scholarly journals NMR Crystallography of Enzyme Active Sites: Probing Chemically Detailed, Three-Dimensional Structure in Tryptophan Synthase

2013 ◽  
Vol 46 (9) ◽  
pp. 2008-2017 ◽  
Author(s):  
Leonard J. Mueller ◽  
Michael F. Dunn
Keyword(s):  
Active Sites ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Tryptophan Synthase ◽  
Three Dimensional Structure ◽  
Enzyme Active Sites ◽  
Nmr Crystallography

scholarly journals Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate

2022 ◽  
Vol 119 (2) ◽  
pp. e2109235119
Author(s):  
Jacob B. Holmes ◽  
Viktoriia Liu ◽  
Bethany G. Caulkins ◽  
Eduardo Hilario ◽  
Rittik K. Ghosh ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Reaction Pathway ◽  
Three Dimensional ◽  
Integrated Approach ◽  
Nucleophilic Attack ◽  
Dimensional Structure ◽  
Tryptophan Synthase ◽  
Hydrogen Bond Donor ◽  
Nmr Crystallography

NMR-assisted crystallography—the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry—holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5′-phosphate–dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid–base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cβ and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

scholarly journals Three-Dimensional Structure of PANI/CdS NRs-SiO2 Hydrogel for Photocatalytic Hydrogen Evolution with High Activity and Stability

Nanomaterials ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 427 ◽  
Author(s):  
Jinrong Lu ◽  
Xin Zhang ◽  
Huiyuan Gao ◽  
Wenquan Cui
Keyword(s):  
Hydrogen Production ◽  
Active Sites ◽  
Production Efficiency ◽  
Conductive Polymer ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Composite Catalyst ◽  
Binary Complex ◽  
Three Dimensional Structure ◽  
Cds Nanorods

Three-dimensional PANI/CdSNRs-SiO2 hydrogel (CdS NRs-PANI-SiO2) was synthesized by loading polyaniline (PANI) onto the semiconductor CdS nanorods (NRs) surface and loading the binary complex on SiO2 gel. The structure, optical properties, and electrochemical properties of the composite were studied in detail. The hydrogen production amount of CdS NRs-PANI (3%)-SiO2 (20%) increased in comparison with CdS NRs and reached 43.25 mmol/g in 3 h under visible light. The three-dimensional structure of SiO2 hydrogel increased the specific surface area of the catalyst, which was conducive to exposing more active sites of the catalyst. In addition, the conductive polymer PANI coated on CdS NRs played the role of conductive charge and effectively inhibited the photo-corrosion of CdS NRs. In addition, the recovery experiment showed that the recovery rate of the composite catalyst reached 90% and hydrogen production efficiency remained unchanged after five cycles, indicating that the composite catalyst had excellent stability.

Structure–function relationships in Gan42B, an intracellular GH42 β-galactosidase fromGeobacillus stearothermophilus

2015 ◽  
Vol 71 (12) ◽  
pp. 2433-2448 ◽  
Author(s):  
Hodaya V. Solomon ◽  
Orly Tabachnikov ◽  
Shifra Lansky ◽  
Rachel Salama ◽  
Hadar Feinberg ◽  
...  
Keyword(s):  
Active Sites ◽  
Plant Cell Wall ◽  
Three Dimensional ◽  
Gel Permeation Chromatography ◽  
Structural Data ◽  
Dimensional Structure ◽  
Cell Wall Polysaccharides ◽  
Small Opening ◽  
Three Dimensional Structure ◽  
Tim Barrel

Geobacillus stearothermophilusT-6 is a Gram-positive thermophilic soil bacterium that contains a battery of degrading enzymes for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. A 9.4 kb gene cluster has recently been characterized inG. stearothermophilusthat encodes a number of galactan-utilization elements. A key enzyme of this degradation system is Gan42B, an intracellular GH42 β-galactosidase capable of hydrolyzing short β-1,4-galactosaccharides into galactose units, making it of high potential for various biotechnological applications. The Gan42B monomer is made up of 686 amino acids, and based on sequence homology it was suggested that Glu323 is the catalytic nucleophile and Glu159 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Gan42B (at 2.45 Å resolution) and its catalytic mutant E323A (at 2.50 Å resolution), as determined by X-ray crystallography, are reported. These structures demonstrate that the three-dimensional structure of the Gan42B monomer generally correlates with the overall fold observed for GH42 proteins, consisting of three main domains: an N-terminal TIM-barrel domain, a smaller mixed α/β domain, and the smallest all-β domain at the C-terminus. The two catalytic residues are located in the TIM-barrel domain in a pocket-like active site such that their carboxylic functional groups are about 5.3 Å from each other, consistent with a retaining mechanism. The crystal structure demonstrates that Gan42B is a homotrimer, resembling a flowerpot in general shape, in which each monomer interacts with the other two to form a cone-shaped tunnel cavity in the centre. The cavity is ∼35 Å at the wide opening and ∼5 Å at the small opening and ∼40 Å in length. The active sites are situated at the interfaces between the monomers, so that every two neighbouring monomers participate in the formation of each of the three active sites of the trimer. They are located near the small opening of the cone tunnel, all facing the centre of the cavity. The biological relevance of this trimeric structure is supported by independent results obtained from gel-permeation chromatography. These data and their comparison to the structural data of related GH42 enzymes are used for a more general discussion concerning structure–activity aspects in this GH family.

