Consensus Mutagenesis and Ancestral Reconstruction Provide Insight into the Substrate Specificity and Evolution of the Front-End Delta6-Desaturase Family.

Marine algae are a major source of omega-3 long-chain polyunsaturated fatty acids (omega3-LCPUFAs), which are conditionally essential nutrients in humans and a target for industrial production. The biosynthesis of these molecules in marine algae requires the desaturation of fatty acids by Delta6-desaturases, and enzymes from different species display a range of specificities toward omega3- and omega6-LCPUFA precursors. In the absence of a molecular structure, the structural basis for the variable substrate specificity of Delta6-desaturases is poorly understood. Here we have conducted a consensus mutagenesis and ancestral protein reconstruction-based analysis of the Delta6-desaturase family, focusing on the omega3-specific Delta6-desaturase from Micromonas pusilla (MpDelta6des) and the bispecific (omega3/omega6) Delta6-desaturase from Ostreococcus tauri (OtDelta6des). Our characterization of consensus amino acid substitutions in MpDelta6des revealed that residues in diverse regions of the protein, such as the N-terminal cytochrome b5 domain, can make important contributions to determining substrate specificity. Ancestral protein reconstruction also suggests that some extant Delta6-desaturases, such as OtDelta6des, could have adapted to different environmental conditions by losing specificity for omega3-LCPUFAs. This data set provides a map of regions within Delta6-desaturases that contribute to substrate specificity and could facilitate future attempts to engineer these proteins for use in biotechnology.