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1.
Acta Crystallogr D Struct Biol ; 78(Pt 8): 975-985, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35916222

RESUMO

Fixed-target serial crystallography allows the high-throughput collection of diffraction data from small crystals at room temperature. This methodology is particularly useful for difficult samples that have sensitivity to radiation damage or intolerance to cryoprotection measures; fixed-target methods also have the added benefit of low sample consumption. Here, this method is applied to the structure determination of the circadian photoreceptor cryptochrome (CRY), previous structures of which have been determined at cryogenic temperature. In determining the structure, several data-filtering strategies were tested for combining observations from the hundreds of crystals that contributed to the final data set. Removing data sets based on the average correlation coefficient among equivalent reflection intensities between a given data set and all others was most effective at improving the data quality and maintaining overall completeness. CRYs are light sensors that undergo conformational photoactivation. Comparisons between the cryogenic and room-temperature CRY structures reveal regions of enhanced mobility at room temperature in loops that have functional importance within the CRY family of proteins. The B factors of the room-temperature structure correlate well with those predicted from molecular-dynamics simulations.


Assuntos
Criptocromos , Drosophila , Animais , Criptocromos/metabolismo , Cristalografia , Cristalografia por Raios X , Drosophila/metabolismo , Síncrotrons , Temperatura
2.
Acta Crystallogr D Struct Biol ; 78(Pt 8): 986-996, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35916223

RESUMO

Single-wavelength anomalous diffraction (SAD) is a routine method for overcoming the phase problem when solving macromolecular structures. This technique requires the accurate measurement of intensities to determine differences between Bijvoet pairs. Although SAD experiments are commonly conducted at cryogenic temperatures to mitigate the effects of radiation damage, such temperatures can alter the conformational ensemble of the protein and may impede the merging of data from multiple crystals due to non-uniform freezing. Here, a strategy is presented to obtain high-quality data from room-temperature, single-crystal experiments. To illustrate the strengths of this approach, native SAD phasing at 6.55 keV was used to solve four structures of three model systems at 295 K. The resulting data sets allow automatic phasing and model building, and reveal alternate conformations that reflect the structure of proteins at room temperature.


Assuntos
Proteínas , Cristalização/métodos , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Proteínas/química , Temperatura
3.
Acta Crystallogr D Struct Biol ; 78(Pt 8): 997-1009, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35916224

RESUMO

Protein crystals grown in microfluidic droplets have been shown to be an effective and robust platform for storage, transport and serial crystallography data collection with a minimal impact on diffraction quality. Single macromolecular microcrystals grown in nanolitre-sized droplets allow the very efficient use of protein samples and can produce large quantities of high-quality samples for data collection. However, there are challenges not only in growing crystals in microfluidic droplets, but also in delivering the droplets into X-ray beams, including the physical arrangement, beamline and timing constraints and ease of use. Here, the crystallization of two human gut microbial hydrolases in microfluidic droplets is described: a sample-transport and data-collection approach that is inexpensive, is convenient, requires small amounts of protein and is forgiving. It is shown that crystals can be grown in 50-500 pl droplets when the crystallization conditions are compatible with the droplet environment. Local and remote data-collection methods are described and it is shown that crystals grown in microfluidics droplets and housed as an emulsion in an Eppendorf tube can be shipped from the US to the UK using a FedEx envelope, and data can be collected successfully. Details of how crystals were delivered to the X-ray beam by depositing an emulsion of droplets onto a silicon fixed-target serial device are provided. After three months of storage at 4°C, the crystals endured and diffracted well, showing only a slight decrease in diffracting power, demonstrating a suitable way to grow crystals, and to store and collect the droplets with crystals for data collection. This sample-delivery and data-collection strategy allows crystal droplets to be shipped and set aside until beamtime is available.


