Cloned (Comment) | Organism |
---|---|
amplified from the pJHD500 plasmid and ligated into a pET21b vector, expressed from pZCH2 in an Escherichia coli BL21 (lambdaDE3) cell line after growth to an OD600 of 0.5 | Vibrio harveyi |
Crystallization (Comment) | Organism |
---|---|
bacterial luciferase/FMN complex, by the hanging drop method, at 2.3 A resolution. Crystals of recombinant luciferase are grown at room temperature prior to soaking with millimolar concentrations of FMN. Belongs to space group P212121. The isoalloxazine ring is coordinated by an unusual cis-Ala-Ala peptide bond. The reactive sulfhydryl group of Cys106 projects toward position C-4a, the site of flavin oxygenation. Mobile loop that is crystallographically disordered, appears to be a boundary between solvent and the active center. Within this portion of the protein, there is a single contact between Phe272 of the R subunit and Tyr151 of the beta subunit | Vibrio harveyi |
Protein Variants | Comment | Organism |
---|---|---|
Y151A | binds FMNH2 more weakly in comparison to the wild-type, substitution at position 151 on the beta subunit causes reductions in activity and total quantum yield | Vibrio harveyi |
Y151D | binds FMNH2 more weakly in comparison to the wild-type, substitution at position 151 on the beta subunit causes reductions in activity and total quantum yield | Vibrio harveyi |
Y151K | binds FMNH2 more weakly in comparison to the wild-type, substitution at position 151 on the beta subunit causes reductions in activity and total quantum yield | Vibrio harveyi |
Y151R | binds FMNH2 more weakly in comparison to the wild-type, substitution at position 151 on the beta subunit causes reductions in activity and total quantum yield | Vibrio harveyi |
Y151T | binds FMNH2 more weakly in comparison to the wild-type, substitution at position 151 on the beta subunit causes reductions in activity and total quantum yield | Vibrio harveyi |
Y151W | least active mutant, binds reduced flavin with wild-type affinity, substitution at position 151 on the beta subunit causes reductions in activity and total quantum yield | Vibrio harveyi |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Vibrio harveyi | P07740 | - |
- |
Purification (Comment) | Organism |
---|---|
on a nickel affinity column, to more than 90% purity | Vibrio harveyi |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
decanal + FMNH + O2 | - |
Vibrio harveyi | decanoic acid + FMN + H2O + light | - |
? | |
dodecanal + FMNH + O2 | - |
Vibrio harveyi | dodecanoic acid + FMN + H2O + light | - |
? | |
octanal + FMNH + O2 | - |
Vibrio harveyi | octanoic acid + FMN + H2O + light | - |
? |
Synonyms | Comment | Organism |
---|---|---|
bacterial luciferase | - |
Vibrio harveyi |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
wild-type enzyme has a 90% reduction in activity after 92 min. Comparable loss for the Y151W mutant after only 11 min | Vibrio harveyi |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FMN | - |
Vibrio harveyi |