Biochemical Properties and Substrate Specificity of Two Acyl-CoA:Lysophosphatidic Acid Acyltransferases (PtATS2a and PtATS2b) from Diatom Phaeodactylum tricornutum

Microsomal fractions from yeast Δ<i>ale1</i> cells harbouring the empty plasmid pYES2/CT and from yeast cells overexpressing <i>Pt</i>ATS2a (Phatr3_J11916) or <i>Pt</i>ATS2b (Phatr3_J43099) were used in the studies. When <i>sn</i>-1-18:1-LPA and [¹⁴C]16:0-CoA were used as exogenous substrates, both <i>Pt</i>ATS2a and <i>Pt</i>ATS2b showed the highest activity at 23 °C in the range of temperatures tested from 10 to 60 °C. Both enzymes showed the highest activity in alkaline pH. For <i>Pt</i>ATS2a, it was pH 10 while for <i>Pt</i>ATS2b, it was pH 11. At pH 6 and pH 12, the activities of both enzymes were very low. The calcium ions at concentrations of 0.05-1 mM drastically decreased the activity of both enzymes. The magnesium ions at a concentration of 0.05 mM had a little effect on the activity of both enzymes, while higher concentrations (0.5 mM and 1 mM) significantly inhibited their activity. To study the substrate specificity, seventeen different acyl-CoAs in combinations with <i>sn</i>-1-[¹⁴C]18:1-LPA were used. <i>Pt</i>ATS2a showed the highest preference for 18:4-CoA n-3 while <i>Pt</i>ATS2b for 18:1-CoA. The pattern of utilisation of other acyl-CoAs tested also differed between the two enzymes. The presented studies, for the first time, characterised LPAAT type enzymes from diatoms, organisms that naturally produced very-long-chain polyunsaturated fatty acids (VLC-PUFA).