Cloning and characterization of an active fragment of luciferase from a luminescent marine alga, Pyrocystis lunula.
Two marine dinoflagellates, Lingulodinium polyedrum and Pyrocystis lunula, emit light in a reaction involving the enzymatic oxidation of its tetrapyrrole luciferin by molecular oxygen. The characteristic properties of P. lunula luciferase have not been clarified, whereas L. polyedrum luciferase, which has three active domains, has been characterized. A cloned partial cDNA of the P. lunula luciferase encodes an active fragment corresponding to part of domain 2 and all of domain 3 of L. polyedrum luciferase. The homology of the amino acid sequence between the two luciferases in domain 3 is about 84.3%. A recombinant His-tagged luciferase fragment containing domain 3 (Mr = 46 kDa) catalyzed the light-emitting oxidation of luciferin (lambdamax = 474 nm). This protein was purified by a single affinity-chromatography procedure. The pH-activity profile and the bioluminescence spectrum of the recombinant enzyme having a third domain are almost identical to those of an extract from P. lunula cultured in vitro. The recombinant enzyme is active at pH 8.0, although the recombinant enzyme derived from the second domain of L. polyedrum luciferase is inactive at pH 8.0. Substitution of Glu-201 by histidine in the third domain of P. lunula luciferase showed a decrease of activity above pH 7.0, suggesting that histidine residues could be responsible for pH-sensitivity in dinoflagellate luciferase.
Morishita
Hisashi
H
Faculty of Education, Shizuoka University, Japan.
Ohashi
Sayumi
S
Oku
Takashi
T
Nakajima
Yoshihiro
Y
Kojima
Satoshi
S
Ryufuku
Masayuki
M
Nakamura
Hideshi
H
Ohmiya
Yoshihiro
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004141:Dinoflagellida / Q000201:enzymology*; D008156:Luciferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D049449:Luminescence; D008386:Marine Biology; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D008156:Luciferases