A 49-kDa mini-lipoxygenase from Anabaena sp. PCC 7120 retains catalytically complete functionality

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Anabaena sp. PCC 7120 possesses a unique lipoxygenase (LOX) gene encoding a mini-LOX fused to a catalase-like hemoprotein.
• This prokaryotic LOX is distinct from plant and animal LOX, featuring an unusually short C-terminal domain.

Purpose of the Study:

• To characterize the catalytic activity and substrate specificity of the Anabaena sp. PCC 7120 mini-LOX.
• To elucidate the stereochemical mechanism and evolutionary relationship of this prokaryotic LOX.

Main Methods:

• Recombinant expression and purification of the mini-LOX.
• Enzymatic assays using linoleic and alpha-linolenic acids, including stereospecifically labeled substrates.
• Site-directed mutagenesis of active site residues.
• Oxygenation studies with modified fatty acids and phosphatidylcholine.

Main Results:

• The recombinant mini-LOX exhibits high activity with linoleic and alpha-linolenic acids, producing 9R-hydroperoxides.
• Stereochemical analysis confirmed a typical antarafacial mechanism for hydrogen abstraction and oxygenation.
• Mutagenesis studies identified Ala215 as a key residue determining oxygenation specificity (9R vs. 13S).
• The enzyme demonstrated control over oxygenation at positions further down the fatty acid chain.

Conclusions:

• The Anabaena sp. PCC 7120 mini-LOX is a catalytically complete enzyme despite lacking certain domains found in eukaryotic LOX.
• This prokaryotic LOX may represent an evolutionary link between different LOX families, with the beta-barrel domain acquired independently in plants and animals.