From protease to decarboxylase: The molecular metamorphosis of phosphatidylserine decarboxylase [Membrane Biology]

February 26th, 2015 by Choi, J.-Y., Duraisingh, M. T., Marti, M., Ben Mamoun, C., Voelker, D. R.

Phosphatidylserine decarboxylases (PSDs) play a central role in the synthesis of phosphatidylethanolamine in numerous species of prokaryotes and eukaryotes. PSDs are unusual decarboxylases containing a pyruvoyl prosthetic group within the active site. The covalently attached pyruvoyl moiety is formed in a concerted reaction when the PSD proenzyme undergoes an endoproteolytic cleavage into a large β-subunit, and a smaller α-subunit, which harbors the prosthetic group at its N-terminus. The mechanism of PSD proenzyme cleavage has long been unclear. Using a coupled in vitro transcription/ translation system with the soluble Plasmodium knowlesi enzyme (PkPSD), we demonstrate that the post-translational processing is inhibited by the serine protease inhibitor, phenylmethylsulfonyl fluoride. Comparison of PSD sequences across multiple phyla reveals a uniquely conserved aspartic acid within an FFxRx6Rx12PxD motif, two uniquely conserved histidine residues within a PxxYHxxHxP motif, and a uniquely conserved serine residue within a GSS/T motif, suggesting that PSDs belong to the D-H-S serine protease family. The function of the conserved D-H-S residues was probed using site directed mutagenesis of PkPSD. The results from these mutagenesis experiments reveal that D139, H198 and S308 are all essential for endoproteolytic processing of PkPSD, which occurs in cis. In addition, within the GSS/T motif found in all PSDs, the G307 residue is also essential, but the S/T 309 is non-essential. These results define the mechanism whereby PSDs begin their biochemical existence as proteases that execute one autoendoproteolytic cleavage reaction to give rise to a mature PSD harboring a pyruvoyl prosthetic group.