A new catalytic mechanism of bacterial ferredoxin‐NADP + reductases due to a particular NADP + binding mode

2021 ◽  
Author(s):  
Paula Monchietti ◽  
Arleth S. López Rivero ◽  
Eduardo A. Ceccarelli ◽  
Daniela L. Catalano‐Dupuy
Keyword(s):  
Catalytic Mechanism ◽  
Binding Mode ◽  
Bacterial Ferredoxin

scholarly journals Structural and Functional Analyses of Human ChaC2 in Glutathione Metabolism

Biomolecules ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 31 ◽  
Author(s):  
Yen T. K. Nguyen ◽  
Joon Sung Park ◽  
Jun Young Jang ◽  
Kyung Rok Kim ◽  
Tam T. L. Vo ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Catalytic Mechanism ◽  
Docking Study ◽  
Binding Mode ◽  
Structural Features ◽  
Glutathione Metabolism ◽  
Binding Assays ◽  
Biochemical Analyses ◽  
Functional Analyses

Glutathione (GSH) degradation plays an essential role in GSH homeostasis, which regulates cell survival, especially in cancer cells. Among human GSH degradation enzymes, the ChaC2 enzyme acts on GSH to form 5-l-oxoproline and Cys-Gly specifically in the cytosol. Here, we report the crystal structures of ChaC2 in two different conformations and compare the structural features with other known γ-glutamylcyclotransferase enzymes. The unique flexible loop of ChaC2 seems to function as a gate to achieve specificity for GSH binding and regulate the constant GSH degradation rate. Structural and biochemical analyses of ChaC2 revealed that Glu74 and Glu83 play crucial roles in directing the conformation of the enzyme and in modulating the enzyme activity. Based on a docking study of GSH to ChaC2 and binding assays, we propose a substrate-binding mode and catalytic mechanism. We also found that overexpression of ChaC2, but not mutants that inhibit activity of ChaC2, significantly promoted breast cancer cell proliferation, suggesting that the GSH degradation by ChaC2 affects the growth of breast cancer cells. Our structural and functional analyses of ChaC2 will contribute to the development of inhibitors for the ChaC family, which could effectively regulate the progression of GSH degradation-related cancers.

scholarly journals Structural dynamics and catalytic mechanism of ATP13A2 (PARK9) from simulations

2021 ◽  
Author(s):  
Teodora Mateeva ◽  
Marco Klaehn ◽  
Edina Rosta
Keyword(s):  
Barrier Height ◽  
Catalytic Reaction ◽  
Active Site ◽  
Electrostatic Interactions ◽  
Catalytic Mechanism ◽  
Atp Hydrolysis ◽  
Binding Mode ◽  
Full Length ◽  
Dimensional Structure ◽  
Missense Mutations

ATP13A2 is a gene encoding a protein of the P5B subfamily of ATPases and is a PARK gene. Molecular defects of the gene are mainly associated with variations of Parkinson's disease (PD). Despite the established importance of the protein in regulating neuronal integrity, the three-dimensional structure of the protein currently remains unresolved crystallographically. We have modelled the structure and reactivity of the full-length protein in its E1-ATP state. Using Molecular Dynamics (MD), Quantum cluster and Quantum Mechanical/Molecular mechanical (QM/MM) methods, we aimed at describing the main catalytic reaction, leading to the phosphorylation of Asp513. Our MD simulations suggest that two positively charged Mg2+ cations are present at the active site during the catalytic reaction, stabilizing a specific triphosphate binding mode. Using QM/MM calculations, we subsequently calculated the reaction profiles for the phosphate transfer step in the presence of one and two Mg2+ cations. The calculated barrier heights in both cases are found to be around 10.5 and 13.0 kcal mol-1, respectively. We elucidated details of the catalytically competent ATP conformation and the binding mode of the second Mg2+ cofactor. We also examined the role of the conserved Arg686 and Lys859 catalytic residues. We observed that by lowering significantly the barrier height of the ATP hydrolysis reaction, Arg686 had significant effect on the reaction. The removal of Arg686 increased the barrier height for the ATP hydrolysis by ~3.5 kcal mol-1 while the removal of key electrostatic interactions created by Lys859 to the gamma-phosphate and Arg513 destabilizes the reactant state. When missense mutations occur in close proximity to an active site residue, they can interfere with the barrier height of the reaction, which can halt the normal enzymatic rate of the protein. We also identified the main binding pockets in the full-length structure, including the pocket in the transmembrane region, which is likely where ATP13A2 cargo binds.

