Isoform-Specific Destabilization of the Active Site Reveals Molecular Mechanism of Intrinsic Activation of KRas G13D

2019 ◽  
Author(s):  
Christian W. Johnson ◽  
Yi-Jang Lin ◽  
Derion Reid ◽  
Jillian Parker ◽  
Patrick Dischinger ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Active Site

scholarly journals Directly repeated insertion of 9-nucleotide sequence detected in penicillin-binding protein 2B gene of penicillin-resistant Streptococcus pneumoniae.

1996 ◽  
Vol 40 (5) ◽  
pp. 1257-1259 ◽  
Author(s):  
A Yamane ◽  
H Nakano ◽  
Y Asahi ◽  
K Ubukata ◽  
M Konno
Keyword(s):  
Streptococcus Pneumoniae ◽  
Nucleotide Sequence ◽  
Molecular Mechanism ◽  
Active Site ◽  
Binding Protein ◽  
Direct Repeat ◽  
Penicillin Binding Protein ◽  
Serine Residue ◽  
Class A ◽  
Class B

We investigated the molecular mechanism of 50 penicillin-resistant Streptococcus pneumoniae strains (penicillin: MIC, > or = 0.125 microgram/ml) having neither class A nor class B mutations in the penicillin-binding protein 2B gene (pbp2b). An analysis of the nucleotide sequences of the pbp2b genes from seven strains revealed an unique direct repeat of 9 nucleotides (TGGTATACT) between active-site serine (residue 385) and Ser-X-Asn (residues 442 to 444) motifs. The same insertion was detected in 13 strains.

scholarly journals Biochemical and structural characterization of oxygen-sensitive 2-thiouridine synthesis catalyzed by an iron-sulfur protein TtuA

2017 ◽  
Vol 114 (19) ◽  
pp. 4954-4959 ◽  
Author(s):  
Minghao Chen ◽  
Shin-ichi Asai ◽  
Shun Narai ◽  
Shusuke Nambu ◽  
Naoki Omura ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Enzymatic Activity ◽  
Molecular Mechanism ◽  
Active Site ◽  
Thermophilic Bacteria ◽  
Oxygen Sensitive ◽  
Sulfur Protein ◽  
Combined Action

Two-thiouridine (s2U) at position 54 of transfer RNA (tRNA) is a posttranscriptional modification that enables thermophilic bacteria to survive in high-temperature environments. s2U is produced by the combined action of two proteins, 2-thiouridine synthetase TtuA and 2-thiouridine synthesis sulfur carrier protein TtuB, which act as a sulfur (S) transfer enzyme and a ubiquitin-like S donor, respectively. Despite the accumulation of biochemical data in vivo, the enzymatic activity by TtuA/TtuB has rarely been observed in vitro, which has hindered examination of the molecular mechanism of S transfer. Here we demonstrate by spectroscopic, biochemical, and crystal structure analyses that TtuA requires oxygen-labile [4Fe-4S]-type iron (Fe)-S clusters for its enzymatic activity, which explains the previously observed inactivation of this enzyme in vitro. The [4Fe-4S] cluster was coordinated by three highly conserved cysteine residues, and one of the Fe atoms was exposed to the active site. Furthermore, the crystal structure of the TtuA-TtuB complex was determined at a resolution of 2.5 Å, which clearly shows the S transfer of TtuB to tRNA using its C-terminal thiocarboxylate group. The active site of TtuA is connected to the outside by two channels, one occupied by TtuB and the other used for tRNA binding. Based on these observations, we propose a molecular mechanism of S transfer by TtuA using the ubiquitin-like S donor and the [4Fe-4S] cluster.

Molecular mechanism of chromogenic substrate hydrolysis in the active site of human carboxylesterase-1

2021 ◽  
Vol 67 (3) ◽  
pp. 300-305
Author(s):  
A.M. Kulakova ◽  
M.G. Khrenova ◽  
A.V. Nemukhin
Keyword(s):  
Molecular Mechanism ◽  
Active Site ◽  
Molecular Mechanisms ◽  
Kinetic Equations ◽  
Binding Constants ◽  
Forward Direction ◽  
Rational Drug Design ◽  
Molecular Mechanics Calculations ◽  
Elementary Steps ◽  
Substrate Complex

Human carboxylesterases are involved in the protective processes of detoxification during the hydrolytic metabolism of xenobiotics. Knowledge of the molecular mechanisms of substrates hydrolysis in the enzymes active site is necessary for the rational drug design. In this work, the molecular mechanism of the hydrolysis reaction of para-nitrophenyl acetate in the active site of human carboxylesterase was determined using modern methods of molecular modeling. According to the combined method of quantum mechanics/molecular mechanics calculations, the chemical reaction occurs within four elementary steps, including two steps of the acylation stage, and two steps of the deacylation stage. All elementary steps have low energy barriers, with the gradual lowering of the intermediate energies that stimulates reaction in the forward direction. The molecular docking was used to estimate the binding constants of the enzyme-substrate complex and the dissociation constant of enzyme-product complexes. The effective kinetic parameters of the enzymatic hydrolysis in the active site of carboxylesterase are determined by numerical solution of the differential kinetic equations.

