Protein Dynamics Influence the Enzymatic Activity of Phospholipase A/Acyltransferases 3 and 4

Abstract

Phospholipase A/acyltransferase 3 (PLAAT3) and PLAAT4 are enzymes involved in the synthesis of bioactive lipids. Despite sequential and structural similarities, the two enzymes differ in activity and specificity. The relation between the activity and dynamics of the N-terminal domains of PLAAT3 and PLAAT4 was studied. PLAAT3 has a much higher melting temperature and exhibits less nanosecond and millisecond dynamics in the active site, in particular in loop L2(B6), as shown by NMR spectroscopy and molecular dynamics calculations. Swapping the L2(B6) loops between the two PLAAT enzymes results in strongly increased phospholipase activity in PLAAT3 but no reduction in PLAAT4 activity, indicating that this loop contributes to the low activity of PLAAT3. The results show that, despite structural similarity, protein dynamics differ substantially between the PLAAT variants, which can help to explain the activity and specificity differences.

Defunctionalizing intracellular organelles such as mitochondria and peroxisomes with engineered phospholipase A/acyltransferases

“…1a ). To test if the TM domain is required for the organelle deformation, we mitochondrially targeted PLAAT4 with the truncated TM (PLAAT4-dTM) which still retains the phospholipase activity 26 . The truncated PLAAT4 did not induce organelle deformation (Supplementary Fig.…”

Defunctionalizing Intracellular Organelles with Genetically-Encoded Molecular Tools Based on Engineered Phospholipase A/Acyltransferases (PLAATs)

“…1b ). We additionally tested PLAAT4 this time with the truncated TM (PLAAT4-dTM), as this simpler form reportedly retains the phospholipase activity in vitro 23 . Unexpectedly, we did not observe mitochondrial deformation ( Supplementary Fig.…”

Crystal structure of the phospholipase A and acyltransferase 4 (PLAAT4) catalytic domain

“…In particular, the differential dynamics of the L2 (B6) loop (residues 100-109), which precedes the active site Cys113 and is in close proximity of the main L1 loop, is a critical determinant of enzymatic activity. Loop-swapped PLAAT3 (with the L2(B6) loop from PLAAT4) gains appreciably in phospholipase activity even in truncated form, while the reverse swap did not inhibit PLAAT4 (Chatterjee et al, 2021).…”

“…In isolation, the recombinant N-terminal domains (NTD) of the family members PLAAT2-4, are enzymatically competent, as evidenced by their self-acylation when exposed to short-chain phosphatidyl choline (Golczak et al, 2012). However, PLAAT4 shows robust phospholipase activity even when truncated (Chatterjee et al, 2021;Golczak et al, 2012), while PLAAT3 and PLAAT2 require the CTD for (appreciable) interfacial phospholipase activity (Pang et al, 2012;Uyama et al, 2009aUyama et al, , 2009b. Furthermore, studies have shown that the CTD also plays a critical role for the targeting of the enzyme to different intracellular membrane compartments (Deucher et al, 2000;Jans et al, 2008;Morishita et al, 2021;Staring et al, 2017;Uyama et al, 2015;Wei et al, 2015).…”

“…This loss in cell-death inducing ability observed for PLAAT3 is restored upon truncation of the main L1 loop (residues 38-56; (Wei et al, 2015)), which is also predicted to interact with the membrane and help orient/tether the enzyme during catalysis (Golczak et al, 2012). A recent study of PLAAT3 and PLAAT4 NTD dynamics in solution (Chatterjee et al, 2021) demonstrated that despite structural similarity, these proteins exhibit different dynamics overall, which might help explain the difference in activity of their truncated form. In particular, the differential dynamics of the L2 (B6) loop (residues 100-109), which precedes the active site Cys113 and is in close proximity of the main L1 loop, is a critical determinant of enzymatic activity.…”