Figure 4. Enzyme Properties of Murine and Human ADPN Variants.
(A) LPAAT activity of mAdpn and mI148M was monitored as a function of [1-14C] oleoyl-CoA concentration and kinetic parameters were calculated by Michaelis-Menten plot using GraphPad Prism software.
(B) Structure around the putative “catalytic dyad” (Ser-47 and Asp-166) involved in the proposed lipase activity of ADPN. The structure was deduced from an alignment of Leu10-Phe175 of ADPN to Leu32-Ala225 of Pat17 (PDB ID 1OWX), the best-scoring template with known 3D structure using the Phyre2 server (Kelley and Sternberg, 2009), and PyMOL (The PyMOL Molecular Graphics System, Version 1.4.1, Schrödinger, LLC). The model reveals close spatial proximity of Ile148 (highlighted in yellow) and Cys15 (highlighted in green) to the proposed active site (highlighted in red).
(C) LPAAT activity of hADPN and hADPN mutants with point mutations at Cys-15 to Ser (hC15S), Ser-47 to Ala (hS47A), Asp-206 to Ala (hD206A), and Pro-311 to Gly (hP311G). Mutants were generated by site-directed mutagenesis and purified under conditions identical to those of the wild-type protein. The inset shows a Coomassie blue-stained SDS-PAGE gel with various purified recombinant ADPN variants (2 μg protein/lane). LPAAT activity was measured using 2 μg of purified proteins or TF as described in the Experimental Procedures.
(D) LPAAT activity was determined in bacterial total lysates (10 μg lysate protein) overexpressing TF, hADPN, or hADPN mutants. Protein overexpression was confirmed by western blotting in bacterial total lysates using anti-His monoclonal antibody (inset). Experiments were done in duplicates and repeated twice. Data are presented as mean ± SD. Statistical significance was determined by a two-tailed Student’s t test, **p < 0.01, ***p < 0.001.