scholarly journals Triazole ureas covalently bind to strigolactone receptors and regulate signaling

2018 ◽  
Author(s):  
Hidemitsu Nakamura ◽  
Kei Hirabayashi ◽  
Takuya Miyakawa ◽  
Ko Kikuzato ◽  
Wenqian Hu ◽  
...  
Keyword(s):  
Potent Inhibitor ◽  
Catalytic Triad ◽  
Yeast Two Hybrid ◽  
Multiple Functions ◽  
X Ray ◽  
X Ray Crystallography ◽  
Agricultural Application ◽  
Two Hybrid ◽  
Complicated Mechanism ◽  
Urea Compounds

AbstractStrigolactones (SLs), a class of plant hormones with multiple functions, mediate plant-plant and plant-microorganism communications in the rhizosphere. In this study, we developed potent strigolactone antagonists, which covalently bind to the strigolactone receptor D14, by preparing an array of triazole urea compounds. Using yeast two-hybrid assays and rice tillering assays, we identified a triazole urea compound KK094 as a potent inhibitor of strigolactone receptors. The LC-MS/MS analysis and X-ray crystallography concluded that KK094 was hydrolyzed by D14, and that a reaction product of this degradation covalently binds to the Ser residue of the catalytic triad of D14. We also identified KK052 and KK073, whose effects on D14–D53/D14–SLR1 complex formation were opposite due to a trifluoromethyl group on its benzene ring. These results demonstrate that triazole urea compounds are potentially powerful tools for agricultural application and may be useful for the elucidation of the complicated mechanism underlying SL-perception.

The RING domain of TRIM69 promotes higher-order assembly

2020 ◽  
Vol 76 (10) ◽  
pp. 954-961 ◽  
Author(s):  
Jeremy R. Keown ◽  
Joy Yang ◽  
Moyra M. Black ◽  
David C. Goldstone
Keyword(s):  
Virus Infection ◽  
Viral Infections ◽  
Potent Inhibitor ◽  
Higher Order ◽  
Ring Domain ◽  
X Ray ◽  
X Ray Crystallography ◽  
Trim Protein ◽  
Vesicular Stomatitis ◽  
Trim Proteins

Members of the TRIM protein family have been shown to inhibit a range of viral infections. Recently, TRIM69 was identified as a potent inhibitor of Vesicular stomatitis Indiana virus infection, with its inhibition being dependent upon multimerization. Using SEC-MALLS analysis, it is demonstrated that the assembly of TRIM69 is mediated through the RING domain and not the Bbox domain as has been shown for other TRIM proteins. Using X-ray crystallography, the structure of the TRIM69 RING domain has been determined to a resolution of 2.1 Å, the oligomerization interface has been identified and regions outside the four-helix bundle have been observed to form interactions that are likely to support assembly.

scholarly journals Investigation of the catalytic triad of arylamine N-acetyltransferases: essential residues required for acetyl transfer to arylamines

2005 ◽  
Vol 390 (1) ◽  
pp. 115-123 ◽  
Author(s):  
James Sandy ◽  
Adeel Mushtaq ◽  
Simon J. Holton ◽  
Pamela Schartau ◽  
Martin E. M. Noble ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Structural Model ◽  
Sequence Data ◽  
Structural Features ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
X Ray ◽  
X Ray Crystallography ◽  
Acetyl Transfer ◽  
Insight Into

The NATs (arylamine N-acetyltransferases) are a well documented family of enzymes found in both prokaryotes and eukaryotes. NATs are responsible for the acetylation of a range of arylamine, arylhydrazine and hydrazine compounds. We present here an investigation into the catalytic triad of residues (Cys-His-Asp) and other structural features of NATs using a variety of methods, including site-directed mutagenesis, X-ray crystallography and bioinformatics analysis, in order to investigate whether each of the residues of the catalytic triad is essential for catalytic activity. The catalytic triad of residues, Cys-His-Asp, is a well defined motif present in several families of enzymes. We mutated each of the catalytic residues in turn to investigate the role they play in catalysis. We also mutated a key residue, Gly126, implicated in acetyl-CoA binding, to examine the effects on acetylation activity. In addition, we have solved the structure of a C70Q mutant of Mycobacterium smegmatis NAT to a resolution of 1.45 Å (where 1 Å=0.1 nm). This structure confirms that the mutated protein is correctly folded, and provides a structural model for an acetylated NAT intermediate. Our bioinformatics investigation analysed the extent of sequence conservation between all eukaryotic and prokaryotic NAT enzymes for which sequence data are available. This revealed several new sequences, not yet reported, of NAT paralogues. Together, these studies have provided insight into the fundamental core of NAT enzymes, and the regions where sequence differences account for the functional diversity of this family. We have confirmed that each of the three residues of the triad is essential for acetylation activity.

