Any feedback?
Literature summary for 1.13.12.5 extracted from
- Molecular basis of thermostability enhancement of Renilla luciferase at higher temperatures by insertion of a disulfide bridge into the structure (2017), Biochim. Biophys. Acta, 1865, 252-259 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| recombinant expression of engineered His-tagged enzyme C-SRLuc8 in Escherichia coli, method optimization | Renilla reniformis |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| N45C/A71C | site-directed mutagenesis at the N-terminal of the enzyme, the engineered luciferase C-SRLuc8, improvement of the stability of super Renilla luciferase 8 (SRLuc8), which is a red-emitter variety of RLuc at higher temperatures, by introduction of a disulfide bridge into its structure. Evaluation of the proper disulfide bond formation based on computational methods, structure-function analysis, overview. The kinetic stability of C-SRLuc8 increases significantly at 60°C to 70°C as compared to SRLuc8. The N45C/A71C crosslink in C-SRLuc8 is involved in a hotspot foldon which seems to be the rate-limiting step of conformational collapse at higher temperatures. Molecular dynamic simulation studies to analyze the molecular basis of the structural changes after the introduction of the disulfide bridge. Increasing the local stability of several regions at this domain significantly improves the kinetic stability of C-SRLuc8, but the disulfide bridge in C-SRLuc8 does not delay the initial temperature of enzyme inactivation. The results of the thermal inactivation at 37°C and 65°C indicate that although CSRLuc8 shows a slight increase in stability during the first thirty minutes of incubation at 37°C, C-SRLuc8 shows a significant increase in thermostability at 65°C and increased activity as compared with SRLuc8 | Renilla reniformis |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.00728 | - |
coelenterazine | pH 7.2, 37°C, recombinant modified enzyme C-SRLuc8 | Renilla reniformis |  |
| 0.00997 | - |
coelenterazine | pH 7.2, 37°C, recombinant wild-type enzyme SRLuc8 | Renilla reniformis | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| coelenterazine + O2 | Renilla reniformis | - |
coelenteramide + CO2 + hv | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Renilla reniformis | P27652 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged engineered enzyme C-SRLuc8 in Escherichia coli by nickel affinity chromatography | Renilla reniformis |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| coelenterazine + O2 | - |
Renilla reniformis | coelenteramide + CO2 + hv | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| ? | x * 36000, modified recombinant His-tagged enzyme C-SRLuc8, SDS-PAGE | Renilla reniformis |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| Renilla luciferase | - |
Renilla reniformis |
| RLuc | - |
Renilla reniformis |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
modified enzyme C-SRLuc8 and wild-type enzyme SRLuc8 | Renilla reniformis |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
C-SRLuc8 and SRLuc8 start to collapse at temperatures close to 37°C | Renilla reniformis |
| 50 | 70 | engineered enzyme C-SRLuc8 shows significant stability, retaining about 20% of its initial activity after incubation at 70°C for 5 min, while wild-type SRLuc8 completely loses its activity after incubation at this temperature | Renilla reniformis |
| 65 | - |
the residual activity of modified enzyme C-SRLuc8 is approximately 20% after incubation at 65°C for 5 min | Renilla reniformis |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.2 | - |
assay at | Renilla reniformis |
General Information
| General Information | Comment | Organism |
|---|---|---|
| malfunction | a collapse of alpha/beta hydrolase fold domain may trigger the irreversible inactivation of the enzyme at higher temperatures. In contrast to wild-type SRLuc8, the alpha-helices in the alpha/beta hydrolase fold domain of engineered C-SRLuc8 have lower perturbations and do not collapse, while some cap domain residues have more perturbations | Renilla reniformis |
Use of this online version of BRENDA is free under the CC BY 4.0 license. See terms of use for full details.
Release 2023.1 (January 2023)
Legal
Curated at
Member of
