195-200 ◽  
Author(s):  
Serena Rinaldo ◽  
Giorgio Giardina ◽  
Nicoletta Castiglione ◽  
Valentina Stelitano ◽  
Francesca Cutruzzolà
Keyword(s):  
Nitric Oxide ◽  
Pseudomonas Aeruginosa ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Product Inhibition ◽  
Electron Donors ◽  
Nitrite Reduction ◽  
Nitrite Reductases ◽  
Shed Light ◽  
Denitrification Process

The cd1 NiRs (nitrite reductases) are enzymes catalysing the reduction of nitrite to NO (nitric oxide) in the bacterial energy conversion denitrification process. These enzymes contain two distinct redox centres: one covalently bound c-haem, which is reduced by external electron donors, and another peculiar porphyrin, the d1-haem (3,8-dioxo-17-acrylate-porphyrindione), where nitrite is reduced to NO. In the present paper, we summarize the most recent results on the mechanism of nitrite reduction by the cd1 NiR from Pseudomonas aeruginosa. We discuss the essential catalytic features of this enzyme, with special attention to the allosteric regulation of the enzyme's activity and to the mechanism employed to avoid product inhibition, i.e. trapping of the active-site reduced haem by the product NO. These results shed light on the reactivity of cd1 NiRs and assign a central role to the unique d1-haem, present only in this class of enzymes.

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