scholarly journals Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme

RNA ◽  
2018 ◽  
Vol 24 (11) ◽  
pp. 1542-1554 ◽  
Author(s):  
Neil A. White ◽  
Minako Sumita ◽  
Victor E. Marquez ◽  
Charles G. Hoogstraten
Keyword(s):  
Active Site ◽  
Conformational Dynamics ◽  
Catalytic Function

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

scholarly journals Crystal Structure of Escherichia coli Agmatinase: Catalytic Mechanism and Residues Relevant for Substrate Specificity

2021 ◽  
Vol 22 (9) ◽  
pp. 4769
Author(s):  
Pablo Maturana ◽  
María S. Orellana ◽  
Sixto M. Herrera ◽  
Ignacio Martínez ◽  
Maximiliano Figueroa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Conformational Dynamics ◽  
High Specificity ◽  
Arginine Decarboxylase ◽  
Space Groups ◽  
X Ray Crystallography ◽  
High Dynamics ◽  
Dynamics Simulations

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer’s, Parkinson’s, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.

In Vivo Consequences of Putative Active Site Mutations in Yeast DNA Polymerases α, ε, δ, and ζ

Genetics ◽  
2001 ◽  
Vol 159 (1) ◽  
pp. 47-64 ◽  
Author(s):  
Youri I Pavlov ◽  
Polina V Shcherbakova ◽  
Thomas A Kunkel
Keyword(s):  
Active Site ◽  
Dna Polymerases ◽  
Base Substitution ◽  
Loss Of Function ◽  
Chromosomal Dna ◽  
Strong Base ◽  
Dna Mismatch ◽  
Catalytic Function ◽  
Base Substitutions

Abstract Several amino acids in the active site of family A DNA polymerases contribute to accurate DNA synthesis. For two of these residues, family B DNA polymerases have conserved tyrosine residues in regions II and III that are suggested to have similar functions. Here we replaced each tyrosine with alanine in the catalytic subunits of yeast DNA polymerases α, δ, ε, and ζ and examined the consequences in vivo. Strains with the tyrosine substitution in the conserved SL/MYPS/N motif in region II in Polδ or Polε are inviable. Strains with same substitution in Rev3, the catalytic subunit of Polζ, are nearly UV immutable, suggesting severe loss of function. A strain with this substitution in Polα (pol1-Y869A) is viable, but it exhibits slow growth, sensitivity to hydroxyurea, and a spontaneous mutator phenotype for frameshifts and base substitutions. The pol1-Y869A/pol1-Y869A diploid exhibits aberrant growth. Thus, this tyrosine is critical for the function of all four eukaryotic family B DNA polymerases. Strains with a tyrosine substitution in the conserved NS/VxYG motif in region III in Polα, -δ, or -ε are viable and a strain with the homologous substitution in Rev3 is UV mutable. The Polα mutant has no obvious phenotype. The Polε (pol2-Y831A) mutant is slightly sensitive to hydroxyurea and is a semidominant mutator for spontaneous base substitutions and frameshifts. The Polδ mutant (pol3-Y708A) grows slowly, is sensitive to hydroxyurea and methyl methanesulfonate, and is a strong base substitution and frameshift mutator. The pol3-Y708A/pol3-Y708A diploid grows slowly and aberrantly. Mutation rates in the Polα, -δ, and -ε mutant strains are increased in a locus-specific manner by inactivation of PMS1-dependent DNA mismatch repair, suggesting that the mutator effects are due to reduced fidelity of chromosomal DNA replication. This could result directly from relaxed base selectivity of the mutant polymerases due to the amino acid changes in the polymerase active site. In addition, the alanine substitutions may impair catalytic function to allow a different polymerase to compete at the replication fork. This is supported by the observation that the pol3-Y708A mutation is recessive and its mutator effect is partially suppressed by disruption of the REV3 gene.