Assuntos
Microfluídica , Proteínas , Cristalização , Cristalografia por Raios X , Coleta de Dados , Emulsões , Humanos
4.
Acta Crystallogr D Struct Biol ; 78(Pt 8): 1010-1020, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35916225

RESUMO

The availability of atomic resolution experimental maps of electrostatic potential from 3D electron diffraction (3D ED) extends the possibility of investigating the electrostatic potential beyond the determination of non-H-atom positions. However, accurate tools to calculate this potential for macromolecules, without the use of expensive quantum calculations, are lacking. The University at Buffalo Data Bank (UBDB) gathers atom types that can be used to calculate accurate electrostatic potential maps via structure-factor calculations. Here, the transferable aspherical atom model (TAAM) is applied with UBDB to investigate theoretically obtained electrostatic potential maps of lysozyme and proteinase K, and compare them with experimental maps from 3D ED. UBDB better reproduces the molecular electrostatic potential of molecules within their entire volume compared with the neutral spherical models used in the popular independent atom model (IAM). Additionally, the theoretical electron-density maps of the studied proteins are shown and compared with the electrostatic potential maps. The atomic displacement parameters (B factors) may affect the electrostatic potential maps in a different way than in the case of electron-density maps. The computational method presented in this study could potentially facilitate the interpretation of the less resolved regions of cryo-electron microscopy density maps and pave the way for distinguishing between different ions/water molecules in the active sites of macromolecules in high-resolution structures, which is of interest for drug-design purposes.


Assuntos
Elétrons , Proteínas , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Substâncias Macromoleculares , Proteínas/química , Eletricidade Estática
5.
Acta Crystallogr D Struct Biol ; 78(Pt 8): 1021-1031, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35916226

RESUMO

The application of sulfur single-wavelength anomalous dispersion (S-SAD) to determine the crystal structures of macromolecules can be challenging if the asymmetric unit is large, the crystals are small, the size of the anomalously scattering sulfur structure is large and the resolution at which the anomalous signals can be accurately measured is modest. Here, as a study of such a case, approaches to the SAD phasing of orthorhombic Ric-8A crystals are described. The structure of Ric-8A was published with only a brief description of the phasing process [Zeng et al. (2019), Structure, 27, 1137-1141]. Here, alternative approaches to determining the 40-atom sulfur substructure of the 103 kDa Ric-8A dimer that composes the asymmetric unit are explored. At the data-collection wavelength of 1.77 Šmeasured at the Frontier micro-focusing Macromolecular Crystallography (FMX) beamline at National Synchrotron Light Source II, the sulfur anomalous signal strength, |Δano|/σΔano (d''/sig), approaches 1.4 at 3.4 Šresolution. The highly redundant, 11 000 000-reflection data set measured from 18 crystals was segmented into isomorphous clusters using BLEND in the CCP4 program suite. Data sets within clusters or sets of clusters were scaled and merged using AIMLESS from CCP4 or, alternatively, the phenix.scale_and_merge tool from the Phenix suite. The latter proved to be the more effective in extracting anomalous signals. The HySS tool in Phenix, SHELXC/D and PRASA as implemented in the CRANK2 program suite were each employed to determine the sulfur substructure. All of these approaches were effective, although HySS, as a component of the phenix.autosol tool, required data from all crystals to find the positions of the sulfur atoms. Critical contributors in this case study to successful phase determination by SAD included (i) the high-flux FMX beamline, featuring helical-mode data collection and a helium-filled beam path, (ii) as recognized by many authors, a very highly redundant, multiple-crystal data set and (iii) the inclusion within that data set of data from crystals that were scanned over large ω ranges, yielding highly isomorphous and highly redundant intensity measurements.