Structure–specificity relationships in Abp, a GH27 β-L-arabinopyranosidase fromGeobacillus stearothermophilusT6

2014 ◽  
Vol 70 (11) ◽  
pp. 2994-3012 ◽  
Author(s):  
Shifra Lansky ◽  
Rachel Salama ◽  
Hodaya V. Solomon ◽  
Hadar Feinberg ◽  
Hassan Belrhali ◽  
...  
Keyword(s):  
Active Sites ◽  
Critical Role ◽  
Three Dimensional ◽  
Structural Data ◽  
Size Exclusion ◽  
Dimensional Structure ◽  
Structural Basis ◽  
X Ray ◽  
Three Dimensional Structure ◽  
Tim Barrel

L-Arabinose sugar residues are relatively abundant in plants and are found mainly in arabinan polysaccharides and in other arabinose-containing polysaccharides such as arabinoxylans and pectic arabinogalactans. The majority of the arabinose units in plants are present in the furanose form and only a small fraction of them are present in the pyranose form. The L-arabinan-utilization system inGeobacillus stearothermophilusT6, a Gram-positive thermophilic soil bacterium, has recently been characterized, and one of the key enzymes was found to be an intracellular β-L-arabinopyranosidase (Abp). Abp, a GH27 enzyme, was shown to remove β-L-arabinopyranose residues from synthetic substrates and from the native substrates sugar beet arabinan and larch arabinogalactan. The Abp monomer is made up of 448 amino acids, and based on sequence homology it was suggested that Asp197 is the catalytic nucleophile and Asp255 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Abp (at 2.28 Å resolution) and its catalytic mutant Abp-D197A with (at 2.20 Å resolution) and without (at 2.30 Å resolution) a bound L-arabinose product are reported as determined by X-ray crystallography. These structures demonstrate that the three-dimensional structure of the Abp monomer correlates with the general fold observed for GH27 proteins, consisting of two main domains: an N-terminal TIM-barrel domain and a C-terminal all-β domain. The two catalytic residues are located in the TIM-barrel domain, such that their carboxylic functional groups are about 5.9 Å from each other, consistent with a retaining mechanism. An isoleucine residue (Ile67) located at a key position in the active site is shown to play a critical role in the substrate specificity of Abp, providing a structural basis for the high preference of the enzyme towards arabinopyranoside over galactopyranoside substrates. The crystal structure demonstrates that Abp is a tetramer made up of two `open-pincers' dimers, which clamp around each other to form a central cavity. The four active sites of the Abp tetramer are situated on the inner surface of this cavity, all opening into the central space of the cavity. The biological relevance of this tetrameric structure is supported by independent results obtained from size-exclusion chromatography (SEC), dynamic light-scattering (DLS) and small-angle X-ray scattering (SAXS) experiments. These data and their comparison to the structural data of related GH27 enzymes are used for a more general discussion concerning structure–selectivity aspects in this glycoside hydrolase (GH) family.

A unique octameric structure of Axe2, an intracellular acetyl-xylooligosaccharide esterase fromGeobacillus stearothermophilus

2014 ◽  
Vol 70 (2) ◽  
pp. 261-278 ◽  
Author(s):  
Shifra Lansky ◽  
Onit Alalouf ◽  
Hodaya Vered Solomon ◽  
Anat Alhassid ◽  
Lata Govada ◽  
...  
Keyword(s):  
Active Sites ◽  
Quaternary Structure ◽  
Gel Filtration ◽  
Three Dimensional ◽  
Structural Data ◽  
Catalytic Triad ◽  
Dimensional Structure ◽  
Internal Cavity ◽  
Three Dimensional Structure ◽  
New Family

