Oxyluciferin, a luminescence product of firefly luciferase, is enzymatically regenerated into luciferin.
The activity regenerating luciferin from the luminescent product oxyluciferin was found in the protein fraction of a lantern extract from Photinus pyralis. The protein, luciferin-regenerating enzyme (LRE), was purified to homogeneity by ammonium sulfate precipitation followed by successive column chromatography on Ultrogel AcA34, S-Sepharose FF, Q-Sepharose FF, TSKgel super Q 5pw and TSKgel G3000 SW(XL). This enzyme was a single polypeptide with a molecular mass of 38 kDa. LRE converted oxyluciferin to 2-cyano-6-hydroxybenzothiazole and thioglycolic acid. In the presence of d-cysteine, 2-cyano-6-hydroxybenzothiazole was turned over into luciferin. The same activities were detected in the extracts from two Japanese fireflies, Luciola cruciata and Luciola lateralis. We have cloned a cDNA encoding LRE from poly(A)+ RNA of the lantern of P. pyralis using reverse transcription-polymerase chain reaction, 5'-RACE (rapid amplification of cDNA ends) and 3'-RACE. The primary structure of LRE from P. pyralis deduced from the nucleotide sequence was shown to consist of 308 amino acids with a molecular weight of 33,619. The cDNA was successfully expressed under the control of the tac promoter in Escherichia coli.
Gomi
K
K
Research and Development Division, Kikkoman Corporation, Noda-shi, Chiba 278-0037 Japan.
Kajiyama
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002845:Chromatography; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D001517:Coleoptera / Q000201:enzymology*; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D049454:Firefly Luciferin / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D007211:Indoles; D008156:Luciferases / Q000096:biosynthesis* / Q000737:chemistry*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011719:Pyrazines; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013696:Temperature; D013864:Thioglycolates / Q000737:chemistry; D013997:Time Factors
D018076:DNA, Complementary; D007211:Indoles; D011719:Pyrazines; D013864:Thioglycolates; C014730:oxyluciferin; D049454:Firefly Luciferin; D008156:Luciferases; D003545:Cysteine