In silico and in vitro characterization of phospholipase A2 isoforms from soybean (Glycine max)

MARIANI EM; VILLARREAL MA; CHEUNG F; LEIVA EPM; MADOERY RR; FIDELIO GD

BIOCHIMIE

ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER

Lugar: Paris; Año: 2012 p. 2608 - 2619

0300-9084

At the present, no secreted phospholipase A2 (sPLA2) from soybean (Glycine max) was investigated indetail. In this work we identified five sequences of putative secreted sPLA2 from soybean after a BLASTsearch in G. max database. Sequence analysis showed a conserved PA2c domain bearing the Ca2þ bindingloop and the active site motif. All the five mature proteins contain 12 cysteine residues, which arecommonly conserved in plant sPLA2s. We propose a phylogenetic tree based on sequence alignment ofreported plant sPLA2s including the novel enzymes from G. max. According to PLA2 superfamily, two of G.max sPLA2s are grouped as XIA and the rest of sequences as XIB, on the basis of differences found in theirmolecular weights and deviating sequences especially in the N- and C-terminal regions of the isoenzymes.Furthermore, we report the cloning, expression and purification of one of the putative isoenzymedenoted as GmsPLA2-XIA-1. We demonstrate that this mature sPLA2 of 114 residues had PLA2 activity onTriton:phospholipid mixed micelles and determine the kinetic parameters for this system. We generatea model based on the known crystal structure of sPLA2 from rice (isoform II), giving first insights into thethree-dimensional structure of folded GmsPLA2-XIA-1. Besides describing the spatial arrangement ofhighly conserved pair HIS-49/ASP-50 and the Caþ2 loop domains, we propose the putative amino acidsinvolved in the interfacial recognition surface. Additionally, molecular dynamics simulations indicatethat calcium ion, besides its key function in the catalytic cycle, plays an important role in the overallstability of GmsPLA2-XIA-1 structure.