Assuntos
Enxofre , Síncrotrons , Cristalografia por Raios X , Conformação Proteica , Enxofre/química
6.
Acta Crystallogr C Struct Chem ; 78(Pt 8): 437-448, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35924362

RESUMO

Creatinine, a biologically important compound, is used to analyze kidney function and kidney diseases in the human body. The salt form of creatinine is used in the formation of drug materials like anti-HIV, antifungal, antiprotozoal, antiviral and antitumour compounds. Here we report the solid-state structures of three new crystalline salts, namely, creatininium (2-amino-1-methyl-4-oxo-4,5-dihydro-1H-imidazol-3-ium) bromide, C4H8N3O+·Br-, (I), creatininium 3-aminobenzoate, C4H8N3O+·C7H6NO2-, (II), and creatininium 3,5-dinitrobenzoate, C4H8N3O+·C7H3N2O6-, (III). These salts have been synthesized and characterized by single-crystal X-ray diffraction and Hirshfeld surface analysis. The structural chemistry of salts (I)-(III) and their crystal packing are discussed in detail. The primary interaction between the creatinine cation and the acid anion in the three salts is N-H...Br/O hydrogen bonds. In salt (I), the creatinine cation and bromide anion are connected through a pair of N-H...Br hydrogen bonds forming R42(8) and R42(12) ring motifs. In salts (II) and (III), the creatinine cation interacts with the corresponding anion via a pair of N-H...O hydrogen bonds. The crystal structure is further stabilized by C-H...O and O-H...O hydrogen bonds with the ring motifs R22(8), R21(7) and R21(6). Furthermore, the crystal structures are stabilized by π-π, C-H...π, C-O...π and N-O...π stacking interactions. The contributions made by each hydrogen bond in maintaining the crystal structure stability has been quantified by Hirshfeld surface analysis.


Assuntos
Ácido Bromídrico , Sais , Brometos , Creatinina , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Estrutura Molecular , Nitrobenzoatos , Sais/química , meta-Aminobenzoatos
7.
Acta Crystallogr D Struct Biol ; 78(Pt 8): 945-963, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35916220

RESUMO

Cryo-cooling has been nearly universally adopted to mitigate X-ray damage and facilitate crystal handling in protein X-ray crystallography. However, cryo X-ray crystallographic data provide an incomplete window into the ensemble of conformations that is at the heart of protein function and energetics. Room-temperature (RT) X-ray crystallography provides accurate ensemble information, and recent developments allow conformational heterogeneity (the experimental manifestation of ensembles) to be extracted from single-crystal data. Nevertheless, high sensitivity to X-ray damage at RT raises concerns about data reliability. To systematically address this critical issue, increasingly X-ray-damaged high-resolution data sets (1.02-1.52 Šresolution) were obtained from single proteinase K, thaumatin and lysozyme crystals at RT (277 K). In each case a modest increase in conformational heterogeneity with X-ray damage was observed. Merging data with different extents of damage (as is typically carried out) had negligible effects on conformational heterogeneity until the overall diffraction intensity decayed to ∼70% of its initial value. These effects were compared with X-ray damage effects in cryo-cooled crystals by carrying out an analogous analysis of increasingly damaged proteinase K cryo data sets (0.9-1.16 Šresolution). X-ray damage-associated heterogeneity changes were found that were not observed at RT. This property renders it difficult to distinguish real from artefactual conformations and to determine the conformational response to changes in temperature. The ability to acquire reliable heterogeneity information from single crystals at RT, together with recent advances in RT data collection at accessible synchrotron beamlines, provides a strong motivation for the widespread adoption of RT X-ray crystallography to obtain conformational ensemble information.


Assuntos
Endopeptidase K/química , Proteínas , Cristalografia por Raios X , Proteínas/química , Reprodutibilidade dos Testes , Temperatura , Raios X
8.
Acta Crystallogr F Struct Biol Commun ; 78(Pt 8): 306-312, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35924598

RESUMO

Elizabethkingia bacteria are globally emerging pathogens that cause opportunistic and nosocomial infections, with up to 40% mortality among the immunocompromised. Elizabethkingia species are in the pipeline of organisms for high-throughput structural analysis at the Seattle Structural Genomics Center for Infectious Disease (SSGCID). These efforts include the structure-function analysis of potential therapeutic targets. Glutamyl-tRNA synthetase (GluRS) is essential for tRNA aminoacylation and is under investigation as a bacterial drug target. The SSGCID produced, crystallized and determined high-resolution structures of GluRS from E. meningosepticum (EmGluRS) and E. anopheles (EaGluRS). EmGluRS was co-crystallized with glutamate, while EaGluRS is an apo structure. EmGluRS shares ∼97% sequence identity with EaGluRS but less than 39% sequence identity with any other structure in the Protein Data Bank. EmGluRS and EaGluRS have the prototypical bacterial GluRS topology. EmGluRS and EaGluRS have similar binding sites and tertiary structures to other bacterial GluRSs that are promising drug targets. These structural similarities can be exploited for drug discovery.


