Change in Substrate Binding Specificity of the Tandem Acyl Carrier Protein Domains of Polyunsaturated Fatty Acid Synthesis

Abstract

Polyunsaturated fatty acids synthases are large enzymatic complexes that produce PUFAs, such as eicosapentaenoic acid (EPA, 20:5ω3) and docosahexaenoic acid (DHA, 22:6ω3). This omega-3 fatty acids are essential for the human health and nutrition. In general, ω-3 fatty acids are found in fish, seeds, and some unicellular organisms like marine gammaproteobacteria, myxobacteria or schizochytrium. PUFA synthases are analogous to polyketide synthases (PKS), which are proteins involve in the bacterial antibiotic production mechanism. In marine proteobacteria, PUFA synthases enzymology and biochemistry understanding continues to be rudimentary. The system contains a main conserved long protein known as PfaA, which features multiple acyl carrier protein (ACP) domains. In this project, PfaA domain, contained in the pfa cluster of Moritella marina, has been studied to understand the mechanism in which PfaA initiates the synthesis by the selection of malonyl units. Also, the objective of this study was to observe the change in substrate binding specificity of the five tandem acyl carrier proteins when a onepoint mutation is introduced in the serine 703 of the active center of the AT domain. The results performed by radioactivity assays showed that when serine 703 is mutated, malonyl-coA cannot bind to the protein and so the synthesis cannot be performed. However, when the radiolabeled acetyl-coA was introduced with the mutated serine 703, it was observed that the acetyl-coA was capable of binding to the protein. Moreover, ATS703A was able to acetylate PfaA ACP domain in vitro. At the same time, other one-point mutations were tried in other two near serine residues (serine 607 and 863) to identify with accuracy the binding site of acetyl-coA. Further experiments must be carried on in order to find why acetyl-coA binds the active center of AT mutated.