scholarly journals Effect of Divalent Metal Ion on the Structure, Stability and Function of Klebsiella pneumoniae Nicotinate-Nucleotide Adenylyltransferase: Empirical and Computational Studies

2021 ◽  
Vol 23 (1) ◽  
pp. 116
Author(s):  
Olamide Jeje ◽  
Reabetswe Maake ◽  
Ruan van Deventer ◽  
Veruschka Esau ◽  
Emmanuel Amarachi Iwuchukwu ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Conformational Changes ◽  
Metal Ion ◽  
Therapeutic Potential ◽  
Divalent Cations ◽  
Homology Modelling ◽  
Substrate Binding ◽  
Structure Stability ◽  
Atp Binding ◽  
The Impact

The continuous threat of drug-resistant Klebsiella pneumoniae justifies identifying novel targets and developing effective antibacterial agents. A potential target is nicotinate nucleotide adenylyltransferase (NNAT), an indispensable enzyme in the biosynthesis of the cell-dependent metabolite, NAD+. NNAT catalyses the adenylation of nicotinamide/nicotinate mononucleotide (NMN/NaMN), using ATP to form nicotinamide/nicotinate adenine dinucleotide (NAD+/NaAD). In addition, it employs divalent cations for co-substrate binding and catalysis and has a preference for different divalent cations. Here, the biophysical structure of NNAT from K. pneumoniae (KpNNAT) and the impact of divalent cations on its activity, conformational stability and substrate-binding are described using experimental and computational approaches. The experimental study was executed using an enzyme-coupled assay, far-UV circular dichroism, extrinsic fluorescence spectroscopy, and thermal shift assays, alongside homology modelling, molecular docking, and molecular dynamic simulation. The structure of KpNNAT revealed a predominately α-helical secondary structure content and a binding site that is partially hydrophobic. Its substrates ATP and NMN share the same binding pocket with similar affinity and exhibit an energetically favourable binding. KpNNAT showed maximum activity and minimal conformational changes with Mg2+ as a cofactor compared to Zn2+, Cu2+ and Ni2+. Overall, ATP binding affects KpNNAT dynamics, and the dynamics of ATP binding depend on the presence and type of divalent cation. The data obtained from this study would serve as a basis for further evaluation towards designing structure-based inhibitors with therapeutic potential.

scholarly journals Zinc limitation in Klebsiella pneumoniae profiled by quantitative proteomics influences transcriptional regulation and cation transporter-associated capsule production

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
A. Sukumaran ◽  
S. Pladwig ◽  
J. Geddes-McAlister
Keyword(s):  
Klebsiella Pneumoniae ◽  
Metal Ion ◽  
Ion Homeostasis ◽  
Cellular Protein ◽  
Pcr Analysis ◽  
Phenotypic Analysis ◽  
Metal Ion Homeostasis ◽  
Capsule Production ◽  
The Impact

Abstract Background Microbial organisms encounter a variety of environmental conditions, including changes to metal ion availability. Metal ions play an important role in many biological processes for growth and survival. As such, microbes alter their cellular protein levels and secretion patterns in adaptation to a changing environment. This study focuses on Klebsiella pneumoniae, an opportunistic bacterium responsible for nosocomial infections. By using K. pneumoniae, we aim to determine how a nutrient-limited environment (e.g., zinc depletion) modulates the cellular proteome and secretome of the bacterium. By testing virulence in vitro, we provide novel insight into bacterial responses to limited environments in the presence of the host. Results Analysis of intra- and extracellular changes identified 2380 proteins from the total cellular proteome (cell pellet) and 246 secreted proteins (supernatant). Specifically, HutC, a repressor of the histidine utilization operon, showed significantly increased abundance under zinc-replete conditions, which coincided with an expected reduction in expression of genes within the hut operon from our validating qRT-PCR analysis. Additionally, we characterized a putative cation transport regulator, ChaB that showed significantly higher abundance under zinc-replete vs. -limited conditions, suggesting a role in metal ion homeostasis. Phenotypic analysis of a chaB deletion strain demonstrated a reduction in capsule production, zinc-dependent growth and ion utilization, and reduced virulence when compared to the wild-type strain. Conclusions This is first study to comprehensively profile the impact of zinc availability on the proteome and secretome of K. pneumoniae and uncover a novel connection between zinc transport and capsule production in the bacterial system.