What Accounts for the Different Function in Photolyases and Cryptochromes: A Computational Study of Critical Events in the Protein Active Site

2019 ◽  
Author(s):  
Daniel Holub ◽  
Thilo Mast ◽  
Tomáš Kubař ◽  
Marcus Elstner ◽  
Natacha Gillet
Keyword(s):  
Experimental Data ◽  
Proton Transfer ◽  
Molecular Mechanism ◽  
Active Site ◽  
Computational Study ◽  
Great Influence ◽  
Functional Differentiation ◽  
E Coli ◽  
Comprehensive Picture ◽  
Protein Active Site

In the current work, we present a combination of various classical and quantum computational protocols to unveil the molecular mechanism of FAD protonation in <i>E. coli</i> photolyase and its mutant. A direct comparison to our previous study on the plant cryptochrome clearly shows the great influence of the electrostatic environment and the flexibility of the FAD pocket on the proton transfer mechanism. Additionally, we propose a proton transfer pathway for WT E. coli photolyase consistent with experimental observations. Taken together, our results and previous experimental data provide a comprehensive picture about the functional differentiation in the cryptochrome-photolyase family.

scholarly journals Isoform-Specific Destabilization of the Active Site Reveals a Molecular Mechanism of Intrinsic Activation of KRas G13D

Cell Reports ◽  
2019 ◽  
Vol 28 (6) ◽  
pp. 1538-1550.e7 ◽  
Author(s):  
Christian W. Johnson ◽  
Yi-Jang Lin ◽  
Derion Reid ◽  
Jillian Parker ◽  
Spiro Pavlopoulos ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Active Site

scholarly journals Molecular mechanism for vitamin C-derived C5-glyceryl-methylcytosine DNA modification catalyzed by algal TET homologue CMD1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wenjing Li ◽  
Tianlong Zhang ◽  
Mingliang Sun ◽  
Yu Shi ◽  
Xiao-Jie Zhang ◽  
...  
Keyword(s):  
Vitamin C ◽  
Molecular Mechanism ◽  
Active Site ◽  
Biochemical Data ◽  
Dna Modification ◽  
Binding Affinities ◽  
Activity Assay ◽  
Tet Proteins ◽  
Key Residues ◽  
Positively Charged

AbstractC5-glyceryl-methylcytosine (5gmC) is a novel DNA modification catalyzed by algal TET homologue CMD1 using vitamin C (VC) as co-substrate. Here, we report the structures of CMD1 in apo form and in complexes with VC or/and dsDNA. CMD1 exhibits comparable binding affinities for DNAs of different lengths, structures, and 5mC levels, and displays a moderate substrate preference for 5mCpG-containing DNA. CMD1 adopts the typical DSBH fold of Fe2+/2-OG-dependent dioxygenases. The lactone form of VC binds to the active site and mono-coordinates the Fe2+ in a manner different from 2-OG. The dsDNA binds to a positively charged cleft of CMD1 and the 5mC/C is inserted into the active site and recognized by CMD1 in a similar manner as the TET proteins. The functions of key residues are validated by mutagenesis and activity assay. Our structural and biochemical data together reveal the molecular mechanism for the VC-derived 5gmC DNA modification by CMD1.

scholarly journals Structural insights into the molecular mechanism of the m6A writer complex

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Paweł Śledź ◽  
Martin Jinek
Keyword(s):  
Molecular Mechanism ◽  
Active Site ◽  
Evolutionary History ◽  
Rna Binding ◽  
Biological Processes ◽  
Catalytic Core ◽  
Genome Regulation ◽  
History Of ◽  
Structural Insights ◽  
Critical Aspects

Methylation of adenosines at the N6 position (m6A) is a dynamic and abundant epitranscriptomic mark that regulates critical aspects of eukaryotic RNA metabolism in numerous biological processes. The RNA methyltransferases METTL3 and METTL14 are components of a multisubunit m6A writer complex whose enzymatic activity is substantially higher than the activities of METTL3 or METTL14 alone. The molecular mechanism underpinning this synergistic effect is poorly understood. Here we report the crystal structure of the catalytic core of the human m6A writer complex comprising METTL3 and METTL14. The structure reveals the heterodimeric architecture of the complex and donor substrate binding by METTL3. Structure-guided mutagenesis indicates that METTL3 is the catalytic subunit of the complex, whereas METTL14 has a degenerate active site and plays non-catalytic roles in maintaining complex integrity and substrate RNA binding. These studies illuminate the molecular mechanism and evolutionary history of eukaryotic m6A modification in post-transcriptional genome regulation.