scholarly journals High Consistency of Structure-Based Design and X-Ray Crystallography: Design, Synthesis, Kinetic Evaluation and Crystallographic Binding Mode Determination of Biphenyl-N-acyl-β-d-Glucopyranosylamines as Glycogen Phosphorylase Inhibitors

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1322 ◽  
Author(s):  
Thomas Fischer ◽  
Symeon M. Koulas ◽  
Anastasia S. Tsagkarakou ◽  
Efthimios Kyriakis ◽  
George A. Stravodimos ◽  
...  
Keyword(s):  
Glycogen Phosphorylase ◽  
Catalytic Mechanism ◽  
Liver Glycogen ◽  
Binding Mode ◽  
Structural Water ◽  
Design Synthesis ◽  
Kinetic Evaluation ◽  
X Ray ◽  
X Ray Crystallography ◽  
Design And Synthesis

Structure-based design and synthesis of two biphenyl-N-acyl-β-d-glucopyranosylamine derivatives as well as their assessment as inhibitors of human liver glycogen phosphorylase (hlGPa, a pharmaceutical target for type 2 diabetes) is presented. X-ray crystallography revealed the importance of structural water molecules and that the inhibitory efficacy correlates with the degree of disturbance caused by the inhibitor binding to a loop crucial for the catalytic mechanism. The in silico-derived models of the binding mode generated during the design process corresponded very well with the crystallographic data.

scholarly journals Structural and functional effect of Trp-62→Gly and Asp-101→Gly substitutions on substrate-binding modes of mutant hen egg-white lysozymes

1998 ◽  
Vol 333 (1) ◽  
pp. 71-76 ◽  
Author(s):  
Katsumi MAENAKA ◽  
Masaaki MATSUSHIMA ◽  
Gota KAWAI ◽  
Akinori KIDERA ◽  
Kimitsuna WATANABE ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Protein Structures ◽  
Binding Mode ◽  
Hydrolytic Activity ◽  
Inhibitory Effect ◽  
Egg White ◽  
Binding Modes ◽  
Wild Type ◽  
Hen Egg White ◽  
Hen Egg

In order to clarify the structural role of subsite B of hen egg-white lysozyme in hydrolytic activity towards a carbohydrate substrate, we analysed the structures of Trp-62 → Gly and Asp-101 → Gly mutant hen lysozymes, which have no side chain at positions 62 or 101, complexed with a substrate analogue, (N-acetyl-d-glucosamine)3 [(GlcNAc)3], using X-ray crystallography. The overall protein structures in the mutant lysozyme complexes were almost identical to those in the wild type. In the crystals of all the mutant complexes, the (GlcNAc)3 molecule, which is an inhibitor of wild-type lysozyme, had no inhibitory effect, but was hydrolysed as a substrate. One of the products, (GlcNAc)2, the reducing end of which is an α-anomer, was bound in an unproductive binding mode, protruding from the active-site cleft, and was able to act as an inhibitor. Hydrolysis of the synthetic substrate by the mutants occurred in a β-anomer-retaining manner, and so the α-anomer product was converted from the β-anomer product. Thus the interactions of Asp-101 and Trp-62 in subsite B are not essential for the catalytic mechanism, but co-operatively enhance the affinity of the substrate in the productive binding mode, other than the inhibitor in the unproductive mode.

scholarly journals Structural aspects of the inhibition and catalytic mechanism of thymidylate synthase.

1995 ◽  
Vol 42 (4) ◽  
pp. 367-380 ◽  
Author(s):  
L W Hardy
Keyword(s):  
Conformational Changes ◽  
Thymidylate Synthase ◽  
Catalytic Mechanism ◽  
Isotope Effects ◽  
Binding Mode ◽  
Amino Acid Residues ◽  
Structure Based Drug Design ◽  
E Coli ◽  
Mechanistic Basis ◽  
Covalent Intermediate

