INTRODUCTION: Lipoxygenase-1 (linoleate:oxygen oxidoreductase, EC 1.13.11.12; LOX-1) belongs to a family of non-heme, non-sulfur iron dioxygenases that take part in the metabolism of polyunsatured fatty acids catalysing their conversion into conjugates hydroperoxides. Soybean LOX-1 has been widely used as a model for studying the functional and structural properties of the homologous family of LOXs. The crystallographic structure of soybean LOX-1 revealed that the protein is organised in two domains (1), a beta-sheet N-terminal domain and a larger, mostly helical C-terminal domain. Mammalian LOXs lack the 30 kDa N-terminal domain present in soybean LOX-1 and other related plant lipoxygenases. Limited proteolysis by tryptic digestion of the native soybean LOX-1 is known to produce a catalytically active 60 kDa fragment, which has been named “mini-LOX” (2). Here, we describe the overall structural characterisation of native soybean LOX-1 and of mini-LOX using the Small Angle X-Ray Scattering (SAXS) approach. The native enzyme and the 60 kDa active fragment were studied both in the presence and in the absence of the inhibitor eicosatetraynoic acid (ETYA) and of glycerol, qualifying the low resolution structures of the enzyme in this different conditions. In parallel, the kinetic properties of the different forms were determined.MATERIALS AND METHODS: Soybean LOX-1 was purified from seeds as described elsewhere (3). Limited proteolysis by tryptic digestion was carried out as reported (2). The isolation of miniLOX was obtained by fast-protein liquid chromatography (FPLC, size exclusion column Superdex-75) using an AKTA Explorer apparatus (Parmacia, Uppsala, Sweden). Dioxygenase activity was calculated as already described (4). SAXS measurements were performed at the synchrotron radiation beam line D24 in the DCI storage ring of LURE (Laboratoire pour l’Utilisation du Rayonnement Electromagnetique, Orsay-Paris). The radius of gyration (Rg) was determined by interpolating the SAXS data on the basis of Guinier approximation I(Q) = I(0) exp(-Rg2Q2/3), where Q = (4sin)/ and I(0) is the scattering intensity at zero scattering angle. The distance distribution function p(r) and the maximum dimension of the protein (Dmax) have been determined using the indirect transform method as implemented in the program GNOM. The theoretical scattering intensities were computed from the atomic coordinates of the crystal structure using the program CRYSOL. The ab initio shape determinations were performed with the dummy atom model (DAM) method (5) using the program DAMMIN. RESULTS: The analysis of the SAXS patterns of the native soybean LOX-1 yielded a value of the radius of gyration, calculated from the Guinier analysis of the scattering intensity, of Rg = 30.0  0.3 Å. Further calculations, using the pair distribution function p(r) of the native enzyme, yielded a value for the maximum dimension of the molecule (Dmax) of 95  5 Å and a value of the radius of gyration of 30.1  0.2 Å, very close to that derived from the Guinier approximation. The comparison between the experimental SAXS data and the theoretical one, obtained from the atomic coordinates of soybean LOX-1 [1F8N.pdb], yields a very good agreement ( =1.747). In parallel to the above-mentioned modelling, ab initio calculations of the overall shape of the protein from the SAXS pattern were performed using the program DAMMIN, no particular conditions of oblateness and symmetry of the particle shape were imposed as constraints. Moreover, the superposition of the solution structure of soybean LOX-1 obtained with the crystal structure clearly shows the strong similarity between the two models. Taken together, these data indicate that the overall shape of the protein does not undergo any significant[...]

STRUCTURAL AND FUNCTIONAL PROPERTIES OF LIPOXYGENASE IN SOLUTION

DAINESE, Enrico;Angelucci C;GIACOMINELLI STUFFLER, Roberto;SABATUCCI, Annalaura;
2004

Abstract

INTRODUCTION: Lipoxygenase-1 (linoleate:oxygen oxidoreductase, EC 1.13.11.12; LOX-1) belongs to a family of non-heme, non-sulfur iron dioxygenases that take part in the metabolism of polyunsatured fatty acids catalysing their conversion into conjugates hydroperoxides. Soybean LOX-1 has been widely used as a model for studying the functional and structural properties of the homologous family of LOXs. The crystallographic structure of soybean LOX-1 revealed that the protein is organised in two domains (1), a beta-sheet N-terminal domain and a larger, mostly helical C-terminal domain. Mammalian LOXs lack the 30 kDa N-terminal domain present in soybean LOX-1 and other related plant lipoxygenases. Limited proteolysis by tryptic digestion of the native soybean LOX-1 is known to produce a catalytically active 60 kDa fragment, which has been named “mini-LOX” (2). Here, we describe the overall structural characterisation of native soybean LOX-1 and of mini-LOX using the Small Angle X-Ray Scattering (SAXS) approach. The native enzyme and the 60 kDa active fragment were studied both in the presence and in the absence of the inhibitor eicosatetraynoic acid (ETYA) and of glycerol, qualifying the low resolution structures of the enzyme in this different conditions. In parallel, the kinetic properties of the different forms were determined.MATERIALS AND METHODS: Soybean LOX-1 was purified from seeds as described elsewhere (3). Limited proteolysis by tryptic digestion was carried out as reported (2). The isolation of miniLOX was obtained by fast-protein liquid chromatography (FPLC, size exclusion column Superdex-75) using an AKTA Explorer apparatus (Parmacia, Uppsala, Sweden). Dioxygenase activity was calculated as already described (4). SAXS measurements were performed at the synchrotron radiation beam line D24 in the DCI storage ring of LURE (Laboratoire pour l’Utilisation du Rayonnement Electromagnetique, Orsay-Paris). The radius of gyration (Rg) was determined by interpolating the SAXS data on the basis of Guinier approximation I(Q) = I(0) exp(-Rg2Q2/3), where Q = (4sin)/ and I(0) is the scattering intensity at zero scattering angle. The distance distribution function p(r) and the maximum dimension of the protein (Dmax) have been determined using the indirect transform method as implemented in the program GNOM. The theoretical scattering intensities were computed from the atomic coordinates of the crystal structure using the program CRYSOL. The ab initio shape determinations were performed with the dummy atom model (DAM) method (5) using the program DAMMIN. RESULTS: The analysis of the SAXS patterns of the native soybean LOX-1 yielded a value of the radius of gyration, calculated from the Guinier analysis of the scattering intensity, of Rg = 30.0  0.3 Å. Further calculations, using the pair distribution function p(r) of the native enzyme, yielded a value for the maximum dimension of the molecule (Dmax) of 95  5 Å and a value of the radius of gyration of 30.1  0.2 Å, very close to that derived from the Guinier approximation. The comparison between the experimental SAXS data and the theoretical one, obtained from the atomic coordinates of soybean LOX-1 [1F8N.pdb], yields a very good agreement ( =1.747). In parallel to the above-mentioned modelling, ab initio calculations of the overall shape of the protein from the SAXS pattern were performed using the program DAMMIN, no particular conditions of oblateness and symmetry of the particle shape were imposed as constraints. Moreover, the superposition of the solution structure of soybean LOX-1 obtained with the crystal structure clearly shows the strong similarity between the two models. Taken together, these data indicate that the overall shape of the protein does not undergo any significant[...]
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11575/10589
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