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 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.

scholarly journals Rational synthesis of an atomically precise carboncone under mild conditions

2019 ◽  
Vol 5 (8) ◽  
pp. eaaw0982 ◽  
Author(s):  
Zheng-Zhong Zhu ◽  
Zuo-Chang Chen ◽  
Yang-Rong Yao ◽  
Cun-Hao Cui ◽  
Shu-Hui Li ◽  
...  
Keyword(s):  
Apex Angle ◽  
Facile Synthesis ◽  
Reaction Conditions ◽  
X Ray ◽  
Carbon Allotropes ◽  
X Ray Crystallography ◽  
Mild Conditions ◽  
Research Opportunity ◽  
Insight Into ◽  
Structural Strain

Carboncones, a special family of all-carbon allotropes, are predicted to have unique properties that distinguish them from fullerenes, carbon nanotubes, and graphenes. Owing to the absence of methods to synthesize atomically well-defined carboncones, however, experimental insight into the nature of pure carboncones has been inaccessible. Herein, we describe a facile synthesis of an atomically well-defined carboncone[1,2] (C70H20) and its soluble penta-mesityl derivative. Identified by x-ray crystallography, the carbon skeleton is a carboncone with the largest possible apex angle. Much of the structural strain is overcome in the final step of converting the bowl-shaped precursor into the rigid carboncone under mild reaction conditions. This work provides a research opportunity for investigations of atomically precise single-layered carboncones having even higher cone walls and/or smaller apex angles.

How big is big? Separation by conventional methods, X-ray and electronic structures of positional isomers of bis-tert-butylisocyano adduct of 2(3),9(10),16(17),23(24)-tetrachloro-3(2),10(9),17(16),24(23)-tetra(2,6-di-iso-propylphenoxy)-phthalocyaninato iron(II) complex

2016 ◽  
Vol 20 (01n04) ◽  
pp. 337-351 ◽  
Author(s):  
Derrick R. Anderson ◽  
Pavlo V. Solntsev ◽  
Hannah M. Rhoda ◽  
Victor N. Nemykin
Keyword(s):  
Electronic Structures ◽  
Density Functional ◽  
Positional Isomers ◽  
Excitation Energies ◽  
Functional Theory ◽  
X Ray ◽  
Vertical Excitation ◽  
X Ray Crystallography ◽  
Tddft Calculations ◽  
Insight Into

A presence of bulky 2,6-di-iso-propylphenoxy groups in bis-tert-butylisocyano adduct of 2(3),9(10),16(17),23(24)-tetrachloro-3(2),10(9),17(16),24(23)-tetra(2,6-di-iso-propylphenoxy)-phthalocyaninato iron(II) complex allows separation of two individual positional isomers and a mixture of the remaining two isomers using conventional chromatography. X-ray structures of “[Formula: see text]” and “[Formula: see text]” isomers were confimed by X-ray crystallography. Density functional theory (DFT) and time-dependent DFT (TDDFT) calculations of each individual positional isomer allowed insight into their electronic structures and vertical excitation energies, which were correlated with the experimental UV-vis and MCD spectra.

scholarly journals PHF13 is a molecular reader and transcriptional co-regulator of H3K4me2/3

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Ho-Ryun Chung ◽  
Chao Xu ◽  
Alisa Fuchs ◽  
Andreas Mund ◽  
Martin Lange ◽  
...  
Keyword(s):  
Dna Damage Response ◽  
Size Exclusion ◽  
X Ray ◽  
Chip Sequencing ◽  
X Ray Crystallography ◽  
Genome Wide ◽  
Damage Response ◽  
Exclusion Chromatography ◽  
Insight Into ◽  
Binding Cell

