Purification and Characterization of Drosophila Taffazin: Discovery of the First Phospholipid Transacylase

Taffazin (TAZ) is a mitochondrial enzyme involved in cardiolipin (CL) metabolism and mutations in TAZ lead to Barth Syndrome. Unavailability of the purified enzyme has hampered direct mechanistic studies. To identify the biochemical reaction catalyzed by tafazzin we expressed a fusion construct of TAZ tagged with maltose‐binding protein (MBP) in E.coli ‐BL21 cells, followed by affinity‐purification on an amylose column. SDS‐PAGE followed by Coomassie Blue staining showed purification to apparent homogeniety. Western blot analysis using TAZ antibody confirmed the 85 kDa band as MBP‐TAZ. Enzymatic analysis demonstrated that the purified enzyme was able to convert [ 14 C] Lyso‐phosphatidylcholine (LPC) into [ 14 C] Phosphatidylcholine (PC) only in the presence of cardiolipin, but not in the presence of linoleoyl‐CoA. Addition of CoA did not stimulate the transfer of acyl groups from CL to [ 14 C] LPC, demonstrating the CoA‐independent nature of the phospholipid transacylation by tafazzin. MBP‐TAZ transferred [ 14 C] linoleoyl residues from [ 14 C] PC to Monolysocardiolipin (MLCL) but not to CoA, forming [ 14 C] CL but not [ 14 C] linoleoyl‐CoA. The formation of [ 14 C] CL was strictly dependent on the presence of MLCL and was not affected by CoA. Also, purified tafazzin did not show phospholipase A 2 activity because it did not release [ 14 C] linoleic acid from [ 14 C] PC, even in the absence of an acyl acceptor. Taken together these results suggest that tafazzin is the first CoA‐independent phospholipid transacylase that does not follow a phospholipase A type mechanism.