Assuntos
Anopheles , Infecções por Flavobacteriaceae , Sequência de Aminoácidos , Animais , Anopheles/metabolismo , Cristalografia por Raios X , Glutamato-tRNA Ligase/química , Glutamato-tRNA Ligase/genética , Glutamato-tRNA Ligase/metabolismo
9.
Acta Crystallogr D Struct Biol ; 78(Pt 8): 964-974, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35916221

RESUMO

Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of 3D protein structures, which has recently been further augmented by revolutionary advances in cryoEM. However, a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts, limiting the overview of inherent protein physiological dynamics, which play a critical role in the biological functions of proteins. Here, a room-temperature X-ray crystallographic method using temperature as a trigger to record movie-like structural snapshots has been developed. The method has been used to show how TL00150, a 175.15 Da fragment, undergoes binding-mode changes in endothiapepsin. A surprising fragment-binding discrepancy was observed between the cryo-cooled and physiological temperature structures, and multiple binding poses and their interplay with DMSO were captured. The observations here open up new promising prospects for structure determination and interpretation at physiological temperatures with implications for structure-based drug discovery.


Assuntos
Proteínas , Ácido Aspártico Endopeptidases , Cristalografia por Raios X , Ligantes , Substâncias Macromoleculares , Proteínas/química , Temperatura
10.
Acta Crystallogr F Struct Biol Commun ; 78(Pt 8): 289-296, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35924596

RESUMO

Levansucrases are biotechnologically interesting fructosyltransferases due to their potential use in the enzymatic or chemo-enzymatic synthesis of glycosides of non-natural substrates relevant to pharmaceutical applications. The structure of Erwinia tasmaniensis levansucrase in complex with (S)-1,2,4-butanetriol and its biochemical characterization suggests the possible application of short aliphatic moieties containing polyols with defined stereocentres in fructosylation biotechnology. The structural information revealed that (S)-1,2,4-butanetriol mimics the natural substrate. The preference of the protein towards a specific 1,2,4-butanetriol enantiomer was assessed using microscale thermophoresis binding assays. Furthermore, the results obtained and the structural comparison of levansucrases and inulosucrases suggest that the fructose binding modes could differ in fructosyltransferases from Gram-positive and Gram-negative bacteria.


Assuntos
Antibacterianos , Bactérias Gram-Negativas , Butanóis , Cristalografia por Raios X , Erwinia , Bactérias Gram-Positivas , Hexosiltransferases
11.
Acta Crystallogr F Struct Biol Commun ; 78(Pt 8): 297-305, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35924597

RESUMO

The bacterial amino-acid transporter MhsT from the SLC6A family has been crystallized in complex with different substrates in order to understand the determinants of the substrate specificity of the transporter. Surprisingly, crystals of the different MhsT-substrate complexes showed interrelated but different crystal-packing arrangements. Space-group assignment and structure determination of these different crystal forms present challenging combinations of pseudosymmetry, twinning and translational noncrystallographic symmetry.


Assuntos
Cristalografia por Raios X
12.
Chempluschem ; 87(7): e202200169, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35789218

RESUMO

This work describes the synthesis of 4-(4-AcPy) and 3-acetylpyridine (3-AcPy) copper soluble complexes for the activation of hydrogen peroxide and the concomitant generation of reactive oxygen species (ROS). Given the paramagnetic effects of copper ions in the Nuclear Magnetic Resonance (NMR) lines, we aimed at demonstrating that the combination of high-resolution 2D solid-state NMR experiments, Electron Paramagnetic Resonance (EPR), single-crystal X-ray crystallography and Density Functional Theory (DFT) calculations allows a detailed study of the chemical structure of the ligands and the surrounding metal ions. The copper complexes synthesized with CuCl2 were useful for the activation of H2 O2 during which the only ROS was the hydroxyl one, as demonstrated by EPR experiments. A removal of methyl orange (MO) azo-dye higher than 85 % was achieved in 200 minutes, combining 1.7 mM of copper complexes with 60 mM of H2 O2 and 40 µM of MO.


