Mutagenesis of Key Catalytic Residues in a Dehydratase Domain from a Bacterial Polyunsaturated Fatty Acid Synthase

Polyunsaturated fatty acids (PUFAs) are components of human health and nutrition. In some species of deep-sea bacteria, PUFAs are synthesized by a modular polyketide synthase, which contains two conserved dehydratase (DH) enzyme domains responsible for the introduction of double bonds into the fatty acid product. We are interested in how the two DH domains act in concert to generate the double bonds observed in the final product. We had previously reported the expression, purification and in vitro enzymatic activity of a fragment comprising the DH domains. In this work, we now report the generation of site-specific mutants in which we have replaced key amino acid residues for alanine. This mutagenesis strategy will enable the detailed interrogation of the individual functions within the multidomain protein. Both histidine and glutamic acid residues in positions 70 and 84’, respectively, were mutated in each DH domain, since they are thought to be involved in catalysis. In order to generate the mutants, two different site-specific mutagenesis protocols were employed: 1) Site-Specific Megaprimer Mutagenesis and 2) QuickChange Site-Directed Mutagenesis (Stratagene). While the Quick Change Method was effective in generating Glu mutants, it did not work for the His mutants. Thus, a different method was employed. Initial results show that both His70 and Glu84’ residues are important for DH activity. Our results also underscore that the effectiveness of the different mutagenesis methodologies tends to vary according to the position and nature of the mutation. This work was funded by Grant CHE0953254 from the NSF and MBRS-RISE Program (R25GM061838) of the UPR-MSC.