PHF13 is a chromatin affiliated protein with a functional role in differentiation, cell division, DNA damage response and higher chromatin order. To gain insight into PHF13's ability to modulate these processes, we elucidate the mechanisms targeting PHF13 to chromatin, its genome wide localization and its molecular chromatin context. Size exclusion chromatography, mass spectrometry, X-ray crystallography and ChIP sequencing demonstrate that PHF13 binds chromatin in a multivalent fashion via direct interactions with H3K4me2/3 and DNA, and indirectly via interactions with PRC2 and RNA PolII. Furthermore, PHF13 depletion disrupted the interactions between PRC2, RNA PolII S5P, H3K4me3 and H3K27me3 and resulted in the up and down regulation of genes functionally enriched in transcriptional regulation, DNA binding, cell cycle, differentiation and chromatin organization. Together our findings argue that PHF13 is an H3K4me2/3 molecular reader and transcriptional co-regulator, affording it the ability to impact different chromatin processes.

scholarly journals Structural characterization of the N-terminal part of the MERS-CoV nucleocapsid by X-ray diffraction and small-angle X-ray scattering

2016 ◽  
Vol 72 (2) ◽  
pp. 192-202 ◽  
Author(s):  
Nicolas Papageorgiou ◽  
Julie Lichière ◽  
Amal Baklouti ◽  
François Ferron ◽  
Marion Sévajol ◽  
...  
Keyword(s):  
Structural Model ◽  
Structural Features ◽  
Terminal Region ◽  
X Ray Diffraction ◽  
Terminal Part ◽  
Multifunctional Protein ◽  
X Ray ◽  
N Protein ◽  
Intrinsically Disordered ◽  
X Ray Scattering

The N protein of coronaviruses is a multifunctional protein that is organized into several domains. The N-terminal part is composed of an intrinsically disordered region (IDR) followed by a structured domain called the N-terminal domain (NTD). In this study, the structure determination of the N-terminal region of the MERS-CoV N proteinviaX-ray diffraction measurements is reported at a resolution of 2.4 Å. Since the first 30 amino acids were not resolved by X-ray diffraction, the structural study was completed by a SAXS experiment to propose a structural model including the IDR. This model presents the N-terminal region of the MERS-CoV as a monomer that displays structural features in common with other coronavirus NTDs.

scholarly journals A hybrid approach reveals the allosteric regulation of GTP cyclohydrolase I

2020 ◽  
Vol 117 (50) ◽  
pp. 31838-31849
Author(s):  
Rebecca Ebenhoch ◽  
Simone Prinz ◽  
Susann Kaltwasser ◽  
Deryck J. Mills ◽  
Robert Meinecke ◽  
...  
Keyword(s):  
Crystal Packing ◽  
Substrate Binding ◽  
Regulatory Protein ◽  
Allosteric Regulation ◽  
Hybrid Approach ◽  
Site Directed Mutagenesis ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
X Ray ◽  
X Ray Crystallography

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1−GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.

scholarly journals Structural insight into the Staphylococcus aureus ATP-driven exporter of virulent peptide toxins

2020 ◽  
Vol 6 (40) ◽  
pp. eabb8219
Author(s):  
N. Zeytuni ◽  
S. W. Dickey ◽  
J. Hu ◽  
H. T. Chou ◽  
L. J. Worrall ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Bound State ◽  
Broad Spectrum Antibiotic ◽  
X Ray ◽  
X Ray Crystallography ◽  
Open Conformation ◽  
Mechanistic Insight ◽  
Cytolytic Peptides ◽  
Peptide Toxins ◽  
Insight Into

Staphylococcus aureus is a major human pathogen that has acquired alarming broad-spectrum antibiotic resistance. One group of secreted toxins with key roles during infection is the phenol-soluble modulins (PSMs). PSMs are amphipathic, membrane-destructive cytolytic peptides that are exported to the host-cell environment by a designated adenosine 5′-triphosphate (ATP)–binding cassette (ABC) transporter, the PSM transporter (PmtABCD). Here, we demonstrate that the minimal Pmt unit necessary for PSM export is PmtCD and provide its first atomic characterization by single-particle cryo-EM and x-ray crystallography. We have captured the transporter in the ATP-bound state at near atomic resolution, revealing a type II ABC exporter fold, with an additional cytosolic domain. Comparison to a lower-resolution nucleotide-free map displaying an “open” conformation and putative hydrophobic inner chamber of a size able to accommodate the binding of two PSM peptides provides mechanistic insight and sets the foundation for therapeutic design.

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