Biochemical Consequences of a Leucine-to-Cysteine Clamp Substitution in Lipoxygenases.

Lipoxygenases (LOXs) are a family of metalloenzymes that oxidize polyunsaturated fatty acids producing cell-signaling hydroperoxides. Fungal LOXs have drawn interest because of their roles in plant and animal pathogenesis. A new subfamily of annotated fungal LOXs has been predicted. One of its unique structural features is the presence of a cysteine amino acid encoded at the invariant leucine clamp. Herein, we isolate three representatives of this LOX subfamily from recombinant expressions in both yeast and bacterial cultures. Metal analysis indicates that the proteins accommodate a mononuclear manganese ion center, similar to other eukaryotic LOXs, but have nominal LOX activity. The functional consequence of the non-conservative mutation is further explored using a Leu-to-Cys (L546C) variant of soybean lipoxygenase, a model plant orthologue. While this L546C variant has comparable structural integrity and metal content to the native enzyme, the variant is associated with a 50-fold decrease in the first-order rate constant. The presence of cysteine at 546, compared to leucine, alanine, or serine, also results in a distinctive kinetic lag phase and product inhibition. The collective data highlight that Cys encoded at the Leu clamp is detrimental to LOX activity. Potential biological functions of these annotated fungal LOXs are discussed.