scholarly journals Chaperonins

1998 ◽  
Vol 333 (2) ◽  
pp. 233-242 ◽  
Author(s):  
Neil A. RANSON ◽  
Helen E. WHITE ◽  
Helen R. SAIBIL
Keyword(s):  
Conformational Changes ◽  
Substrate Binding ◽  
Atp Binding ◽  
Dependent Manner ◽  
Oligomeric Proteins ◽  
Double Ring ◽  
Substrate Protein ◽  
Hydrophobic Binding ◽  
Substrate Binding Sites

The molecular chaperones are a diverse set of protein families required for the correct folding, transport and degradation of other proteins in vivo. There has been great progress in understanding the structure and mechanism of action of the chaperonin family, exemplified by Escherichia coli GroEL. The chaperonins are large, double-ring oligomeric proteins that act as containers for the folding of other protein subunits. Together with its co-protein GroES, GroEL binds non-native polypeptides and facilitates their refolding in an ATP-dependent manner. The action of the ATPase cycle causes the substrate-binding surface of GroEL to alternate in character between hydrophobic (binding/unfolding) and hydrophilic (release/folding). ATP binding initiates a series of dramatic conformational changes that bury the substrate-binding sites, lowering the affinity for non-native polypeptide. In the presence of ATP, GroES binds to GroEL, forming a large chamber that encapsulates substrate proteins for folding. For proteins whose folding is absolutely dependent on the full GroE system, ATP binding (but not hydrolysis) in the encapsulating ring is needed to initiate protein folding. Similarly, ATP binding, but not hydrolysis, in the opposite GroEL ring is needed to release GroES, thus opening the chamber. If the released substrate protein is still not correctly folded, it will go through another round of interaction with GroEL.

scholarly journals Zinc limitation in Klebsiella pneumoniae profiled by quantitative proteomics influences transcriptional regulation and cation transporter-associated capsule production

2019 ◽  
Author(s):  
Arjun Sukumaran ◽  
Jennifer Geddes-McAlister
Keyword(s):  
Klebsiella Pneumoniae ◽  
Environmental Conditions ◽  
Metal Ion ◽  
Cellular Protein ◽  
Pcr Analysis ◽  
Phenotypic Analysis ◽  
Growth And Survival ◽  
Expression Of Genes ◽  
Capsule Production ◽  
The Impact

Abstract Background: Microbial organisms encounter a variety of environmental conditions, including changes to metal ion availability. Metals ions play an important role in many biological processes for growth and survival. As such, microbes alter their cellular protein regulation and secretion patterns in adaptation to changing environmental conditions. This study focuses on Klebsiella pneumoniae, an opportunistic bacterium responsible for nosocomial infections and by using K. pneumoniae, we aim to determine how a nutrient-limited environment (e.g., zinc) modulates the cellular proteome and secretome of the bacteria. This information will inform on protein-level regulation of bacterial responses to nutritional immunity within the host and improve our understanding of the dynamic and complex relationship between host and pathogen during infection. Results: Analysis of intra- and extracellular changes identified 2,380 proteins from the total cellular proteome (cell pellet) and 246 secreted proteins (supernatant). Specifically, hutC, a repressor of the histidine utilization operon, showed significantl increases abundance under replete conditions, which coincided with an expected reduction in expression of genes within the hut operon from our validation qRT-PCR analysis. Additionally, we characterized a putative cation transport regulator, chaB that was significantly abundant under zinc-replete conditions. Phenotypic analysis of a chaBdeletion strain observe a reduction in capsule production, greater tolerance to high extracellular zinc concentrations, and unimpaired virulence when compared to the WT strain. Conclusions: This is first study to comprehensively profile the impact of zinc availability on the proteome and secretome of K. pneumoniae and uncover a novel connection between zinc transport and capsule production in the bacterial system.