scholarly journals The Active Site of a Prototypical “Rigid” Drug Target is Marked by Extensive Conformational Dynamics

2020 ◽  
Vol 59 (51) ◽  
pp. 22916-22921
Author(s):  
Himanshu Singh ◽  
Chandan K. Das ◽  
Suresh K. Vasa ◽  
Kristof Grohe ◽  
Lars V. Schäfer ◽  
...  
Keyword(s):  
Active Site ◽  
Drug Target ◽  
Conformational Dynamics

scholarly journals Resolving the complex role of enzyme conformational dynamics in catalytic function

2012 ◽  
Vol 109 (15) ◽  
pp. 5699-5704 ◽  
Author(s):  
U. Doshi ◽  
L. C. McGowan ◽  
S. T. Ladani ◽  
D. Hamelberg
Keyword(s):  
Conformational Dynamics ◽  
Catalytic Function

A unique aromatic cluster near the active site of H. pylori CPA is essential for catalytic function

2021 ◽  
Author(s):  
Ditsa Sarkar ◽  
Ramachandran Vijayan ◽  
Samudrala Gourinath ◽  
Apurba Kumar Sau
Keyword(s):  
Active Site ◽  
Catalytic Function ◽  
H Pylori ◽  
Aromatic Cluster

scholarly journals Dynamism and Cooperativity Essential for Efficient Catalysis in Aspergillus Niger Monoamine Oxidase

2019 ◽  
Author(s):  
Christian Curado-Carballada ◽  
Ferran Feixas ◽  
Sílvia Osuna
Keyword(s):  
Aspergillus Niger ◽  
Monoamine Oxidase ◽  
Active Site ◽  
Conformational Dynamics ◽  
Catalytic Efficiency ◽  
Building Blocks ◽  
Chiral Amine ◽  
Evolutionary Pathway ◽  
Accelerated Molecular Dynamics ◽  
Open States

<p><b> </b><i>Aspergillus niger </i>Monoamine Oxidase (MAO-N) is a homodimeric enzyme responsible for the oxidation of amines into the corresponding imine. Laboratory evolved variants of MAO-N in combination with a non-selective chemical reductant represents a powerful strategy for the deracemisation of chiral amine mixtures and, thus, is of interest for obtaining chiral amine building blocks. MAO-N presents a rich conformational dynamics with a flexible ß-hairpin region that can adopt closed, partially closed and open states. Despite the ß-hairpin conformational dynamics is altered along the laboratory evolutionary pathway of MAO-N, the connection between the ß-hairpin conformational dynamics and active site catalysis still remains unclear. In this work, we use accelerated molecular dynamics to elucidate the potential interplay between the ß-hairpin conformational dynamics and catalytic activity in MAO-N wild type and its evolved D5 variant. Our study reveals a delicate communication between both MAO-N subunits that impacts the active site architecture, and thus its catalytic efficiency. In both MAO-N WT and the laboratory evolved D5 variant, the ß-hairpin conformation in one of the monomers affects the productive binding of the substrate in the active site of the other subunit. However, both MAO-N WT and D5 variants show a quite different behaviour due to the distal mutations introduced experimentally with Directed Evolution. </p>

scholarly journals A Single Point Mutation Controls the Rate of Interconversion Between the g+ and g− Rotamers of the Histidine 189 χ2 Angle That Activates Bacterial Enzyme I for Catalysis

2021 ◽  
Vol 8 ◽  
Author(s):  
Jeffrey A. Purslow ◽  
Jolene N. Thimmesch ◽  
Valeria Sivo ◽  
Trang T. Nguyen ◽  
Balabhadra Khatiwada ◽  
...  
Keyword(s):  
Protein Design ◽  
Active Site ◽  
Single Point ◽  
Conformational Dynamics ◽  
Phosphorylation Site ◽  
Phosphoryl Group ◽  
Single Point Mutation ◽  
Phosphotransferase System ◽  
Function Relationship ◽  
Enzyme I