Assuntos
Cobre , Cobre/química , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Ligantes , Espécies Reativas de Oxigênio
13.
J Med Chem ; 65(13): 9281-9294, 2022 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-35776775

RESUMO

A series of new (tricarbonyl)rhenium(I) complexes were synthesized using chiral bidentate ligands (+)/(-)-iminopyridines (LR/LS). The reaction yielded a mixture of mononuclear Re(I) diastereoisomers, formulated as fac-[Br(CO)3Re(S/R)L(S/R)]. Each single diastereoisomer was isolated and fully characterized. X-ray crystallography and circular dichroism spectra verified their enantiomeric nature. The cytotoxicity of each complex was evaluated against six cancer cell lines. The effect of the two complexes on viability, proliferation, and migration was analyzed on glioblastoma cell lines (U251 and LN229). Changes in the expression of histones, apoptotic, and key signaling proteins, as well as alterations in DNA structure, were also observed. These experiments showed that the chirality associated with both metal and ligand has a strong influence on cytotoxicity.


Assuntos
Glioblastoma , Rênio , Cristalografia por Raios X , Glioblastoma/tratamento farmacológico , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Rênio/química
14.
Inorg Chem ; 61(28): 10965-10976, 2022 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-35793494

RESUMO

Two particular fumaric acid bridging lanthanide-encapsulated selenotungstates [H2N(CH3)2]16Na8[Ln3(H2O)7]2 [W4O8(C4H2O4) (C4H3O4)]2[SeW6O25]2[B-α-SeW9O33]4·46H2O [Ln = Ce3+ (1), La3+ (2)] were acquired by the deliberately designed step-by-step synthetic strategy, which are composed of four trilacunary Keggin [B-α-SeW9O33]8- and two original [SeW6O25]10- building units together with one fumaric acid bridging heterometallic [Ln3(H2O)7]2[W4O8(C4H2O4) (C4H3O4)]228+ entity. Particularly, this heterometallic cluster contains four fumaric acid ligands, which play two different roles: one works as the pendant decorating the cluster and the other acts as the linker connecting the whole structure. In addition, the 1@DDA hybrid material was produced through the cation exchange of 1 and dimethyl distearylammonium chloride (DDA·Cl) and its beehive-shaped film of 1@DDA was prepared by the breath figure method, which can be further used to establish an electrochemical biosensor for detecting a kind of mycotoxin-ochratoxin A (OX-A). The 1@DDA beehive-shaped film-based electrochemical biosensor exhibits good reproducibility and specific sensing toward OX-A with a low detection limit of 29.26 pM. These results highlight the huge feasibility of long-chain flexible ligands in building lanthanide-encapsulated selenotungstates with structural complexity and further demonstrate great electrochemical application potentiality of polyoxometalate-involved materials in bioanalysis, tumor diagnosis, and iatrology.


Assuntos
Elementos da Série dos Lantanídeos , Micotoxinas , Ânions , Cristalografia por Raios X , Técnicas Eletroquímicas , Fumaratos , Elementos da Série dos Lantanídeos/química , Ligantes , Modelos Moleculares , Micotoxinas/análise , Polieletrólitos , Reprodutibilidade dos Testes
15.
Dalton Trans ; 51(29): 11086-11097, 2022 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-35796232

