Structural analysis of a membrane-associated delta-6 fatty acid desaturase from Prorocentrum micans.

Delta-6 fatty acid desaturases, which play key roles in the biosynthesis of polyunsaturated fatty acids (PUFAs), are membrane-associated enzymes that present significant challenges for isolation and purification, complicating their structural characterization. Here we report the identification and structure-function analysis of a novel Δ6 fatty acid desaturase (PmD6) from the marine alga Prorocentrum micans with substrate preference to α-linolenic acid (18:3n-3). Structural modeling revealed a mushroom-like structure of PmD6 formed by four transmembrane α-helices as a stem and three cytoplasmic domains as a cap. Structural alignment identified several key residues positioned around the substrate tunnel and catalytic center in PmD6. Functional analysis of these residues by site-directed mutagenesis showed that Tyr226, Trp235, Phe345, and Ser349, facing the middle region of the substrate tunnel of PmD6, played critical roles in defining the structure for acceptance of substrates. Thr200, Leu391, and Met389, surrounding the end of the substrate tunnel, had roles in interaction with the methyl end of substrates. Asp255, close to a metal iron in the catalytic center, was essential for catalytic reaction by supporting the regional structure. These results have provided mechanistic insights into the structure-function relationship of membrane-bound front-end fatty acid desaturases.