scholarly journals Structural characterization of a GNAT family acetyltransferase from Elizabethkingia anophelis bound to acetyl-CoA reveals a new dimeric interface

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
P. Shirmast ◽  
S. M. Ghafoori ◽  
R. M. Irwin ◽  
J. Abendroth ◽  
S. J. Mayclin ◽  
...  
Keyword(s):  
High Resolution ◽  
Tertiary Structure ◽  
Crystallographic Structure ◽  
Acetyl Coa ◽  
Diverse Range ◽  
In Silico Docking ◽  
Dimeric Interface ◽  
High Resolution Structure ◽  
Acetyl Group

AbstractGeneral control non-repressible 5 (GCN5)-related N-acetyltransferases (GNATs) catalyse the acetylation of a diverse range of substrates, thereby orchestrating a variety of biological processes within prokaryotes and eukaryotes. GNAT enzymes can catalyze the transfer of an acetyl group from acetyl coenzyme A to substrates such as aminoglycoside antibiotics, amino acids, polyamines, peptides, vitamins, catecholamines, and large macromolecules including proteins. Although GNATs generally exhibit low to moderate sequence identity, they share a conserved catalytic fold and conserved structural motifs. In this current study we characterize the high-resolution X-ray crystallographic structure of a GNAT enzyme bound with acetyl-CoA from Elizabethkingia anophelis, an important multi-drug resistant bacterium. The tertiary structure is comprised of six α-helices and nine β-strands, and is similar with other GNATs. We identify a new and uncharacterized GNAT dimer interface, which is conserved in at least two other unpublished GNAT structures. This suggests that GNAT enzymes can form at least five different types of dimers, in addition to a range of other oligomers including trimer, tetramer, hexamer, and dodecamer assemblies. The high-resolution structure presented in this study is suitable for future in-silico docking and structure–activity relationship studies.

scholarly journals High resolution structural and functional analysis of a hemopexin motif protein from Dolichos

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Sarita Chandan Sharma ◽  
Ashish Kumar ◽  
Sharad Vashisht ◽  
Dinakar M. Salunke
Keyword(s):  
High Resolution ◽  
Peroxidase Activity ◽  
Tertiary Structure ◽  
Seed Proteins ◽  
X Ray Crystallography ◽  
Surface Charge Distribution ◽  
2S Albumins ◽  
High Resolution Structure ◽  
Conserved Water Molecules ◽  
Germinating Seeds

AbstractIt is increasingly evident that seed proteins exhibit specific functions in plant physiology. However, many proteins remain yet to be functionally characterized. We have screened the seed proteome of Dolichos which lead to identification and purification of a protein, DC25. The protein was monomeric and highly thermostable in extreme conditions of pH and salt. It was crystallized and structure determined at 1.28 Å resolution using x-ray crystallography. The high-resolution structure of the protein revealed a four-bladed β-propeller hemopexin-type fold containing pseudo four-fold molecular symmetry at the central channel. While the structure exhibited homology with 2S albumins, variations in the loops connecting the outermost strands and the differences in surface-charge distribution may be relevant for distinct functions. Comparative study of the protein with other seed hemopexins revealed the presence of four conserved water molecules in between the blades which cross-link them and maintain the tertiary structure. The protein exhibited intrinsic peroxidase activity, which could be inhibited by binding of a heme analog. The identification of redox-sensitive cysteine and inhibition of peroxidase activity by iodoacetamide facilitated characterization of the possible active site. The determined peroxidase activity of DC25 may be responsible for rescuing germinating seeds from oxidative stress.

scholarly journals High-Resolution Structure of the Histidine-Containing Phosphocarrier Protein (HPr) from Staphylococcus aureus and Characterization of Its Interaction with the Bifunctional HPr Kinase/Phosphorylase

2004 ◽  
Vol 186 (17) ◽  
pp. 5906-5918 ◽  
Author(s):  
Till Maurer ◽  
Sebastian Meier ◽  
Norman Kachel ◽  
Claudia Elisabeth Munte ◽  
Sonja Hasenbein ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
High Resolution ◽  
Core Protein ◽  
Phosphorylation Site ◽  
Hydroxyl Group ◽  
Nmr Data ◽  
High Resolution Structure ◽  
Structural Restraints ◽  
Resolution Structure

ABSTRACT A high-resolution structure of the histidine-containing phosphocarrier protein (HPr) from Staphylococcus aureus was obtained by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy on the basis of 1,766 structural restraints. Twenty-three hydrogen bonds in HPr could be directly detected by polarization transfer from the amide nitrogen to the carbonyl carbon involved in the hydrogen bond. Differential line broadening was used to characterize the interaction of HPr with the HPr kinase/phosphorylase (HPrK/P) of Staphylococcus xylosus, which is responsible for phosphorylation-dephosphorylation of the hydroxyl group of the regulatory serine residue at position 46. The dissociation constant Kd was determined to be 0.10 ± 0.02 mM at 303 K from the NMR data, assuming independent binding. The data are consistent with a stoichiometry of 1 HPr molecule per HPrK/P monomer in solution. Using transversal relaxation optimized spectroscopy-heteronuclear single quantum correlation, we mapped the interaction site of the two proteins in the 330-kDa complex. As expected, it covers the region around Ser46 and the small helix b following this residue. In addition, HPrK/P also binds to the second phosphorylation site of HPr at position 15. This interaction may be essential for the recognition of the phosphorylation state of His15 and the phosphorylation-dependent regulation of the kinase/phosphorylase activity. In accordance with this observation, the recently published X-ray structure of the HPr/HPrK core protein complex from Lactobacillus casei shows interactions with the two phosphorylation sites. However, the NMR data also suggest differences for the full-length protein from S. xylosus: there are no indications for an interaction with the residues preceding the regulatory Ser46 residue (Thr41 to Lys45) in the protein of S. xylosus. In contrast, it seems to interact with the C-terminal helix of HPr in solution, an interaction which is not observed for the complex of HPr with the core of HPrK/P of L. casei in crystals.