Thymidylate synthase (TS) is a target for anticancer drugs, due to its unique role in the biosynthesis of an essential DNA precursor. The X-ray structures available for several bacterial enzymes have been used to design novel inhibitors of TS, to structurally analyze the binding mode of existing inhibitors, and to propose catalytic roles for amino-acid residues on the protein. The first part of this paper describes some aspects of structure-based drug design, including a recent result from the groups of Montfort and Maley emphasizing the importance of conformational changes in inhibitor binding. The second part of the paper describes the work of the author on the TS mechanism, especially the catalytic roles of active site amino acids Asn177 and Glu58 in TS from Escherichia coli. An important function for Glu58 is proposed to be preventing the excessive stabilization of a covalent intermediate. The use of isotope effects to probe the mechanistic basis for stimulation of E. coli TS by magnesium ions, and to identify differences between the E. coli and human enzymes, is described. The hypothesis that N5 of tetrahydrofolate provides the basicity for deprotonation of the nucleotide is also discussed.

Laue and monochromatic crystallography on carbonic anhydrase

1992 ◽  
Vol 340 (1657) ◽  
pp. 301-309 ◽  
Keyword(s):  
Carbonic Anhydrase ◽  
Structural Changes ◽  
Catalytic Mechanism ◽  
Structural Information ◽  
Binding Mode ◽  
Molecular Structures ◽  
Data Sets ◽  
Time Resolved ◽  
Zinc Enzyme ◽  
Unexpected Findings

We wanted to analyse the binding mode of small anion inhibitors to the zinc enzyme carbonic anhydrase in order to explore the binding of substrates and the catalytic mechanism of the enzyme. This was started by recording two data-sets by Laue diffraction to obtain the wanted structural information. In addition we wanted to test the capacity of the Laue method to show the small structural changes that are often associated with the catalytic activity of m any enzymes. To be able to exploit fully time-resolved crystallography the method should be able to detect such minor structural changes. The obtained Laue results did not agree with the expected molecular structures. Thus we needed to record monochromatic data-sets of the same states of the enzyme to confirm our results. All major findings from the Laue data agree with the monochromatic data. Stimulated by the unexpected findings we have continued the investigations of anion binding to carbonic anhydrase. We have studied both the zinc enzyme and replaced the native metal by cobalt which also yields an active enzyme. The accumulated picture of the ligand binding to the enzyme sheds new light on the substrate binding and on the catalytic mechanism.

scholarly journals A structural study of TatD from Staphylococcus aureus elucidates a putative DNA-binding mode of a Mg2+-dependent nuclease

IUCrJ ◽  
2020 ◽  
Vol 7 (3) ◽  
pp. 509-521
Author(s):  
Kyu-Yeon Lee ◽  
Seung-Ho Cheon ◽  
Dong-Gyun Kim ◽  
Sang Jae Lee ◽  
Bong-Jin Lee
Keyword(s):  
Crystal Structure ◽  
Staphylococcus Aureus ◽  
Dna Binding ◽  
Catalytic Mechanism ◽  
Nuclease Activity ◽  
Binding Mode ◽  
Functional Study ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Key Residues

TatD has been thoroughly investigated as a DNA-repair enzyme and an apoptotic nuclease, and still-unknown TatD-related DNases are considered to play crucial cellular roles. However, studies of TatD from Gram-positive bacteria have been hindered by an absence of atomic detail and the resulting inability to determine function from structure. In this study, an X-ray crystal structure of SAV0491, which is the TatD enzyme from the Gram-positive bacterium Staphylococcus aureus (SaTatD), is reported at a high resolution of 1.85 Å with a detailed atomic description. Although SaTatD has the common TIM-barrel fold shared by most TatD-related homologs, and PDB entry 2gzx shares 100% sequence identity with SAV0491, the crystal structure of SaTatD revealed a unique binding mode of two phosphates interacting with two Ni2+ ions. Through a functional study, it was verified that SaTatD has Mg2+-dependent nuclease activity as a DNase and an RNase. In addition, structural comparison with TatD homologs and the identification of key residues contributing to the binding mode of Ni2+ ions and phosphates allowed mutational studies to be performed that revealed the catalytic mechanism of SaTatD. Among the key residues composing the active site, the acidic residues Glu92 and Glu202 had a critical impact on catalysis by SaTatD. Furthermore, based on the binding mode of the two phosphates and structural insights, a putative DNA-binding mode of SaTatD was proposed using in silico docking. Overall, these findings may serve as a good basis for understanding the relationship between the structure and function of TatD proteins from Gram-positive bacteria and may provide critical insights into the DNA-binding mode of SaTatD.

