Identification, Sequence and Phylogenetic analysis of a new Secretory Phospholipase A2 from Glycine max Soybean.

MARIANI ME; MADOERY RICARDO; FIDELIO GERARDO

QUILMES

Workshop; AABBC; 2010

AABBC

The Phospholipase A2 superfamily is a broad and growing group of enzymes that stereospecifically catalyze the cleavage of the sn-2 acyl ester union of diacyl-phospholipids and liberate 1-acyl-2-lysophospholipids and free fatty acids. In plants, secretory PLA2s (sPLA2s) mediate a variety of cellular processes, including growth, defense and stress response. On the other hand, the enzymatically produced lysoderivatives are strong bioemulsifyers with numerous applications in food and pharmaceutical industries. Although multiple sPLA2s genes have been identified in plants, little is known about these enzymes in opposition to their animal counterparts. We have previously identified phospholipase activity in extracts from soybean (Glycine max) and performed some preliminary biochemical characterization. We intend to perform a detailed structural and functional study of sPLA2 directed to the optimization of procedures for industrial applications. Results A TBLASTN search of  the Glycine max genome mRNA database  using Arabidopsis thaliana, Oryza sativa and Zea mays sPLA2s as templates, showed the existence of five putative sPLA2s isoenzymes, denoted as GmsPLA2-I,GmsPLA2-II, GmsPLA2-III, GmsPLA2-IV and  GmsPLA2-V. Sequence analyses indicated the presence of conserved clusters of amino acids among all the plant sPLA2s (Fig.1). The five sPLA2s sequences contained a PA2c domain having the Ca2+- binding loop (YGKYCGxxxxGC) and the active site motif (DACCxxHDxC) and the His/Asn, His or Ser dyad. These sequences encode proteins with N-terminal signal peptides and contain 12 cys residues. A phylogenetic tree of Glycine max sPLA2s using the PHYLIP program was proposed after sequence alignment with the CLUSTAL X program. The sequences were grouped in two categories (known as group XIA and XIB) on the basis of their differences in molecular weight and deviating sequences especially in the N- and C- terminal regions of the proteins. Enzymes of these groups differ in the third Ca2+ coordinating amino acid and the amino acid composition of the ?dyad? (Fig. 1). The identified GmsPLA2-I cDNA fragment was isolated and sequenced. Nucleotide sequence analysis indicated that the sPLA2 gene consists of a 417 nucleotide open reading frame (ORF), corresponding to a mature protein of 114 amino acids. The sequence analysis using bioinformatic tools allowed us to identify sPLA2 sequences within the Glycine max genome, characterize those motifs relevant for enzyme function and classify the encoded proteins within the PLA2 superfamily. This also helped us to design molecular biology experiments with the aim to obtain the mature proteins for future kinetic studies.