Characterization of the Chitinolytic Machinery of Enterococcus faecalis V583 and High-Resolution Structure of Its Oxidative CBM33 Enzyme

2012 ◽  
Vol 416 (2) ◽  
pp. 239-254 ◽  
Author(s):  
Gustav Vaaje-Kolstad ◽  
Liv Anette Bøhle ◽  
Sigrid Gåseidnes ◽  
Bjørn Dalhus ◽  
Magnar Bjørås ◽  
...  
Keyword(s):  
High Resolution ◽  
Enterococcus Faecalis ◽  
High Resolution Structure ◽  
Resolution Structure

scholarly journals Biochemical and Kinetic Characterization of the Eukaryotic Phosphotransacetylase Class IIa Enzyme from Phytophthora ramorum

2015 ◽  
Vol 14 (7) ◽  
pp. 652-660
Author(s):  
Tonya Taylor ◽  
Cheryl Ingram-Smith ◽  
Kerry S. Smith
Keyword(s):  
Phytophthora Ramorum ◽  
Regulatory Domain ◽  
Kinetic Characterization ◽  
Acetyl Phosphate ◽  
Acetyl Coa ◽  
Content Type ◽  
Regulatory Domains ◽  
Key Enzyme ◽  
Acetyl Group

ABSTRACT Phosphotransacetylase (Pta), a key enzyme in bacterial metabolism, catalyzes the reversible transfer of an acetyl group from acetyl phosphate to coenzyme A (CoA) to produce acetyl-CoA and P i . Two classes of Pta have been identified based on the absence (Pta I ) or presence (Pta II ) of an N-terminal regulatory domain. Pta I has been fairly well studied in bacteria and one genus of archaea; however, only the Escherichia coli and Salmonella enterica Pta II enzymes have been biochemically characterized, and they are allosterically regulated. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Pta from the oomycete Phytophthora ramorum . The two Ptas from P. ramorum , designated PrPta II 1 and PrPta II 2, both belong to class II. PrPta II 1 displayed positive cooperativity for both acetyl phosphate and CoA and is allosterically regulated. We compared the effects of different metabolites on PrPta II 1 and the S. enterica Pta II and found that, although the N-terminal regulatory domains share only 19% identity, both enzymes are inhibited by ATP, NADP, NADH, phosphoenolpyruvate (PEP), and pyruvate in the acetyl-CoA/P i -forming direction but are differentially regulated by AMP. Phylogenetic analysis of bacterial, archaeal, and eukaryotic sequences identified four subtypes of Pta II based on the presence or absence of the P-loop and DRTGG subdomains within the N-terminal regulatory domain. Although the E. coli , S. enterica , and P. ramorum enzymes all belong to the IIa subclass, our kinetic analysis has indicated that enzymes within a subclass can still display differences in their allosteric regulation.

scholarly journals High-resolution structure of AKR1a4 in the apo form and its interaction with ligands

2012 ◽  
Vol 68 (11) ◽  
pp. 1271-1274 ◽  
Author(s):  
Frédérick Faucher ◽  
Zongchao Jia
Keyword(s):  
Crystal Structure ◽  
High Resolution ◽  
Aldehyde Reductase ◽  
Founding Member ◽  
High Resolution Structure ◽  
Ascorbate Biosynthesis ◽  
Resolution Structure

Aldo-keto reductase 1a4 (AKR1a4; EC 1.1.1.2) is the mouse orthologue of human aldehyde reductase (AKR1a1), the founding member of the AKR family. As an NADPH-dependent enzyme, AKR1a4 catalyses the conversion of D-glucuronate to L-gulonate. AKR1a4 is involved in ascorbate biosynthesis in mice, but has also recently been found to interact with SMAR1, providing a novel mechanism of ROS regulation by ATM. Here, the crystal structure of AKR1a4 in its apo form at 1.64 Å resolution as well as the characterization of the binding of AKR1a4 to NADPH and P44, a peptide derived from SMAR1, is presented.

scholarly journals High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage

Cell Reports ◽  
2019 ◽  
Vol 26 (13) ◽  
pp. 3741-3751.e5 ◽  
Author(s):  
Ian M. Slaymaker ◽  
Pablo Mesa ◽  
Max J. Kellner ◽  
Soumya Kannan ◽  
Edward Brignole ◽  
...  
Keyword(s):  
High Resolution ◽  
Biochemical Characterization ◽  
Rna Targeting ◽  
High Resolution Structure ◽  
Resolution Structure

scholarly journals High-resolution structure of cas13b and biochemical characterization of RNA targeting and cleavage

Cell Reports ◽  
2021 ◽  
Vol 34 (10) ◽  
pp. 108865
Author(s):  
Ian M. Slaymaker ◽  
Pablo Mesa ◽  
Max J. Kellner ◽  
Soumya Kannan ◽  
Edward Brignole ◽  
...  
Keyword(s):  
High Resolution ◽  
Biochemical Characterization ◽  
Rna Targeting ◽  
High Resolution Structure ◽  
Resolution Structure
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