The substrate preferences and "counting" mode of the cyanobacterial ω3 (Δ15) acyl-lipid desaturase.

Abstract

Fatty acid desaturases (FADs) belong to of the oxygenase superfamily. They play important roles in metabolic pathways and adaption mechanisms in a wide range of organisms, including bacteria and humans. These enzymes dehydrogenate a single bond in the acyl chain of fatty acids (FAs), forming a double bond. Multiple parameters influence the precise position of double bond formation and acyl chain docking in the catalytic pocket of various FADs, such as the length of an acyl chain, the position of previously generated double bonds, the location of the enzyme's metal catalytic site, and so on. The "counting" mode differs from one FAD to another. The cyanobacterium Synechocystis sp. strain PCC 6803 has four FADs (Δ9, Δ12, Δ6, and Δ15 or ω3) that synthesize mono-, di-, tri-, and tetraenoic FAs. The substrate preferences and "counting" modes for the first three FADs have been identified, but the substrate specificity for the terminal ω3-FAD remains uncertain. We used molecular cloning, heterologous expression with exogenous FAs, and molecular docking to explore the substrate selectivity and counting mode of ω3-FAD. Our results show that ω3-FAD "counts" from the carboxyl (Δ) end, introduces a double bond between 15 and 16 atoms, and requires a specific acyl substrate configuration with two pre-existing double bonds at Δ⁹ and Δ¹² positions.