A 49-kDa mini-lipoxygenase from Anabaena sp. PCC 7120 retains catalytically complete functionality.
Anabaena sp. PCC 7120 is one of the few prokaryotes harboring a lipoxygenase (LOX) gene. The sequence resides in an open reading frame encoding a fusion protein of a catalase-like hemoprotein with an unusually short LOX (approximately 49 kDa) at the C terminus. The recombinant mini-LOX contains a non-heme iron in the active site and is highly active with linoleic and alpha-linolenic acids (which occur naturally in Anabaena) giving the respective 9R-hydroperoxides, the mirror image of the 9S-LOX products of plants. Using stereospecifically labeled [11-(3)H]linoleic acids we show that reaction is catalyzed via a typical antarafacial relationship of initial hydrogen abstraction and oxygenation. The mini-LOX oxygenated C16/C18:2-phosphatidylcholine with 9R specificity, suggesting a "tail first" mode of fatty acid binding. Site-directed mutagenesis of an active site Ala (Ala215), typically conserved as Gly in R-LOX, revealed that substitution with Gly retained 9R specificity, whereas the larger Val substitution switched oxygenation to 13S, implying that Ala215 represents the functional equivalent of the Gly in other R-LOX. Metabolism studies using a synthetic fatty acid with extended double bond conjugation, 9E,11Z,14Z-20:3omega6, showed that the mini-LOX can control oxygenation two positions further along the fatty acid carbon chain. We conclude that the mini-LOX, despite lacking the beta-barrel domain and much additional sequence, is catalytically complete. Interestingly, animal and plant LOX, which undoubtedly share a common ancestor, are related in sequence only in the catalytic domain; it is possible that the prokaryotic LOX represents a common link and that the beta-barrel domain was then acquired independently in the animal and plant kingdoms.
Zheng
Yuxiang
Y
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6602, USA.
Boeglin
William E
WE
Schneider
Claus
C
Brash
Alan R
AR
D019943:Amino Acid Substitution; D017033:Anabaena / Q000201:enzymology* / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D002384:Catalysis; D019143:Evolution, Molecular; D008084:Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D016366:Open Reading Frames / Q000235:genetics; D010944:Plants / Q000201:enzymology / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017962:alpha-Linolenic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D017962:alpha-Linolenic Acid; D008084:Lipoxygenase