Cell-Free Phospholipid Biosynthesis by Gene-Encoded Enzymes Reconstituted in Liposomes
The GPAT and LPAAT enzymes were either produced separately or concurrently in the presence of G3P and p-CoA substrates. The generated lipid products 16:0 LPA and DPPA were detected by LC-MS. (a) End-point measurements of 16:0 LPA and DPPA synthesized under various experimental conditions. Substrate concentrations were 500 μM G3P, 100 μM p-CoA and 100 μM 16:0 LPA. Individual and combined enzymatic reactions were carried out with (inside-out configuration) or without 400-nm liposomes during overnight incubation at 37°C. Samples with liposomes and without p-CoA served as a negative control. Both acyltransferase enzymes showed reduced activity in the absence of SUVs. Higher yield of DPPA is obtained by two-step acyl transfer catalysed by the GPAT and LPAAT enzymes co-reconstituted in proteoliposomes. Data represent mean and s.e.m. of three independent experiments. For each repeat the mean of multiple sample injections was calculated and data are reported as the mean and standard error of three independent trials. Student t-test analysis: *P<0.1, **P<0.12, ***P<0.012. (b-e) Kinetic of acyltransferase activity in single-enzyme and two-enzyme modes. For each reaction scenario the percentage of acyl-CoA conversion to final product is also indicated. Substrate concentrations were 500 μM G3P and 100 μM p-CoA (b), 500 μM G3P, 50 μM p-CoA and 50 μM 16:0 LPA (c), 500 μM G3P and 100 μM p-CoA (d,e). Produced 16:0 LPA does not accumulate beyond 3 μM in the two-step acyl transfer scheme (d) since it is consumed in the second enzymatic reaction. When GPAT and LPAAT are co-expressed, production of DPPA is initially limited by GPAT activity but then it reaches higher concentration (e) than with LPAAT only starting from purified LPA and p-CoA precursors (c). Each data point is mean and s.e.m. of two independent sample preparations. For each replicate the mean of two sample injections was calculated and data are reported as the mean and standard error of independent preparations.