Purification and molecular cloning of an 8R-lipoxygenase from the coral Plexaura homomalla reveal the related primary structures of R- and S-lipoxygenases.
Lipoxygenases that form S configuration fatty acid hydroperoxides have been purified or cloned from plant and mammalian sources. Our objectives were to characterize one of the lipoxygenases with R stereospecificity, many of which are described in marine and freshwater invertebrates. Characterization of the primary structure of an R-specific enzyme should help provide a new perspective to consider the enzyme-substrate interactions that are the basis of the specificity of all lipoxygenases. We purified an 8R-lipoxygenase of the prostaglandin-containing coral Plexaura homomalla by cation and anion exchange chromatography. This yielded a colorless enzyme preparation, a band of approximately 100 kDa on SDS-polyacrylamide gel electrophoresis, and turnover numbers of 4000 min-1 of 8R-lipoxygenase activity in peak chromatographic fractions. The full-length cDNA was cloned by PCR using peptide sequence from the purified protein and by 5'- and 3'-rapid amplification of cDNA ends. The cDNA encodes a polypeptide of 715 amino acids, including over 70 amino acids identified by peptide microsequencing. A peptide presequence of 52 amino acids is cleaved to give the mature protein of 76 kDa; the difference from the estimated size by SDS-PAGE implies a post-translational modification of the P. homomalla enzyme. All of the iron-binding histidines of S-lipoxygenases are conserved in the 8R-lipoxygenase. However, the C-terminal amino acid is a threonine, as opposed to the isoleucine that provides the carboxylate ligand to the iron in all known S-lipoxygenases. These results establish that the 8R-lipoxygenase is related in primary structure to the S-lipoxygenases. A model of the basis of R and S stereospecificity is described.
Brash
A R
AR
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6602, USA.
Boeglin
W E
WE
Chang
M S
MS
Shieh
B H
BH
D000595:Amino Acid Sequence; D000818:Animals; D001091:Arachidonate Lipoxygenases / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003063:Cnidaria / Q000201:enzymology*; D018076:DNA, Complementary / Q000235:genetics; D005796:Genes; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity
D018076:DNA, Complementary; D010446:Peptide Fragments; D001091:Arachidonate Lipoxygenases; C054192:arachidonate 8-lipoxygenase