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

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Marine algae are a major source of omega (ω)-3 long-chain polyunsaturated fatty acids (ω3-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 Δ6-desaturases and enzymes from different species display a range of specificities towards ω3 and ω6 LCPUFAs. In the absence of a molecular structure, the structural basis for the variable substrate specificity of Δ6-desaturases is poorly understood. Here we have conducted a consensus mutagenesis and ancestral protein reconstruction-based analysis of the Δ6-desaturase family, focusing on the ω3-specific Δ6-desaturase from <jats:italic>Micromonas pusilla</jats:italic> (MpΔ6des) and the bispecific (ω3/ω6) Δ6-desaturase from <jats:italic>Ostreococcus tauri</jats:italic> (OtΔ6des). Our characterization of consensus amino acid substitutions in MpΔ6des 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 Δ6-desaturases, such as OtΔ6des, could have adapted to different environmental conditions by losing specificity for ω3-LCPUFAs. This dataset provides a map of regions within Δ6-desaturases that contribute to substrate specificity and could facilitate future attempts to engineer these proteins for use in biotechnology.</jats:p>

Combining ancestral reconstruction with folding-landscape simulations to engineer heterologous protein expression

“…4d) and, according to our working hypothesis, we do not expect its replacement with the corresponding ancestral sequence to improve the efficiency of heterologous folding. The experimental results on CPk- [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] thioredoxin conform to this expectation (Fig. 5c and d).…”

“…This is a reasonable scenario, in particular since co-evolution may have led to the adaptation of the protein to the assistance machinery of its original host. Consequently, the fact that resurrected ancestral proteins often show improved heterologous expression as compared with their modern counterparts [22][23][24][25][26][27][28][29][30][31][32][33][34][35] plausibly reflects an ancient adaptation to unassisted folding. Efficient unassisted folding would no longer be a useful feature after the evolutionary emergence of cellular foldingassistance, thus allowing the evolutionary acceptance of mutations that impair the ancestral feature.…”

“…Finally, a somewhat intriguing result of our extensive experimental study on the stability of CPk thioredoxin variants deserves some attention. While most of the variants show the expected stability enhancement, this is not the case with CPk- [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] thioredoxin. This modern/ancestral chimera involves 10 back-to-ancestor amino acid replacements in the longest a-helix of the thioredoxin structure, which is expected to fold early according to our statistical-mechanical calculations (Fig.…”

“…4). CPk- [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] thioredoxin displays a stability similar to that of the CPk thioredoxin background as shown by both the denaturation temperature values and the chemical denaturation profiles (Fig. S3).…”

“…Moreover, a substantial number of studies have actually reported improved heterologous expression of ancestral proteins as compared with their modern counterparts. Examples include: phosphate-binding protein 22 , periplasmic binding protein 23 , serum paraoxonase 24 , coagulation factor VIII 25 , titin 26 , haloalkane dehalogenases 27 , cytidine and adenine base editors 28 , diterpene cyclase 29 , rubisco 30 , endoglucanases 31 , L-amino acid oxidases 32 , laccases 33 , front-end D6-desaturases 34 and fatty acid photo-decarboxylases 35 . On a related note, recent studies on ancestral proteins have noted an improved capability to yield crystals suitable for X-ray structural determination 36,37 .…”