Enzyme I (EI) of the bacterial phosphotransferase system (PTS) is a master regulator of bacterial metabolism and a promising target for development of a new class of broad-spectrum antibiotics. The catalytic activity of EI is mediated by several intradomain, interdomain, and intersubunit conformational equilibria. Therefore, in addition to its relevance as a drug target, EI is also a good model for investigating the dynamics/function relationship in multidomain, oligomeric proteins. Here, we use solution NMR and protein design to investigate how the conformational dynamics occurring within the N-terminal domain (EIN) affect the activity of EI. We show that the rotameric g+-to-g− transition of the active site residue His189 χ2 angle is decoupled from the state A-to-state B transition that describes a ∼90° rigid-body rearrangement of the EIN subdomains upon transition of the full-length enzyme to its catalytically competent closed form. In addition, we engineered EIN constructs with modulated conformational dynamics by hybridizing EIN from mesophilic and thermophilic species, and used these chimeras to assess the effect of increased or decreased active site flexibility on the enzymatic activity of EI. Our results indicate that the rate of the autophosphorylation reaction catalyzed by EI is independent from the kinetics of the g+-to-g− rotameric transition that exposes the phosphorylation site on EIN to the incoming phosphoryl group. In addition, our work provides an example of how engineering of hybrid mesophilic/thermophilic chimeras can assist investigations of the dynamics/function relationship in proteins, therefore opening new possibilities in biophysics.

scholarly journals The Active Site of a Prototypical “Rigid” Drug Target is Marked by Extensive Conformational Dynamics

2020 ◽  
Vol 132 (51) ◽  
pp. 23116-23121
Author(s):  
Himanshu Singh ◽  
Chandan K. Das ◽  
Suresh K. Vasa ◽  
Kristof Grohe ◽  
Lars V. Schäfer ◽  
...  
Keyword(s):  
Active Site ◽  
Drug Target ◽  
Conformational Dynamics

scholarly journals Design and Characterisation of Inhibitory Peptides against Bleg1_2478, an Evolutionary Divergent B3 Metallo-β-lactamase

Molecules ◽  
2020 ◽  
Vol 25 (24) ◽  
pp. 5797
Author(s):  
Gayathri Selvaraju ◽  
Thean Chor Leow ◽  
Abu Bakar Salleh ◽  
Yahaya M. Normi
Keyword(s):  
Active Site ◽  
Protein Complexes ◽  
Hypothetical Protein ◽  
Titration Calorimetry ◽  
Binding Properties ◽  
Vitro Assay ◽  
In Silico Docking ◽  
Inhibitory Peptides ◽  
Catalytic Function

Previously, a hypothetical protein (HP) termed Bleg1_2437 (currently named Bleg1_2478) from Bacillus lehensis G1 was discovered to be an evolutionary divergent B3 subclass metallo-β-lactamase (MBL). Due to the scarcity of clinical inhibitors for B3 MBLs and the divergent nature of Bleg1_2478, this study aimed to design and characterise peptides as inhibitors against Bleg1_2478. Through in silico docking, RSWPWH and SSWWDR peptides with comparable binding energy to ampicillin were obtained. In vitro assay results showed RSWPWH and SSWWDR inhibited the activity of Bleg1_2478 by 50% at concentrations as low as 0.90 µM and 0.50 µM, respectively. At 10 µM of RSWPWH and 20 µM of SSWWDR, the activity of Bleg1_2478 was almost completely inhibited. Isothermal titration calorimetry (ITC) analyses showed slightly improved binding properties of the peptides compared to ampicillin. Docked peptide–protein complexes revealed that RSWPWH bound near the vicinity of the Bleg1_2478 active site while SSWWDR bound at the center of the active site itself. We postulate that the peptides caused the inhibition of Bleg1_2478 by reducing or blocking the accessibility of its active site from ampicillin, thus hampering its catalytic function.

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