scholarly journals Crystallization and Crystallographic Analysis of a Bradyrhizobium Elkanii USDA94 Haloalkane Dehalogenase Variant with an Eliminated Halide-Binding Site

Crystals ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 375 ◽  
Author(s):  
Tatyana Prudnikova ◽  
Barbora Kascakova ◽  
Jeroen R. Mesters ◽  
Pavel Grinkevich ◽  
Petra Havlickova ◽  
...  
Keyword(s):  
Binding Site ◽  
Data Bank ◽  
Crystallographic Analysis ◽  
Chloride Ions ◽  
Monoclinic Space Group ◽  
Haloalkane Dehalogenase ◽  
Molecular Tagging ◽  
In The Wild ◽  
Bradyrhizobium Elkanii

Haloalkane dehalogenases are a very important class of microbial enzymes for environmental detoxification of halogenated pollutants, for biocatalysis, biosensing and molecular tagging. The double mutant (Ile44Leu + Gln102His) of the haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 (DbeAΔCl) was constructed to study the role of the second halide-binding site previously discovered in the wild-type structure. The variant is less active, less stable in the presence of chloride ions and exhibits significantly altered substrate specificity when compared with the DbeAwt. DbeAΔCl was crystallized using the sitting-drop vapour-diffusion procedure with further optimization by the random microseeding technique. The crystal structure of the DbeAΔCl has been determined and refined to the 1.4 Å resolution. The DbeAΔCl crystals belong to monoclinic space group C121. The DbeAΔCl molecular structure was characterized and compared with five known haloalkane dehalogenases selected from the Protein Data Bank.

Structural and functional analysis of a novel haloalkane dehalogenase with two halide-binding sites

2014 ◽  
Vol 70 (7) ◽  
pp. 1884-1897 ◽  
Author(s):  
Radka Chaloupkova ◽  
Tatyana Prudnikova ◽  
Pavlina Rezacova ◽  
Zbynek Prokop ◽  
Tana Koudelakova ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Substrate Inhibition ◽  
Chloride Ions ◽  
The Novel ◽  
Haloalkane Dehalogenase ◽  
Enzyme Functionality ◽  
Rate Limiting ◽  
Lower Basicity

The crystal structure of the novel haloalkane dehalogenase DbeA fromBradyrhizobium elkaniiUSDA94 revealed the presence of two chloride ions buried in the protein interior. The first halide-binding site is involved in substrate binding and is present in all structurally characterized haloalkane dehalogenases. The second halide-binding site is unique to DbeA. To elucidate the role of the second halide-binding site in enzyme functionality, a two-point mutant lacking this site was constructed and characterized. These substitutions resulted in a shift in the substrate-specificity class and were accompanied by a decrease in enzyme activity, stability and the elimination of substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the rate-limiting hydrolytic step mediated by the lower basicity of the catalytic histidine.

scholarly journals The Potential Role of 6-gingerol and 6-shogaol as ACE Inhibitors in Silico Study

2020 ◽  
Vol 8 (2) ◽  
pp. 210
Author(s):  
Yohanes Bare ◽  
Maria Helvina ◽  
Gabriella Chandrakirana Krisnamurti ◽  
Mansur S
Keyword(s):  
Amino Acid ◽  
Angiotensin Converting Enzyme ◽  
Binding Site ◽  
Potential Role ◽  
Enzyme Inhibitor ◽  
Renin Angiotensin System ◽  
Data Bank ◽  
Amino Acid Residues ◽  
Converting Enzyme

Hypertension has become the third highest cause of death in Indonesia. The condition is correlated with angiotensin-converting enzyme (ACE), and possibly managed with the use of drugs. In addition, some natural compounds, including 6-shogaol and 6-gingerol from ginger, are used to decrease blood pressure. However, the mechanism and binding site of these compounds to ACE protein is currently unclear. This study, therefore, aims to investigate the potential role of these compounds as an angiotensin-converting enzyme inhibitor. The ACE protein was downloaded from Protein Data Bank (PDB) database with the ID: 3bkk, while the 6-shogaol (CID: 5281794) and 6-gingerol (CID: 44559528) ligands were obtained from the PubChem database. Meanwhile, molecular docking was established using HEX 8.0.0 software. The analysis examined the amino acid residues and the bonds formed from these interactions. According to the results, fourteen amino acid residues were formed by the interaction between 6-shogaol and ACE, while the interaction between 6-gingerol and ACE formed eight amino acids. Also, thirteen amino acid residues in the novelty binding site of ACE were discovered to be blocked by the ligands from ginger. Therefore, the compounds have potential roles as inhibitors, and this possibly helps to prevent regulation of the renin-angiotensin system. These interactions also formed hydrogen bonds, as well as electrostatic, unfavorable, and hydrophobic sites, making the binding stronger than others. 

scholarly journals Structural basis of degenerate specificity of an anti-sugar antibody

