Identification and characterization of an arachidonate 11R-lipoxygenase.
11R-Lipoxygenase (11R-LOX) activity has been detected in several marine invertebrates, and here we report the first cloning and expression of the enzyme. The cDNA encoding a protein of 77kDa was isolated by RT-PCR from the soft coral Gersemia fruticosa and expressed in Escherichia coli. Incubations of recombinant enzyme with arachidonic acid yielded a single product, identified by RP-HPLC, GC-MS, and chiral phase-HPLC as 11R-hydroperoxyeicosatetraenoic acid. Other C18, C20, and C22 substrates are also oxygenated, preferentially at the omega10 position. Significantly, both Ca(2+)-ions and a membrane fraction are required for catalytic activity. Calcium effects translocation of the soluble 11R-LOX to the membrane and this association is reversible by Ca(2+) chelation. The enzyme sequence contains some conserved amino acids implicated in calcium activation of mammalian 5-LOX, and with its obligate requirement for membrane interaction the 11R-LOX may thus provide a new model for further analysis of this aspect of lipoxygenase activation.
Mortimer
Monika
M
Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, Tallinn 12618, Estonia.
Järving
Reet
R
Brash
Alan R
AR
Samel
Nigulas
N
Järving
Ivar
I
D000595:Amino Acid Sequence; D000818:Animals; D037421:Anthozoa / Q000201:enzymology*; D001094:Arachidonate 5-Lipoxygenase / Q000235:genetics; D001091:Arachidonate Lipoxygenases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D018076:DNA, Complementary; D016718:Arachidonic Acid; D001091:Arachidonate Lipoxygenases; D001094:Arachidonate 5-Lipoxygenase; D002118:Calcium