Consensus mutagenesis and ancestral reconstruction provide insight into the substrate specificity and evolution of the front-end Δ6-desaturase family

Abstract

Marine algae are a major source of omega ({omega})-3 long-chain polyunsaturated fatty acids ({omega}3-LCPUFAs), which are conditionally essential nutrients in humans and a target for industrial production. The biosynthesis of these molecules in marine algae begins with the desaturation of fatty acids by {Delta}6-desaturases and enzymes from different species display a range of specificities towards {omega}3 and {omega}6 LCPUFAs. In the absence of a molecular structure, the structural basis for the variable substrate specificity of {Delta}6-desaturases is poorly understood. Here we have conducted a consensus mutagenesis and ancestral protein reconstruction-based analysis of the {Delta}6-desaturase family, focusing on the {omega}3-specific {Delta}6-desaturase from Micromonas pusilla (Mp{Delta}6des) and the bispecific ({omega}3/{omega}6) {Delta}6-desaturase from Ostreococcus tauri (Ot{Delta}6des). Our characterization of consensus amino acid substitutions in Mp{Delta}6des 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 {Delta}6-desaturases, such as Ot{Delta}6des, could have adapted to different environmental conditions by losing specificity for {omega}3-LCPUFAs. This dataset provides a map of regions within {Delta}6-desaturases that contribute to substrate specificity and could facilitate future attempts to engineer these proteins for use in biotechnology.