2014 ◽  
Vol 70 (a1) ◽  
pp. C258-C258
Author(s):  
Sharad Vashisht ◽  
Kanwal Kaur ◽  
Dinakar Salunke
Keyword(s):  
Binding Site ◽  
Molecular Mimicry ◽  
Conformational Flexibility ◽  
Carbohydrate Antigen ◽  
Model System ◽  
Crystallographic Analysis ◽  
Reducing Sugar ◽  
Structural Basis ◽  
Immune Recognition

Although remarkable specificity of the acquired immune system was clearly being demonstrated, degenerate specificity in immune recognition is often been observed. We had started working on degenerate specificity of antibodies using peptide and sugar as a model system. Both carbohydrate antigen (me-α-Man) and peptide (DVFYPYPYASGS) were established to be equivalent in polyclonal as well as in mAb (2D10) responses (1). Thermodynamic analysis of Ag-Ab interactions had suggested the role of conformational flexibility (2) while crystallographic analysis indicated the importance of plasticity in the interactions (3) of the antigen combining site in the manifestation of molecular mimicry. It has been pointed out that even if the potential for flexibility existed, it was not being utilized while recognizing both ligands. So, in order to address this conundrum we started looking for other sugars and peptides that can bind to the mAb 2D10 with comparable affinities. Crystallographic analysis of 2D10 binding to five different sugars (me-α-Glc, α-Lac, α1-3-Mannobiose, α1-6-Mannobiose, α1-3, α1-6-Mannotriose) has given insights underlying the basis of specificity in molecular recognition. Comparison of all the structures has demonstrated that the antigen combining site for sugars is constituted of CDR H3, L1 and L3 only. All five sugars have an overlapping primary binding site (equivalent to me-α-Man interacting region). This primary sugar binding site has been shown to accommodate similar as well as dissimilar sugars by utilizing plasticity in the interacting residues available in the antigen combining site. The reducing sugar of the similar disaccharides (α1-3-Mannobiose, α1-6-Mannobiose) have been adjusted in the same direction but with utilizing different sets of interacting residues of the antibody paratope. However, the reducing sugar of a dissimilar disaccharide (α-Lac) exploits different paratope space altogether. The trisaccharide (α1-3, α1-6-Mannotriose) was accommodated in the same site by utilizing the conformational flexibility in the paratope region (mainly in CDR L1). This study had demonstrated that an affinity matured antibody may utilize at least three different strategies in order to accommodate structurally similar/dissimilar sugars.

Cell-specific regulation of IRS-1 gene expression: role of E box and C/EBP binding site in HepG2 cells and CHO cells

Diabetes ◽  
1997 ◽  
Vol 46 (3) ◽  
pp. 354-362 ◽  
Author(s):  
K. Matsuda ◽  
E. Araki ◽  
R. Yoshimura ◽  
K. Tsuruzoe ◽  
N. Furukawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Cho Cells ◽  
Hepg2 Cells ◽  
E Box ◽  
Specific Regulation

scholarly journals Transcriptome Changes in Pseudomonas putida KT2440 during Medium-Chain-Length Polyhydroxyalkanoate Synthesis Induced by Nitrogen Limitation

2020 ◽  
Vol 22 (1) ◽  
pp. 152
Author(s):  
Dorota Dabrowska ◽  
Justyna Mozejko-Ciesielska ◽  
Tomasz Pokój ◽  
Slawomir Ciesielski
Keyword(s):  
Chain Length ◽  
Nitrogen Limitation ◽  
Stringent Response ◽  
Wild Type ◽  
Pseudomonas Putida Kt2440 ◽  
Medium Chain ◽  
Environmental Stimuli ◽  
Medium Chain Length ◽  
In The Wild

Pseudomonas putida’s versatility and metabolic flexibility make it an ideal biotechnological platform for producing valuable chemicals, such as medium-chain-length polyhydroxyalkanoates (mcl-PHAs), which are considered the next generation bioplastics. This bacterium responds to environmental stimuli by rearranging its metabolism to improve its fitness and increase its chances of survival in harsh environments. Mcl-PHAs play an important role in central metabolism, serving as a reservoir of carbon and energy. Due to the complexity of mcl-PHAs’ metabolism, the manner in which P. putida changes its transcriptome to favor mcl-PHA synthesis in response to environmental stimuli remains unclear. Therefore, our objective was to investigate how the P. putida KT2440 wild type and mutants adjust their transcriptomes to synthesize mcl-PHAs in response to nitrogen limitation when supplied with sodium gluconate as an external carbon source. We found that, under nitrogen limitation, mcl-PHA accumulation is significantly lower in the mutant deficient in the stringent response than in the wild type or the rpoN mutant. Transcriptome analysis revealed that, under N-limiting conditions, 24 genes were downregulated and 21 were upregulated that were common to all three strains. Additionally, potential regulators of these genes were identified: the global anaerobic regulator (Anr, consisting of FnrA, Fnrb, and FnrC), NorR, NasT, the sigma54-dependent transcriptional regulator, and the dual component NtrB/NtrC regulator all appear to play important roles in transcriptome rearrangement under N-limiting conditions. The role of these regulators in mcl-PHA synthesis is discussed.

