title: In silico and in vitro characterization of phospholipase A₂ isoforms from soybean (Glycine max).
abstract: At the present, no secreted phospholipase A₂ (sPLA₂) from soybean (Glycine max) was investigated in detail. In this work we identified five sequences of putative secreted sPLA₂ from soybean after a BLAST search in G. max database. Sequence analysis showed a conserved PA2c domain bearing the Ca²⁺ binding loop and the active site motif. All the five mature proteins contain 12 cysteine residues, which are commonly conserved in plant sPLA₂s. We propose a phylogenetic tree based on sequence alignment of reported 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 their molecular 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 isoenzyme denoted as GmsPLA₂-XIA-1. We demonstrate that this mature sPLA₂ of 114 residues had PLA₂ activity on Triton:phospholipid mixed micelles and determine the kinetic parameters for this system. We generate a model based on the known crystal structure of sPLA₂ from rice (isoform II), giving first insights into the three-dimensional structure of folded GmsPLA₂-XIA-1. Besides describing the spatial arrangement of highly conserved pair HIS-49/ASP-50 and the Ca⁺² loop domains, we propose the putative amino acids involved in the interfacial recognition surface. Additionally, molecular dynamics simulations indicate that calcium ion, besides its key function in the catalytic cycle, plays an important role in the overall stability of GmsPLA₂-XIA-1 structure.
authors.affiliation: Centro de Investigaciones en Química Biológica de Córdoba, (CIQUIBIC, UNCeCONICET), Departamento de Química Biológica, Fac. de Cs. Químicas, Universidad Nacional de Córdoba, Haya de Torre y Medina Allende, Ciudad Universitaria, X5000HUA Córdoba, Argentina.
authors.forename: María Elisa
authors.initials: ME
authors.lastname: Mariani
authors.forename: Marcos Ariel
authors.initials: MA
authors.lastname: Villarreal
authors.forename: Foo
authors.initials: F
authors.lastname: Cheung
authors.forename: Ezequiel Pedro Marcos
authors.initials: EP
authors.lastname: Leiva
authors.forename: Ricardo Román
authors.initials: RR
authors.lastname: Madoery
authors.forename: Gerardo Daniel
authors.initials: GD
authors.lastname: Fidelio
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D055162:Biocatalysis; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism; D006461:Hemolysis; D006801:Humans; D007527:Isoenzymes / Q000145:classification / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D054467:Phospholipases A2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000201:enzymology* / Q000235:genetics*; D013050:Spectrometry, Fluorescence
chemical_list: D007527:Isoenzymes; D010940:Plant Proteins; D054467:Phospholipases A2; D002118:Calcium