scholarly journals Observing force-regulated conformational changes and ligand dissociation from a single integrin on cells

2012 ◽  
Vol 199 (3) ◽  
pp. 497-512 ◽  
Author(s):  
Wei Chen ◽  
Jizhong Lou ◽  
Evan A. Evans ◽  
Cheng Zhu
Keyword(s):  
Conformational Changes ◽  
Dynamic Equilibrium ◽  
Divalent Cations ◽  
Control Cell ◽  
Intercellular Adhesion Molecule ◽  
Intercellular Adhesion Molecule 1 ◽  
Biomembrane Force Probe ◽  
Tensile Forces ◽  
A Cell ◽  
The Impact

As adhesion molecules, integrins connect a cell to its environment and transduce signals across the membrane. Their different functional states correspond to distinct conformations. Using a biomembrane force probe, we observed real-time reversible switches between bent and extended conformations of a single integrin, αLβ2, on the surface of a living cell by measuring its nanometer-scale headpiece displacements, bending and unbending frequencies, and molecular stiffness changes. We determined the stabilities of these conformations, their dynamic equilibrium, speeds and rates of conformational changes, and the impact of divalent cations and tensile forces. We quantified how initial and subsequent conformations of αLβ2 regulate the force-dependent kinetics of dissociation from intercellular adhesion molecule 1. Our findings provide new insights into how integrins function as nanomachines to precisely control cell adhesion and signaling.

scholarly journals Zinc limitation in Klebsiella pneumoniae profiled by quantitative proteomics influences transcriptional regulation and cation transporter-associated capsule production

2021 ◽  
Author(s):  
Arjun Sukumaran ◽  
Samanta Pladwig ◽  
Jennifer Geddes-McAlister
Keyword(s):  
Klebsiella Pneumoniae ◽  
Metal Ion ◽  
Ion Homeostasis ◽  
Cellular Protein ◽  
Pcr Analysis ◽  
Phenotypic Analysis ◽  
Metal Ion Homeostasis ◽  
Capsule Production ◽  
The Impact

Abstract Background: Microbial organisms encounter a variety of environmental conditions, including changes to metal ion availability. Metal ions play an important role in many biological processes for growth and survival. As such, microbes alter their cellular protein levels and secretion patterns in adaptation to a changing environment. This study focuses on Klebsiella pneumoniae, an opportunistic bacterium responsible for nosocomial infections. By using K. pneumoniae, we aim to determine how a nutrient-limited environment (e.g., zinc depletion) modulates the cellular proteome and secretome of the bacterium. By testing virulence in vitro, we provide novel insight into bacterial responses to limited environments in the presence of the host. Results: Analysis of intra- and extracellular changes identified 2,380 proteins from the total cellular proteome (cell pellet) and 246 secreted proteins (supernatant). Specifically, HutC, a repressor of the histidine utilization operon, showed significantly increased abundance under zinc-replete conditions, which coincided with an expected reduction in expression of genes within the hut operon from our validating qRT-PCR analysis. Additionally, we characterized a putative cation transport regulator, ChaB that showed significantly higher abundance under zinc-replete vs. -limited conditions, suggesting a role in metal ion homeostasis. Phenotypic analysis of a chaB deletion strain demonstrated a reduction in capsule production, zinc-dependent growth and ion utilization, and reduced virulence when compared to the wild-type strain. Conclusions: This is first study to comprehensively profile the impact of zinc availability on the proteome and secretome of K. pneumoniae and uncover a novel connection between zinc transport and capsule production in the bacterial system.

