Structural characterization of PfaC: the key protein in PUFA synthesis

Abstract

Polyunsaturated fatty acid synthases are large enzymatic complexes capable of producing polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). Both EPA and DHA are classified as omega-3 fatty acids and are essential for human health and nutrition. These PUFAs are naturally present in fish, seafood, including fish oils, as well as in other organisms like microalgae and proteobacteria from the deep sea. However, due to concerns regarding overpopulation and sustainability, there is a growing need to search for new alternatives for their production. The marine bacteria Moritella marina possesses an EPA and DHA producing cluster that consists of the pfa genes: pfaA, pfaB, pfaC, pfaD and pfaE. Therefore, deciphering the structure and function of these proteins is of great interest. In this work, we studied the PfaC protein, which consists of two ketosynthase-like domains and two dehydratases domains (DHs). We successfully expressed and purified the full-length Moritella marina PfaC protein, as well as the C-terminal domain containing two dehydratases domains (DHs), using E.coli as the expression system. To accomplish this, we tested several overexpression conditions and purification tags. For the DHs domain, we conducted crystal screenings experiments. To complement the experimental work, computer programs such as Alphafold2 were used to predict the protein structure of PfaC. This computational approach has allowed us to predict the positions of the active sites and understand the molecular mechanism of PfaC. We have gained insights into the specialized functions of the PfaC protein that presents domains preserved in other pfa genes but, in this case, is specialized first to elongate the chain above 18 carbon atoms and, secondly, to stabilize double bonds from trans to cis by isomerization.