RESUMO

Multinuclear tungsten complexes are intriguing candidates for new contrast media that can provide substantial improvements in CT imaging diagnostics. Herein, we present a ligand strategy, based on amino acids, and mono- and disubstituted EDTA derivatives, that enables the development of stable complexes with high tungsten content and reasonably low osmolality. Accordingly, a series of neutral and monoanionic di-µ-sulfido W(V) dimers have been synthesized via a convenient procedure utilizing microwave heating in combination with ion-pair HPLC reaction monitoring. The compounds were characterized in detail by various techniques, including ESI-HRMS, NMR spectroscopy, HPLC, elemental analysis, and X-ray crystallography. The aqueous stability of the complexes under physiologically relevant conditions, and during heat sterilization was also examined as an initial assessment of their potential applicability as radiocontrast agents. Monoanionic complexes featuring monosubstituted EDTA derivatives have demonstrated high stability, while producing a lower number of ions in solution (resulting in lower osmolality) in comparison to their bis-anionic EDTA counterparts. Nevertheless, they exhibited insufficient water solubility for application as intravascular contrast agents. However, our study showed that aqueous solubility of this type of complexes can be tuned by small modifications in the ligand structure.


Assuntos
Meios de Contraste , Tungstênio , Meios de Contraste/química , Cristalografia por Raios X , Ácido Edético , Ligantes , Modelos Moleculares , Polímeros , Enxofre , Tomografia Computadorizada por Raios X , Tungstênio/química , Água/química
16.
Dalton Trans ; 51(29): 11125-11134, 2022 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-35797528

RESUMO

The active sites of [FeFe]-hydrogenase promoted by Fe/E (E = S, Se) clusters have attracted considerable interest due to their significance in understanding the interconversion of hydrogen with protons and electrons. As an extension of the study on Fe/Se clusters related to H-cluster model compounds of [FeFe]-hydrogenase, a series of tertiary phosphine substituted Fe/Se carbonyls were successfully prepared. The treatment of Fe2(µ-SePh)2(CO)6 (A) and excess PR3 resulted in the ferrous bis(selenolate) carbonyls Fe(SePh)2(CO)2(PR3)2 (PR3 = PPhMe2, 1; PMe3, 2) in moderate yields. In striking contrast, the reaction of Fe2(µ-SeCH2Ph)2(CO)6 (B) with the same PR3 ligand resulted in the PR3-disubstituted models Fe2(µ-SeCH2Ph)2(CO)4(PR3)2 (PR3 = PPhMe2, 3; PMe3, 4) as the principal products. The more interesting finding is that two independent isomers (anti- and syn-) can be isolated according to different reaction temperatures. Further reactions of 3 or 4 with PR3 under UV irradiation afforded the first PR3-trisubstituted 2Fe2Se derivatives Fe2(µ-SeCH2Ph)2(CO)3(PR3)3 (PR3 = PPhMe2, 5; PMe3, 6). 6 could be further converted into the tetrasubstituted product Fe2(µ-SeCH2Ph)2(CO)2(PMe3)4 (7), while no further substitution was observed with 5 and excess of PPhMe2. All the prepared compounds were fully characterized by elemental analysis, various spectroscopic techniques and X-ray crystallography. In addition, some electrochemical properties of these models were studied by cyclic voltammetry (CV) in MeCN. Compounds 4, 6 and 7 were found to be catalysts for the H2 evolution reaction under electrochemical conditions.


Assuntos
Hidrogenase , Proteínas Ferro-Enxofre , Cristalografia por Raios X , Hidrogênio/química , Hidrogenase/química , Proteínas Ferro-Enxofre/química , Modelos Moleculares , Prótons
17.
Commun Biol ; 5(1): 704, 2022 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-35835834

RESUMO

When overexpressed as an immature enzyme in the mesophilic bacterium Escherichia coli, recombinant homoserine dehydrogenase from the hyperthermophilic archaeon Sulfurisphaera tokodaii (StHSD) was markedly activated by heat treatment. Both the apo- and holo-forms of the immature enzyme were successively crystallized, and the two structures were determined. Comparison among the structures of the immature enzyme and previously reported structures of mature enzymes revealed that a conformational change in a flexible part (residues 160-190) of the enzyme, which encloses substrates within the substrate-binding pocket, is smaller in the immature enzyme. The immature enzyme, but not the mature enzyme, formed a complex that included NADP+, despite its absence during crystallization. This indicates that the opening to the substrate-binding pocket in the immature enzyme is not sufficient for substrate-binding, efficient catalytic turnover or release of NADP+. Thus, specific conformational changes within the catalytic region appear to be responsible for heat-induced activation.