scholarly journals The organomercurial lyase Merb possesses unique metal-binding properties

2014 ◽  
Vol 70 (a1) ◽  
pp. C1680-C1680
Author(s):  
Haytham Wahba ◽  
Ahmed Mansour ◽  
Julien Vanasse ◽  
Laurent Cappadocia ◽  
Jurgen Sygusch ◽  
...  
Keyword(s):  
Heavy Metals ◽  
Metal Binding ◽  
Catalytic Triad ◽  
Bacterial Strains ◽  
Paramagnetic Resonance ◽  
Mer Operon ◽  
X Ray ◽  
E Coli ◽  
X Ray Crystallography ◽  
Organomercurial Lyase

Select bacterial strains survive in mercury-contaminated environments due to acquisition of a transferable genetic element known as the mer operon. The mer operon typically encodes for a series of proteins that includes two enzymes, MerA and MerB. The organomercurial lyase (MerB) cleaves carbon-mercury bonds of organomercurial compounds yielding ionic mercury Hg (II) and a reduced-carbon compound. The Hg (II) ion product remains bounds until it is shuttled directly to the mercuric ion reductase (MerA) to be reduced. Based on NMR spectroscopy and X-ray crystallography studies1, we have determined that Cys96, Asp99 and Cys159 of E. Coli MerB form a catalytic triad required for cleavage of the carbon-Hg bond and binding of the Hg (II) ion product. The three catalytic residues are conserved in 61 of 65 known variants of MerB and the four remaining variants retain both cysteine residues, but contain a serine in place of Asp99. Given its unique activity, we have examined the role of serine as a catalytic residue and the ability of MerB to cleave other organometals such as organotin (known substrates or inhibitors) and organolead compounds. Soaking MerB crystals with either dimethyltindibromide or trimethylleadchloride compound indicates that MerB crystals have the capacity to cleave both carbon-Sn and carbon-Pb bonds, and we have determined crystal structures of a MerB-Sn and a MerB-Pb complex. Furthermore, substitution of Ser for Asp99 (MerB D99S) in E. coli MerB alters the metal-binding specificity, as MerB D99S chelated an unknown metal during its purification. X-ray crystallography, ICP-MS and electron paramagnetic resonance (EPR) studies were performed to identify the unknown metal and the results of these studies will be presented. Given that mercury contaminated sites are often contaminated with other heavy metals, these studies indicate that other heavy metals may have important implications when using MerA and MerB in bioremediation of organomercurial compounds.

scholarly journals Structure of the Xylan O-Acetyltransferase AtXOAT1 Reveals Molecular Insight into Polysaccharide Acetylation in Plants

2020 ◽  
Author(s):  
Vladimir V. Lunin ◽  
Hsin-Tzu Wang ◽  
Vivek S. Bharadwaj ◽  
Markus Alahuhta ◽  
Maria J. Peña ◽  
...  
Keyword(s):  
Cell Walls ◽  
Catalytic Triad ◽  
Plant Cell Walls ◽  
X Ray ◽  
X Ray Crystallography ◽  
Polymer Interactions ◽  
Biochemical Analyses ◽  
Insight Into ◽  
Enzyme Intermediate

AbstractAcetylation of biomolecules is gaining increased attention due to both the abundance and importance of this modification across all kingdoms of life. Xylans are a major component of plant cell walls and are the third most abundant biopolymer in Nature. O-Acetyl moieties are the dominant backbone substituents of glucuronoxylan in dicots and play a major role in the polymer-polymer interactions that are crucial for proper wall architecture and normal plant development. Here, we describe the biochemical, structural, and mechanistic characterization of Arabidopsis thaliana xylan O-acetyltransferase 1 (AtXOAT1), a member of the plant-specific Trichome Birefrigence Like (TBL) family that catalyzes the 2-O-acetylation of xylan. A multipronged approach involving X-ray crystallography, biochemical analyses, mutagenesis, and molecular simulations show that XOAT1 catalyzes xylan acetylation through formation of an acyl-enzyme intermediate by a double displacement bi-bi mechanism involving a Ser-His-Asp catalytic triad and unconventionally employs an arginine residue in formation of an oxyanion hole.