pH-Dependent Thermal Stability of Vibrio cholerae L-asparaginase

2019 ◽  
Vol 26 (10) ◽  
pp. 743-750 ◽  
Author(s):  
Remya Radha ◽  
Sathyanarayana N. Gummadi
Keyword(s):  
Vibrio Cholerae ◽  
Conformational Changes ◽  
Kinetic Studies ◽  
Structural Features ◽  
Structure Stability ◽  
Kcal Mole ◽  
Scanning Calorimetry ◽  
Wide Range ◽  
Ph Dependent ◽  
Ph Range

Background:pH is one of the decisive macromolecular properties of proteins that significantly affects enzyme structure, stability and reaction rate. Change in pH may protonate or deprotonate the side group of aminoacid residues in the protein, thereby resulting in changes in chemical and structural features. Hence studies on the kinetics of enzyme deactivation by pH are important for assessing the bio-functionality of industrial enzymes. L-asparaginase is one such important enzyme that has potent applications in cancer therapy and food industry.Objective:The objective of the study is to understand and analyze the influence of pH on deactivation and stability of Vibrio cholerae L-asparaginase.Methods:Kinetic studies were conducted to analyze the effect of pH on stability and deactivation of Vibrio cholerae L-asparaginase. Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) studies have been carried out to understand the pH-dependent conformational changes in the secondary structure of V. cholerae L-asparaginase.Results:The enzyme was found to be least stable at extreme acidic conditions (pH< 4.5) and exhibited a gradual increase in melting temperature from 40 to 81 °C within pH range of 4.0 to 7.0. Thermodynamic properties of protein were estimated and at pH 7.0 the protein exhibited ΔG37of 26.31 kcal mole-1, ΔH of 204.27 kcal mole-1 and ΔS of 574.06 cal mole-1 K-1.Conclusion:The stability and thermodynamic analysis revealed that V. cholerae L-asparaginase was highly stable over a wide range of pH, with the highest stability in the pH range of 5.0–7.0.

scholarly journals BOARD INVITED REVIEW: The pig microbiota and the potential for harnessing the power of the microbiome to improve growth and health1

2019 ◽  
Vol 97 (9) ◽  
pp. 3741-3757 ◽  
Author(s):  
Nirosh D Aluthge ◽  
Dana M Van Sambeek ◽  
Erin E Carney-Hinkle ◽  
Yanshuo S Li ◽  
Samodha C Fernando ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Gut Microbiota ◽  
Therapeutic Potential ◽  
Animal Health ◽  
Microbial Colonization ◽  
Critical Importance ◽  
Specific Host ◽  
Microbiome Research ◽  
Health And Disease ◽  
The Impact

Abstract A variety of microorganisms inhabit the gastrointestinal tract of animals including bacteria, archaea, fungi, protozoa, and viruses. Pioneers in gut microbiology have stressed the critical importance of diet:microbe interactions and how these interactions may contribute to health status. As scientists have overcome the limitations of culture-based microbiology, the importance of these interactions has become more clear even to the extent that the gut microbiota has emerged as an important immunologic and metabolic organ. Recent advances in metagenomics and metabolomics have helped scientists to demonstrate that interactions among the diet, the gut microbiota, and the host to have profound effects on animal health and disease. However, although scientists have now accumulated a great deal of data with respect to what organisms comprise the gastrointestinal landscape, there is a need to look more closely at causative effects of the microbiome. The objective of this review is intended to provide: 1) a review of what is currently known with respect to the dynamics of microbial colonization of the porcine gastrointestinal tract; 2) a review of the impact of nutrient:microbe effects on growth and health; 3) examples of the therapeutic potential of prebiotics, probiotics, and synbiotics; and 4) a discussion about what the future holds with respect to microbiome research opportunities and challenges. Taken together, by considering what is currently known in the four aforementioned areas, our overarching goal is to set the stage for narrowing the path towards discovering how the porcine gut microbiota (individually and collectively) may affect specific host phenotypes.

Sign in / Sign up

Export Citation Format

Share Document