scholarly journals Biochemical Characterization and Structural Insight into Interaction and Conformation Mechanisms of Serratia marcescens Lysine Decarboxylase (SmcadA)

Molecules ◽  
2021 ◽  
Vol 26 (3) ◽  
pp. 697
Author(s):  
Tolbert Osire ◽  
Zhina Qiao ◽  
Taowei Yang ◽  
Meijuan Xu ◽  
Xian Zhang ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Structural Changes ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Acid Stress ◽  
Close Relation ◽  
Lysine Decarboxylase ◽  
Wild Type ◽  
Protein Residues ◽  
Insight Into

Inducible lysine decarboxylases (LDCs) are essential in various cellular processes of microorganisms and plants, especially under acid stress, which induces the expression of genes encoding LDCs. In this study, a novel Serratia marcesenes LDC (SmcadA) was successfully expressed in E. coli, purified and characterized. The protein had an optimal pH of 6 and a temperature of 40 °C and phylogenetic analysis to determine the evolution of SmcadA, which revealed a close relation to Enterobacteriaceae, Klebsiella sp., among others. The molecular weight of SmcadA was approximately 75 kDa after observation on SDS-PAGE and structural modeling showed the protein as a decamer, comprised of five interlinked dimers. The biocatalytic activity of the purified wild-type SmcadA (WT) was improved through site directed mutations and the results showed that the Arg595Lys mutant had the highest specific activity of 286.55 U/mg, while the Ser512Ala variant and wild-type SmcadA had 215.72 and 179.01 U/mg, respectively. Furthermore, molecular dynamics simulations revealed that interactions through hydrogen bonds between the protein residues and cofactor pyridoxal-5-phosphate (PLP) are vital for biocatalysis. Molecular Dynamics (MD) simulations also indicated that mutations conferred structural changes on protein residues and PLP hence altered the interacting residues with the cofactor, subsequently influencing substrate bioconversion. Moreover, the temperature also induced changes in orientation of cofactor PLP and amino acid residues. This work therefore demonstrates the successful expression and characterization of the purified novel lysine decarboxylase from Serratia marcesenes and provided insight into the mechanism of protein–cofactor interactions, highlighting the role of protein–ligand interactions in altering cofactor and binding site residue conformations, thus contributing to improved biocatalysis.

scholarly journals Biochemical Characterization of StyAB from Pseudomonas sp. Strain VLB120 as a Two-Component Flavin-Diffusible Monooxygenase

2004 ◽  
Vol 186 (16) ◽  
pp. 5292-5302 ◽  
Author(s):  
Katja Otto ◽  
Karin Hofstetter ◽  
Martina Röthlisberger ◽  
Bernard Witholt ◽  
Andreas Schmid
Keyword(s):  
Biochemical Characterization ◽  
Specific Activity ◽  
Styrene Oxide ◽  
Sole Source ◽  
Nadh Oxidation ◽  
Flavin Mononucleotide ◽  
Wild Type ◽  
Oxidation Activity ◽  
Epoxidation Reaction ◽  
Sequential Binding

ABSTRACT Pseudomonas sp. VLB120 uses styrene as a sole source of carbon and energy. The first step in this metabolic pathway is catalyzed by an oxygenase (StyA) and a NADH-flavin oxidoreductase (StyB). Both components have been isolated from wild-type Pseudomonas strain VLB120 as well as from recombinant Escherichia coli. StyA from both sources is a dimer, with a subunit size of 47 kDa, and catalyzes the enantioselective epoxidation of C═C double bonds. Styrene is exclusively converted to S-styrene oxide with a specific activity of 2.1 U mg−1 (k cat = 1.6 s−1) and Km values for styrene of 0.45 ± 0.05 mM (wild type) and 0.38 ± 0.09 mM (recombinant). The epoxidation reaction depends on the presence of a NADH-flavin adenine dinucleotide (NADH-FAD) oxidoreductase for the supply of reduced FAD. StyB is a dimer with a molecular mass of 18 kDa and a NADH oxidation activity of 200 U mg−1 (k cat [NADH] = 60 s−1). Steady-state kinetics determined for StyB indicate a mechanism of sequential binding of NADH and flavin to StyB. This enzyme reduces FAD as well as flavin mononucleotide and riboflavin. The NADH oxidation activity does not depend on the presence of StyA. During the epoxidation reaction, no formation of a complex of StyA and StyB has been observed, suggesting that electron transport between reductase and oxygenase occurs via a diffusing flavin.

scholarly journals Mechanistic Insights into the Effects of Key Mutations on SARS-CoV-2 RBD-ACE2 Binding

2021 ◽  
Author(s):  
Abhishek Aggarwal ◽  
Supriyo Naskar ◽  
Nikhil Maroli ◽  
Biswajit Gorai ◽  
Narendra M Dixit ◽  
...  
Keyword(s):  
Structural Changes ◽  
Underlying Mechanism ◽  
Free Energy Calculations ◽  
Target Cells ◽  
Original Strain ◽  
Binding Affinities ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Dynamics Simulations ◽  
Insight Into

Some recent SARS-CoV-2 variants appear to have increased transmissibility than the original strain. An underlying mechanism could be the improved ability of the variants to bind receptors on target cells and infect them. In this study, we provide atomic-level insight into the binding of the receptor binding domain (RBD) of the wild-type SARS-CoV-2 spike protein and its single (N501Y), double (E484Q, L452R) and triple (N501Y, E484Q, L452R) mutated variants to the human ACE2 receptor. Using extensive all-atom molecular dynamics simulations and advanced free energy calculations, we estimate the associated binding affinities and binding hotspots. We observe significant secondary structural changes in the RBD of the mutants, which lead to different binding affinities. We find higher binding affinities of the double (E484Q, L452R) and triple (N501Y, E484Q, L452R) mutated variants than the wild type and the N501Y variant, which could contribute to the higher transmissibility of recent variants containing these mutations.

