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Plant phospholipase D mining unravels new conserved residues important for catalytic activity

Yani Arhab 1 Abdelkarim Abousalham 1 Alexandre Noiriel 1
1 MEM² - Métabolisme, Enzymes et Mécanismes Moléculaires
ICBMS - Institut de Chimie et Biochimie Moléculaires et Supramoléculaires
Abstract : Phospholipase D (PLD) is a key enzyme involved in numerous processes in all living organisms. Hydrolysis of phospholipids by PLD allows the release of phosphatidic acid which is a crucial intermediate of multiple pathways and signaling reactions, including tumorigenesis in mammals and defense responses in plants. One common feature found in the plant alpha isoform (PLDα), in some PLD from microbes and in all PLD from eukaryotes, is a duplicated motif named HKD involved in the catalysis. However, other residues are strictly conserved among these organisms and their role remains obscure. To gain further insights into PLD structure and the role of these conserved residues, we first looked for all the plant PLDα sequences available in public databases. With >200 sequences retrieved, a generic sequence was constructed showing that 138 residues are strictly conserved among plant PLDα, with some of them identical to residues found in mammalian PLDs. Using site-directed mutagenesis of the PLDα from Arabidopsis thaliana, we demonstrated that mutation of some of these residues abolished the PLD activity. Moreover, mutation of the residues around both HKD motifs enabled us to re-define the consensus sequence of these motifs. By sequential deletions of the N-terminal extremity, the minimum length of the domain required for catalytic activity was determined. Overall, this work furthers our understanding of the structure of eukaryotic PLDs and it may lead to the discovery of new regions involved in the catalytic reaction that could be targeted by small molecule modulators of PLDs.
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Yani Arhab, Abdelkarim Abousalham, Alexandre Noiriel. Plant phospholipase D mining unravels new conserved residues important for catalytic activity. Biochimica et Biophysica Acta Molecular and Cell Biology of Lipids, Elsevier, 2019, 1864 (5), pp.688-703. ⟨10.1016/j.bbalip.2019.01.008⟩. ⟨hal-02132514⟩

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