scholarly journals Premating barriers in young sympatric snail species

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Arina L. Maltseva ◽  
Marina A. Varfolomeeva ◽  
Arseniy A. Lobov ◽  
Polina O. Tikanova ◽  
Egor A. Repkin ◽  
...  
Keyword(s):  
Gene Flow ◽  
Snail Species ◽  
Sympatric Populations ◽  
Gastropod Species ◽  
Shell Size ◽  
Closely Related Species ◽  
Partial Canonical Correspondence Analysis ◽  
In The Wild ◽  
Isolation Index

AbstractSympatric coexistence of recently diverged species raises the question of barriers restricting the gene flow between them. Reproductive isolation may be implemented at several levels, and the weakening of some, e.g. premating, barriers may require the strengthening of the others, e.g. postcopulatory ones. We analysed mating patterns and shell size of mates in recently diverged closely related species of the subgenus Littorina Neritrema (Littorinidae, Caenogastropoda) in order to assess the role of premating reproductive barriers between them. We compared mating frequencies observed in the wild with those expected based on relative densities using partial canonical correspondence analysis. We introduced the fidelity index (FI) to estimate the relative accuracy of mating with conspecific females and precopulatory isolation index (IPC) to characterize the strength of premating barriers. The species under study, with the exception of L. arcana, clearly demonstrated preferential mating with conspecifics. According to FI and IPC, L. fabalis and L. compressa appeared reliably isolated from their closest relatives within Neritrema. Individuals of these two species tend to be smaller than those of the others, highlighting the importance of shell size changes in gastropod species divergence. L. arcana males were often found in pairs with L. saxatilis females, and no interspecific size differences were revealed in this sibling species pair. We discuss the lack of discriminative mate choice in the sympatric populations of L. arcana and L. saxatilis, and possible additional mechanisms restricting gene flow between them.

scholarly journals Group size and aquatic vegetation modulates male preferences for female shoals in wild zebrafish, Danio rerio

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Aditya Ghoshal ◽  
Anuradha Bhat
Keyword(s):  
Group Size ◽  
Aquatic Vegetation ◽  
Natural Habitat ◽  
Ecological Factors ◽  
Choice Test ◽  
Female Group ◽  
Aquatic Flora ◽  
In The Wild ◽  
The Individual

AbstractShoaling decisions in the wild are determined by a combination of innate preferences of the individual along with the interplay of multiple ecological factors. In their natural habitat as well as in the laboratory, zebrafish is a shoaling fish. Here, we investigate the role of group size and associated vegetation in shaping shoaling preferences of wild male zebrafish. We studied the association preference of males to groups of female shoals in a multi-choice test design. We found that males made greater proportion of visits to an 8-female group compared to 2 and 4-female groups. However, males spent similar proportions of time across the three female-containing groups. When artificial vegetation was incorporated along with female number as an additional factor, we found that males prefer high and moderately vegetated patches compared to low or no-vegetation groups, irrespective of the number of females in these patches. Based on experiments using a novel multi-choice design, our results show that preference for group size can change due to interaction of two separate factors. This work is a first attempt to understand the role of aquatic flora in determining shoaling preferences in zebrafish, using an experimental paradigm consisting of a gradation in female and vegetation densities.

scholarly journals Controlled Transcription of Regulator Gene carS by Tet-on or by a Strong Promoter Confirms Its Role as a Repressor of Carotenoid Biosynthesis in Fusarium fujikuroi

2020 ◽  
Vol 9 (1) ◽  
pp. 71
Author(s):  
Julia Marente ◽  
Javier Avalos ◽  
M. Carmen Limón
Keyword(s):  
Wild Strain ◽  
Ring Finger ◽  
Carotenoid Biosynthesis ◽  
Negative Regulator ◽  
Fusarium Fujikuroi ◽  
Structural Genes ◽  
Gene Encoding ◽  
In The Wild ◽  
Set Up

Carotenoid biosynthesis is a frequent trait in fungi. In the ascomycete Fusarium fujikuroi, the synthesis of the carboxylic xanthophyll neurosporaxanthin (NX) is stimulated by light. However, the mutants of the carS gene, encoding a protein of the RING finger family, accumulate large NX amounts regardless of illumination, indicating the role of CarS as a negative regulator. To confirm CarS function, we used the Tet-on system to control carS expression in this fungus. The system was first set up with a reporter mluc gene, which showed a positive correlation between the inducer doxycycline and luminescence. Once the system was improved, the carS gene was expressed using Tet-on in the wild strain and in a carS mutant. In both cases, increased carS transcription provoked a downregulation of the structural genes of the pathway and albino phenotypes even under light. Similarly, when the carS gene was constitutively overexpressed under the control of a gpdA promoter, total downregulation of the NX pathway was observed. The results confirmed the role of CarS as a repressor of carotenogenesis in F. fujikuroi and revealed that its expression must be regulated in the wild strain to allow appropriate NX biosynthesis in response to illumination.

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