High-Resolution X-ray Crystallography Reveals Precise Binding Interactions between Human Nonpancreatic Secreted Phospholipase A2and a Highly Potent Inhibitor (FPL67047XX)

1996 ◽  
Vol 39 (20) ◽  
pp. 3878-3881 ◽  
Author(s):  
Sun-Shin Cha ◽  
Dennis Lee ◽  
Jerry Adams ◽  
Jeffrey T. Kurdyla ◽  
Christopher S. Jones ◽  
...  
Keyword(s):  
High Resolution ◽  
Potent Inhibitor ◽  
Binding Interactions ◽  
X Ray ◽  
X Ray Crystallography

Difference Fourier Analysis of Glucose Embedded and Frozen Hydrated Purple Membrane

1982 ◽  
Vol 40 ◽  
pp. 74-75
Author(s):  
Jules S. Jaffe ◽  
Robert M. Glaeser
Keyword(s):  
Purple Membrane ◽  
Data Set ◽  
High Resolution Data ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fourier Techniques ◽  
Versus Protein ◽  
The Difference ◽  
Difference Fourier ◽  
Ideal Method

Although difference Fourier techniques are standard in X-ray crystallography it has only been very recently that electron crystallographers have been able to take advantage of this method. We have combined a high resolution data set for frozen glucose embedded Purple Membrane (PM) with a data set collected from PM prepared in the frozen hydrated state in order to visualize any differences in structure due to the different methods of preparation. The increased contrast between protein-ice versus protein-glucose may prove to be an advantage of the frozen hydrated technique for visualizing those parts of bacteriorhodopsin that are embedded in glucose. In addition, surface groups of the protein may be disordered in glucose and ordered in the frozen state. The sensitivity of the difference Fourier technique to small changes in structure provides an ideal method for testing this hypothesis.

3-D reconstruction of molecular shape from microcrystal thin sections

1983 ◽  
Vol 41 ◽  
pp. 748-749
Author(s):  
S. Cusack ◽  
J.-C. Jésior
Keyword(s):  
Three Dimensional ◽  
Molecular Shape ◽  
Biological Molecules ◽  
Fourier Space ◽  
Single Particles ◽  
Thin Sections ◽  
Standard Methods ◽  
X Ray ◽  
X Ray Crystallography ◽  
Dimensional Reconstruction

Three-dimensional reconstruction techniques using electron microscopy have been principally developed for application to 2-D arrays (i.e. monolayers) of biological molecules and symmetrical single particles (e.g. helical viruses). However many biological molecules that crystallise form multilayered microcrystals which are unsuitable for study by either the standard methods of 3-D reconstruction or, because of their size, by X-ray crystallography. The grid sectioning technique enables a number of different projections of such microcrystals to be obtained in well defined directions (e.g. parallel to crystal axes) and poses the problem of how best these projections can be used to reconstruct the packing and shape of the molecules forming the microcrystal.Given sufficient projections there may be enough information to do a crystallographic reconstruction in Fourier space. We however have considered the situation where only a limited number of projections are available, as for example in the case of catalase platelets where three orthogonal and two diagonal projections have been obtained (Fig. 1).

Electron microscopy of rabbit FC crystals

1983 ◽  
Vol 41 ◽  
pp. 730-731
Author(s):  
Robert A. Grant ◽  
Laura L. Degn ◽  
Wah Chiu ◽  
John Robinson
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
High Resolution Electron Microscopy ◽  
Molecular Replacement ◽  
X Ray ◽  
X Ray Crystallography ◽  
Resolution Electron ◽  
Replacement Technique ◽  
Hydrated Crystal ◽  
Molecular Structure Analysis

Proteolytic digestion of the immunoglobulin IgG with papain cleaves the molecule into an antigen binding fragment, Fab, and a compliment binding fragment, Fc. Structures of intact immunoglobulin, Fab and Fc from various sources have been solved by X-ray crystallography. Rabbit Fc can be crystallized as thin platelets suitable for high resolution electron microscopy. The structure of rabbit Fc can be expected to be similar to the known structure of human Fc, making it an ideal specimen for comparing the X-ray and electron crystallographic techniques and for the application of the molecular replacement technique to electron crystallography. Thin protein crystals embedded in ice diffract to high resolution. A low resolution image of a frozen, hydrated crystal can be expected to have a better contrast than a glucose embedded crystal due to the larger density difference between protein and ice compared to protein and glucose. For these reasons we are using an ice embedding technique to prepare the rabbit Fc crystals for molecular structure analysis by electron microscopy.

X-ray Crystallography Shared Resource

2020 ◽  
Author(s):  
Keyword(s):  
Shared Resource ◽  
X Ray ◽  
X Ray Crystallography

An Introduction to X-Ray Crystallography

1998 ◽  
Vol 213 (5) ◽  
Author(s):  
P. Luger
Keyword(s):  
X Ray ◽  
X Ray Crystallography
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