scholarly journals Molecular mechanism of activation of the immunoregulatory amidase NAAA

2018 ◽  
Vol 115 (43) ◽  
pp. E10032-E10040 ◽  
Author(s):  
Alexei Gorelik ◽  
Ahmad Gebai ◽  
Katalin Illes ◽  
Daniele Piomelli ◽  
Bhushan Nagar
Keyword(s):  
Molecular Mechanism ◽  
Active Site ◽  
Lipid Membranes ◽  
Acyl Chain ◽  
Reversible Inhibitor ◽  
Therapeutic Drug ◽  
Inhibitor Binding ◽  
Stable Substrate ◽  
Molecular Mechanism Of Action ◽  
Antiinflammatory Effects

Palmitoylethanolamide is a bioactive lipid that strongly alleviates pain and inflammation in animal models and in humans. Its signaling activity is terminated through degradation by N-acylethanolamine acid amidase (NAAA), a cysteine hydrolase expressed at high levels in immune cells. Pharmacological inhibitors of NAAA activity exert profound analgesic and antiinflammatory effects in rodent models, pointing to this protein as a potential target for therapeutic drug discovery. To facilitate these efforts and to better understand the molecular mechanism of action of NAAA, we determined crystal structures of this enzyme in various activation states and in complex with several ligands, including both a covalent and a reversible inhibitor. Self-proteolysis exposes the otherwise buried active site of NAAA to allow catalysis. Formation of a stable substrate- or inhibitor-binding site appears to be conformationally coupled to the interaction of a pair of hydrophobic helices in the enzyme with lipid membranes, resulting in the creation of a linear hydrophobic cavity near the active site that accommodates the ligand’s acyl chain.

scholarly journals Amylase action pattern on starch polymers

Biologia ◽  
2008 ◽  
Vol 63 (6) ◽  
Author(s):  
Annabel Bijttebier ◽  
Hans Goesaert ◽  
Jan Delcour
Keyword(s):  
Molecular Mechanism ◽  
Active Site ◽  
Action Pattern ◽  
Binding Properties ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Random Manner ◽  
Different Sources ◽  
Multiple Attack

AbstractSeveral decades ago, the first reports on differences in action pattern between amylases from different sources indicated that the starch polymers are not degraded in a completely random manner. We here give an overview of different action patterns of amylases on amylose and amylopectin, focusing on the so-called multiple attack action of the enzymes. Nowadays, the multiple attack action is generally an accepted concept to explain the differences in amylase action pattern. However, the pancreatic α-amylase remains one of the few enzymes known with a considerable level of multiple attack action. Despite some recent studies, the molecular mechanism of the multiple attack action is still largely unclear. Probably, the degree to which the active site architecture and binding properties allow both the reorganization (sliding) of the substrate in the active site and the stabilisation of the productive enzyme/substrate complex mainly determine the multiple attack action of amylases.

scholarly journals The interaction of α2-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism

1973 ◽  
Vol 133 (4) ◽  
pp. 709-724 ◽  
Author(s):  
Alan J. Barrett ◽  
Phyllis M. Starkey
Keyword(s):  
Conformational Change ◽  
Molecular Mechanism ◽  
Active Site ◽  
Peptide Bond ◽  
Serine Proteinases ◽  
Equimolar Ratio ◽  
Binding Characteristics ◽  
Sensitive Region ◽  
Physiological Importance ◽  
Α2 Macroglobulin

1. α2-Macroglobulin is known to bind and inhibit a number of serine proteinases. We show that it binds thiol and carboxyl proteinases, and there is now reason to believe that α2-macroglobulin can bind essentially all proteinases. 2. Radiochemically labelled trypsin, chymotrypsin, cathepsin B1 and papain are bound by α2-macroglobulin in an approximately equimolar ratio. Equimolar binding was confirmed for trypsin by activesite titration. 3. Pretreatment of α2-macroglobulin with a saturating amount of one proteinase prevented the subsequent binding of another. We conclude that each molecule of α2-macroglobulin is able to react with one molecule of proteinase only. 4. α2-Macroglobulin did not react with exopeptidases, non-proteolytic hydrolases or inactive forms of endopeptidases. 5. The literature on binding and inhibition of proteinases by α2-macroglobulin is reviewed, and from consideration of this and our own work several general characteristics of the interaction can be discerned. 6. A model is proposed for the molecular mechanism of the interaction of α2-macroglobulin with proteinases. It is suggested that the enzyme cleaves a peptide bond in a sensitive region of the macroglobulin, and that this results in a conformational change in the α2-macroglobulin molecule that traps the enzyme irreversibly. Access of substrates to the active site of the enzyme becomes sterically hindered, causing inhibition that is most pronounced with large substrate molecules. 7. The possible physiological importance of the unique binding characteristics of α2-macroglobulin is discussed.

Sign in / Sign up

Export Citation Format

Share Document