title: Probing selected structural regions in the secreted phospholipase A₂ from Arabidopsis thaliana for their impact on stability and activity.
abstract: In contrast to the well characterized secreted phospholipases A2 (sPLA2) from animals, their homologues from plants have been less explored. Their production in purified form is more difficult, and no data on their stability are known. In the present paper, different variants of the sPLA2 isoform α from Arabidopsis thaliana (AtPLA2α) were designed using a new homology model with the aim to probe the impact of regions that are assumed to be important for stability and catalysis. Moreover tryptophan residues were introduced in critical regions to enable stability studies by fluorescence spectroscopy. The variants were expressed in Escherichia coli and the purified enzymes were analyzed to get first insights into the peculiarities of structure stability and structure activity relationships in plant sPLA2s in comparison with the well-characterized homologous enzymes from bee venom and porcine pancreas. Stability data of the AtPLA2 variants obtained by fluorescence or CD measurements of the reversible unfolding by guanidine hydrochloride and urea showed that all enzyme variants are less stable than the enzymes from animal sources although a similar tertiary core structure can be assumed based on molecular modeling. More extended loop structures at the N-terminus in AtPLA2α are suggested to be the main reasons for the much lower thermodynamic stabilities and cooperativities of the transition curves. Modifications in the N-terminal region (insertion, deletion, substitution by a Trp residue) exhibited a strong positive effect on activity whereas amino acid exchanges in other regions of the protein such as the Ca(2+)-binding loop and the loop connecting the two central helices were deleterious with respect to activity.
authors.affiliation: Martin-Luther University Halle-Wittenberg, Institute of Biochemistry and Biotechnology, Kurt-Mothes-Str. 3, D-06120 Halle (Saale), Germany. Electronic address: Mansfeld@biochemtech.uni-halle.de.
authors.forename: Johanna
authors.initials: J
authors.lastname: Mansfeld
authors.affiliation: Martin-Luther University Halle-Wittenberg, Institute of Biochemistry and Biotechnology, Kurt-Mothes-Str. 3, D-06120 Halle (Saale), Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schöpfel
authors.affiliation: Martin-Luther University Halle-Wittenberg, Institute of Biochemistry and Biotechnology, Kurt-Mothes-Str. 3, D-06120 Halle (Saale), Germany.
authors.forename: Jessica
authors.initials: J
authors.lastname: Lorenz
authors.affiliation: Martin-Luther University Halle-Wittenberg, Institute of Biochemistry and Biotechnology, Kurt-Mothes-Str. 3, D-06120 Halle (Saale), Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Trutschel
authors.affiliation: Martin-Luther University Halle-Wittenberg, Institute of Biochemistry and Biotechnology, Kurt-Mothes-Str. 3, D-06120 Halle (Saale), Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Heilmann
authors.affiliation: Leibniz Institute of Plant Biochemistry, Department of Bioorganic Chemistry, Weinberg 3, 06120 Halle, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Brandt
authors.affiliation: Martin-Luther University Halle-Wittenberg, Institute of Biochemistry and Biotechnology, Kurt-Mothes-Str. 3, D-06120 Halle (Saale), Germany.
authors.forename: Renate
authors.initials: R
authors.lastname: Ulbrich-Hofmann
mesh_terms: D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D004795:Enzyme Stability; D054513:Group IV Phospholipases A2 / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D040681:Structural Homology, Protein
chemical_list: D029681:Arabidopsis Proteins; D054513:Group IV Phospholipases A2
keywords: Activity; N-Terminal deletion; Secreted phospholipase A(2) from Arabidopsis thaliana; Structure–stability relationship; Trp variants