Geobacillus stearothermophilusT6 is a thermophilic, Gram-positive soil bacterium that possesses an extensive and highly regulated hemicellulolytic system, allowing the bacterium to efficiently degrade high-molecular-weight polysaccharides such as xylan, arabinan and galactan. As part of the xylan-degradation system, the bacterium uses a number of side-chain-cleaving enzymes, one of which is Axe2, a 219-amino-acid intracellular serine acetylxylan esterase that removes acetyl side groups from xylooligosaccharides. Bioinformatic analyses suggest that Axe2 belongs to the lipase GDSL family and represents a new family of carbohydrate esterases. In the current study, the detailed three-dimensional structure of Axe2 is reported, as determined by X-ray crystallography. The structure of the selenomethionine derivative Axe2-Se was initially determined by single-wavelength anomalous diffraction techniques at 1.70 Å resolution and was used for the structure determination of wild-type Axe2 (Axe2-WT) and the catalytic mutant Axe2-S15A at 1.85 and 1.90 Å resolution, respectively. These structures demonstrate that the three-dimensional structure of the Axe2 monomer generally corresponds to the SGNH hydrolase fold, consisting of five central parallel β-sheets flanked by two layers of helices (eight α-helices and five 310-helices). The catalytic triad residues, Ser15, His194 and Asp191, are lined up along a substrate channel situated on the concave surface of the monomer. Interestingly, the Axe2 monomers are assembled as a `doughnut-shaped' homo-octamer, presenting a unique quaternary structure built of two staggered tetrameric rings. The eight active sites are organized in four closely situated pairs, which face the relatively wide internal cavity. The biological relevance of this octameric structure is supported by independent results obtained from gel-filtration, TEM and SAXS experiments. These data and their comparison to the structural data of related hydrolases are used for a more general discussion focusing on the structure–function relationships of enzymes of this category.

Exchange of K+or Cs+for Na+Induces Local and Long-Range Changes in the Three-Dimensional Structure of the Tryptophan Synthase α2β2Complex‡

Biochemistry ◽  
1996 ◽  
Vol 35 (13) ◽  
pp. 4211-4221 ◽  
Author(s):  
Sangkee Rhee ◽  
Kevin D. Parris ◽  
S. Ashraf Ahmed ◽  
Edith Wilson Miles ◽  
David R. Davies
Keyword(s):  
Long Range ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Tryptophan Synthase ◽  
Three Dimensional Structure

The Three-dimensional structure of frozen-hydrated nudaurelia capensis β virus

1987 ◽  
Vol 45 ◽  
pp. 650-651
Author(s):  
N. H. Olson ◽  
T. S. Baker ◽  
Wu Bo Mu ◽  
J. E. Johnson ◽  
D. A. Hendry
Keyword(s):  
South African ◽  
Rna Virus ◽  
Carbon Film ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Electron Dose ◽  
Total Electron ◽  
Three Dimensional Structure ◽  
Negative Stain ◽  
Dimensional Reconstruction

Nudaurelia capensis β virus (NβV) is an RNA virus of the South African Pine Emperor moth, Nudaurelia cytherea capensis (Lepidoptera: Saturniidae). The NβV capsid is a T = 4 icosahedron that contains 60T = 240 subunits of the coat protein (Mr = 61,000). A three-dimensional reconstruction of the NβV capsid was previously computed from visions embedded in negative stain suspended over holes in a carbon film. We have re-examined the three-dimensional structure of NβV, using cryo-microscopy to examine the native, unstained structure of the virion and to provide a initial phasing model for high-resolution x-ray crystallographic studiesNβV was purified and prepared for cryo-microscopy as described. Micrographs were recorded ∼1 - 2 μm underfocus at a magnification of 49,000X with a total electron dose of about 1800 e-/nm2.

Three Dimensional structure and organization of diploid chromosomes by optical section microscopy

1987 ◽  
Vol 45 ◽  
pp. 633-635
Author(s):  
David A. Agard ◽  
Yasushi Hiraoka ◽  
John W. Sedat
Keyword(s):  
Dimensional Space ◽  
Three Dimensional ◽  
Developmental State ◽  
Mitotic Cell ◽  
Biological Properties ◽  
Dimensional Structure ◽  
Specific Gene ◽  
Three Dimensional Structure ◽  
Complementary Techniques ◽  
Dynamic Structures

In an effort to understand the complex relationship between structure and biological function within the nucleus, we have embarked on a program to examine the three-dimensional structure and organization of Drosophila melanogaster embryonic chromosomes. Our overall goal is to determine how DNA and proteins are organized into complex and highly dynamic structures (chromosomes) and how these chromosomes are arranged in three dimensional space within the cell nucleus. Futher, we hope to be able to correlate structual data with such fundamental biological properties as stage in the mitotic cell cycle, developmental state and transcription at specific gene loci.Towards this end, we have been developing methodologies for the three-dimensional analysis of non-crystalline biological specimens using optical and electron microscopy. We feel that the combination of these two complementary techniques allows an unprecedented look at the structural organization of cellular components ranging in size from 100A to 100 microns.

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