scholarly journals High Consistency of Structure-Based Design and X-Ray Crystallography: Design, Synthesis, Kinetic Evaluation and Crystallographic Binding Mode Determination of Biphenyl-N-Acyl-β-D-Glucopyranosylamines as Glycogen Phosphorylase Inhibitors

2019 ◽  
Author(s):  
Thomas Fischer ◽  
Symeon M. Koulas ◽  
Anastasia S. Tsagkarakou ◽  
Efthimios Kyriakis ◽  
George A. Stravodimos ◽  
...  
Keyword(s):  
Glycogen Phosphorylase ◽  
Catalytic Mechanism ◽  
Liver Glycogen ◽  
Binding Mode ◽  
Structural Water ◽  
Design Synthesis ◽  
Kinetic Evaluation ◽  
X Ray ◽  
X Ray Crystallography ◽  
Design And Synthesis

Structure-based design and synthesis of two biphenyl-N-acyl-β-D-glucopyranosylamine derivatives as well as their assessment as inhibitors of human liver glycogen phosphorylase (hlGPa, a pharmaceutical target for type 2 diabetes) is presented. X-ray crystallography revealed the importance of structural water molecules and that the inhibitory efficacy correlates with the degree of disturbance caused by the inhibitor binding to a loop crucial for the catalytic mechanism. The in silico derived models of the binding mode generated during the design process corresponded very well with the crystallographic data.

Probing the structural basis of oxygen binding in a cofactor-independent dioxygenase

2017 ◽  
Vol 73 (7) ◽  
pp. 573-580 ◽  
Author(s):  
Kunhua Li ◽  
Elisha N. Fielding ◽  
Heather L. Condurso ◽  
Steven D. Bruner
Keyword(s):  
Catalytic Mechanism ◽  
Binding Pocket ◽  
Binding Mode ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
General Mechanism ◽  
Oxygen Binding ◽  
Migration Pathway ◽  
And Migration ◽  
Key Residues

The enzyme DpgC is included in the small family of cofactor-independent dioxygenases. The chemistry of DpgC is uncommon as the protein binds and utilizes dioxygen without the aid of a metal or organic cofactor. Previous structural and biochemical studies identified the substrate-binding mode and the components of the active site that are important in the catalytic mechanism. In addition, the results delineated a putative binding pocket and migration pathway for the co-substrate dioxygen. Here, structural biology is utilized, along with site-directed mutagenesis, to probe the assigned dioxygen-binding pocket. The key residues implicated in dioxygen trafficking were studied to probe the process of binding, activation and chemistry. The results support the proposed chemistry and provide insight into the general mechanism of dioxygen binding and activation.

scholarly journals The active site and substrate-binding mode of 1-aminocyclopropane-1-carboxylate oxidase determined by site-directed mutagenesis and comparative modelling studies

2004 ◽  
Vol 380 (2) ◽  
pp. 339-346 ◽  
Author(s):  
Young Sam SEO ◽  
Ahrim YOO ◽  
Jinwon JUNG ◽  
Soon-Kee SUNG ◽  
Dae Ryook YANG ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Binding Mode ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Directed Mutagenesis ◽  
Comparative Modelling

The active site and substrate-binding mode of MD-ACO1 (Malus domestica Borkh. 1-aminocyclopropane-1-carboxylate oxidase) have been determined using site-directed mutagenesis and comparative modelling methods. The MD-ACO1 protein folds into a compact jelly-roll motif comprised of eight α-helices, 12 β-strands and several long loops. The active site is well defined as a wide cleft near the C-terminus. The co-substrate ascorbate is located in cofactor Fe2+-binding pocket, the so-called ‘2-His-1-carboxylate facial triad’. In addition, our results reveal that Arg244 and Ser246 are involved in generating the reaction product during enzyme catalysis. The structure agrees well with the biochemical and site-directed mutagenesis results. The three-dimensional structure together with the steady-state kinetics of both the wild-type and mutant MD-ACO1 proteins reveal how the substrate specificity of MD-ACO1 is involved in the catalytic mechanism, providing insights into understanding the fruit ripening process at atomic resolution.

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