Evaluation of Luciferase Thermal Stability by Arginine Saturation in the Flexible Loops

2020 ◽  
Vol 17 (1) ◽  
pp. 30-39
Author(s):  
Farzane Kargar ◽  
Mojtaba Mortazavi ◽  
Masoud Torkzadeh-Mahani ◽  
Safa Lotfi ◽  
Shahryar Shakeri
Keyword(s):  
Thermal Stability ◽  
Structural Changes ◽  
Specific Activity ◽  
Temperature Stability ◽  
3D Models ◽  
Firefly Luciferase ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Original Activity ◽  
Luciferase Enzyme

Background: The firefly luciferase enzyme is widely used in protein engineering and diverse areas of biotechnology, but the main problem with this enzyme is low-temperature stability. Previous reports indicated that surface areas of thermostable proteins are rich in arginine, which increased their thermal stability. In this study, this aspect of thermophilic proteins evaluated by mutations of surface residues to Arg. Here, we report the construction, purification, and studying of these mutated luciferases. Methods: For mutagenesis, the QuikChange site-directed mutagenesis was used and the I108R, T156R, and N177R mutant luciferases were created. In the following, the expression and purification of wild-type and mutant luciferases were conducted and their kinetic and structural properties were analyzed. To analyze the role of these Arg in these loops, the 3D models of these mutants’ enzymes were constructed in the I-TASSER server and the exact situation of these mutants was studied by the SPDBV and PyMOL software. Results: Overall, the optimum temperature of these mutated enzymes was not changed. However, after 30 min incubation of these mutated enzymes at 30°C, the I108R, T156R, N177R, and wild-type kept the 80%, 50%, 20%, and 20% of their original activity, respectively. It should be noted that substitution of these residues by Arg preserved the specific activity of firefly luciferase. Conclusion: Based on these results, it can be concluded that T156R and N177R mutants by compacting local protein structure, increased the thermostability of luciferase. However, insertion of positively charged residues in these positions create the new hydrogen bonds that associated with a series of structural changes and confirmed by intrinsic and extrinsic fluorescence spectroscopy and homology modeling studies.

scholarly journals Lysine Decarboxylase Expression by Vibrio vulnificus Is Induced by SoxR in Response to Superoxide Stress

2006 ◽  
Vol 188 (24) ◽  
pp. 8586-8592 ◽  
Author(s):  
Ju-Sim Kim ◽  
Sang Ho Choi ◽  
Jeong K. Lee
Keyword(s):  
Oxidative Stress ◽  
Superoxide Dismutase ◽  
Promoter Region ◽  
Methyl Viologen ◽  
Vibrio Vulnificus ◽  
External Medium ◽  
Gene Dosage ◽  
Acid Stress ◽  
Lysine Decarboxylase ◽  
Wild Type

ABSTRACT Lysine decarboxylase expression by Vibrio vulnificus, which is up-regulated by CadC in response to acid stress, is also induced by SoxR in response to superoxide stress. SoxR binds to the promoter region of the cadBA operon, coding for a lysine-cadaverine antiporter (CadB) and a lysine decarboxylase (CadA). The induction of cadBA transcription by SoxR is independent of CadC. Cadaverine, which neutralizes the external medium, also appears to scavenge superoxide radicals, since increasing cellular cadaverine by elevating the gene dosage of cadBA significantly diminished the induction of Mn-containing superoxide dismutase under methyl viologen-induced oxidative stress. Consistently, a lack of cadaverine caused by mutation in cadA resulted in low tolerance to oxidative stress compared with that of the wild type.

scholarly journals Molecular Dynamics of SARS-CoV-2 Nsp1 Structural Changes Associated with Variant Mutations

2021 ◽  
Author(s):  
Shokouh Rezaei ◽  
Yahya Sefidbakht ◽  
Filipe Pereira
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Structural Changes ◽  
Structure And Function ◽  
Dynamics Simulation ◽  
Structural Protein ◽  
Wild Type ◽  
Deletion Mutations ◽  
And Function

Abstract SARS-CoV-2 non-structural protein 1 (Nsp1) is a virulence factor that inhibits the translation of host mRNAs and interact with viral RNA. Despite the relevance of Nsp1, few studies have been conducted to understand the effect of mutations on Nsp1 structure and function. Here, we provide a molecular dynamics simulation of SARS-CoV-2 Nsp1, wild type and variants. We found that SARS-CoV-2 Nsp1 has a more Rg value than SARS-CoV-1 Nsp1, with indicate an effect on the folding protein. This result suggest that SARS-CoV-2 Nsp1 can more easily approach the active site of the ribosome compared to SARS-CoV-1 Nsp1. In addition, we found that the C-terminal of the SARS-CoV-2 Nsp1, in particular residues 164 to 170, are more flexible than other regions of SARS-CoV-2 Nsp1 and SARS-CoV-1 Nsp1, confirming the role of this region in the interaction with the 40S subunit. Moreover, multiple deletion mutations have been found in the N/C-terminal of the SARS-CoV-2 Nsp1, which seems the effect of SARS-CoV-2 Nsp1 multiple deletions is greater than that of substitutions. Among all deletions, D156-158 and D80-90 may destabilize the protein structure and possibly increase the virulence of the SARS-CoV-2. Overall, our findings reinforce the importance of studying Nsp1 conformational changes in new variants and its effect on virulence of SARS-CoV-2.

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