A New Thermophilic Ene-Reductase from the Filamentous Anoxygenic Phototrophic Bacterium Chloroflexus aggregans
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sequence Analysis and Cloning
2.2. Expression, Analysis and Purification of Recombinant Protein
2.3. Native Gel and NBT Staining
2.4. Analytical Gel Filtration
2.5. Thermofluor Measurements
2.6. Activity Assay and Kinetics
2.7. Determination of pH Optimum
2.8. Crystallization and Data Collection
2.9. Model Building and Refinement
3. Results
3.1. Identification of New Putative CaOYE and Sequence Analysis
3.2. Purification of Recombinant CaOYE and Evaluation of Its Oligomeric State
3.3. Evaluation of CaOYE Thermal Stability and pH Optimum
3.4. Evaluation of CaOYE Activity with Standard Substrates and Steady-State Kinetic Parameters
3.5. CaOYE Crystal Structure
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Substrate | Specific Activity | Kinetic Parameters | |||
---|---|---|---|---|---|
Structure | Name | U/mg | Km (mM) | kcat (s−1) | kcat/Km (mM−1 s−1) |
2-Cyclopenten-1-one | 1.1 ± 0.09 | N.D. | N.D. | N.D. | |
2-Cyclohexen-1-one | 5.25 ± 0.22 | 5.520 ± 0.627 | 3.82 ± 0.176 | 0.69 | |
2-Methyl-cyclohexen-1-one | 1.19 ± 0.07 | N.D. | N.D. | N.D. | |
4-Ketoisophorone | 8.3 ± 0.50 | 0.013 ± 0.002 | 2.27 ± 0.055 | 174.6 | |
Maleimide | 2.2 ± 0.37 | N.D. | N.D. | N.D. | |
2-Methyl-pentenal | 2.0 ± 0.15 | N.D. | N.D. | N.D. | |
trans-2-Hexen-1-al | 1.1 ± 0.09 | N.D. | N.D. | N.D. | |
1-Octen-3-one | 9.4 ± 0.40 | 10.32 ± 1.438 | 12.13 ± 0.661 | 1.17 |
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Robescu, M.S.; Niero, M.; Loprete, G.; Cendron, L.; Bergantino, E. A New Thermophilic Ene-Reductase from the Filamentous Anoxygenic Phototrophic Bacterium Chloroflexus aggregans. Microorganisms 2021, 9, 953. https://doi.org/10.3390/microorganisms9050953
Robescu MS, Niero M, Loprete G, Cendron L, Bergantino E. A New Thermophilic Ene-Reductase from the Filamentous Anoxygenic Phototrophic Bacterium Chloroflexus aggregans. Microorganisms. 2021; 9(5):953. https://doi.org/10.3390/microorganisms9050953
Chicago/Turabian StyleRobescu, Marina Simona, Mattia Niero, Giovanni Loprete, Laura Cendron, and Elisabetta Bergantino. 2021. "A New Thermophilic Ene-Reductase from the Filamentous Anoxygenic Phototrophic Bacterium Chloroflexus aggregans" Microorganisms 9, no. 5: 953. https://doi.org/10.3390/microorganisms9050953