Assuntos
Escherichia coli/enzimologia , Homosserina Desidrogenase/química , Homosserina Desidrogenase/metabolismo , Temperatura Alta , Sulfolobaceae/enzimologia , Domínio Catalítico/fisiologia , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , NADP/química , NADP/metabolismo
18.
J Am Chem Soc ; 144(28): 12632-12637, 2022 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-35786956

RESUMO

The scission of a C(sp3)-H bond to form a new metal-alkyl bond is a fundamental step in coordination chemistry and catalysis. However, the extent of C-H bond weakening when this moiety interacts with a transition metal is poorly understood and quantifying this phenomenon could provide insights into designing more efficient C-H functionalization catalysts. We present a nickel complex with a robust adamantyl reporter ligand that enables the measurement of C-H acidity (pKa) and bond dissociation free energy (BDFE) for a C(sp3)-H agostic interaction, showing a decrease in pKa by dozens of orders of magnitude and BDFE decrease of about 30 kcal/mol upon coordination. X-ray crystallographic data is provided for all molecules, including a distorted square planar NiIII metalloradical and "doubly agostic" NiII(κ2-CH2) complex.


Assuntos
Metais , Níquel , Catálise , Cristalografia por Raios X , Ligantes , Níquel/química
19.
J Agric Food Chem ; 70(28): 8653-8661, 2022 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-35791917

RESUMO

Eleven new chlorinated cyclopentene derivatives, periconsins A-K, and a new diketopiperazine, periconzin, were found from Periconia sp. cultured in three different media by the one strain many compounds strategy. Additionally, the C-1 methyl hydroxylation of chlorinated cyclopentene was found for the first time in the host plant culture. The structures were identified by extensive spectroscopic analyses, electronic circular dichroism (ECD) and 13C NMR calculations, and single-crystal X-ray diffraction. Compounds 3, 5, 7-11, 15, and 17 showed significant antifungal activities against the plant pathogens Periconia sp., Altemaria sp., and Nigrospora oryzae with MICs ≤2 µg/mL. Other compounds had antifungal activities with MICs ≤8 µg/mL. The antifungal structure-activity relationship of these metabolites indicated that the chlorine at C-5 can increase the activity, but the hydroxy group at C-1 lowered the activity.


Assuntos
Antifúngicos , Ciclopentanos , Antifúngicos/química , Antifúngicos/farmacologia , Dicroísmo Circular , Cristalografia por Raios X , Ciclopentanos/farmacologia , Estrutura Molecular
20.
Phytochemistry ; 201: 113292, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35780923

RESUMO

Marine-derived actinomycetes from the genus Streptomycete have a huge potential for the production of metabolites with structural and bioactive uniqueness and diversity. This study described the isolation and structural elucidation of twenty metabolites, including seven previously unreported compounds galbonolide H, galbonolide I, streptophenylpropionic acid A, treptophenylpropyl ester A, streptophenvaleramide A, seco-geldanamycin A and streptorapamycin A, from the marine-associated Streptomycete sp. ZZ1944. Structures of the isolated compounds were elucidated by a combination of extensive NMR spectroscopic analyses, HRESIMS data, optical rotation and ECD calculations. The structure of galbonolide H was also confirmed by a single crystal X-ray diffraction. Both autolytimycin and seco-geldanamycin A showed potent activity against the proliferation of glioma, lung cancer, colorectal cancer and breast cancer cells. Autolytimycin blocked cell cycle of glioma cells and seco-geldanamycin A induced apoptosis of glioma cells.


Assuntos
Antineoplásicos , Glioma , Streptomyces , Antineoplásicos/farmacologia , Cristalografia por Raios X , Glioma/tratamento farmacológico , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Streptomyces/química
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