Lipoxygenases are non-heme, non-sulfur iron containing enzymes that catalyze the dioxygenation of polyunsatured fatty acids containing one or more pentadiene systems to the corresponding hydroperoxy derivatives. Structural studies in solution of the mammalian and plant enzyme revealed that the latter has a more stable and compact conformation1. As yet, metal atom extraction, reconstitution and substitution with vicariate metals have not been successfully applied to soybean lipoxygenase-1, because of the highly buried position of the iron atom within the active site. Tryptic digestion of lipoxygenase-1 and the subsequent isolation of the 60 kDa C-terminal region allowed to generate a “mini-lipoxygenase-1 (miniLOX)” that retains the catalytically active iron, but in a more accessible position2. In this study, we investigated by near-UV-circular dichroism and fluorescence spectroscopies the structural and functional effects of iron removal, reconstitution and vicariation in miniLOX. Moreover, we report the kinetic analysis and the membrane binding ability of the apo- and metal-substituted forms of miniLOX, using fluorescence resonance energy transfer and monolamellar vesicles. Taken together, these data demonstrate an unprecedented structural role of iron, which is involved not only in the catalytic activity but also in the membrane binding ability of lipoxygenase-1.1. Dainese E. Sabatucci A. van Zadelhoff G. Angelucci C. B. Vachette P., Veldink G. Finazzi Agrò A. and Maccarrone M. (2005). J. Mol. Biol. 349, 143-152.2. Maccarrone M. Salucci M. L. van Zadelhoff G. Malatesta F. Veldink, G. Vliegenthart J. F. and Finazzi Agrò A. (2001). Biochemistry 40, 6819-6827.[...]
MODULATION OF THE ENZYMATIC ACTIVITY AND MEMBRANE BINDING PROPERTIES OF SOYBEAN LIPOXYGENASE-1 THROUGH LIMITED PROTEOLYSIS AND METAL SUBSTITUTION
DAINESE, Enrico;Angelucci C;SABATUCCI, Annalaura;
2008-01-01
Abstract
Lipoxygenases are non-heme, non-sulfur iron containing enzymes that catalyze the dioxygenation of polyunsatured fatty acids containing one or more pentadiene systems to the corresponding hydroperoxy derivatives. Structural studies in solution of the mammalian and plant enzyme revealed that the latter has a more stable and compact conformation1. As yet, metal atom extraction, reconstitution and substitution with vicariate metals have not been successfully applied to soybean lipoxygenase-1, because of the highly buried position of the iron atom within the active site. Tryptic digestion of lipoxygenase-1 and the subsequent isolation of the 60 kDa C-terminal region allowed to generate a “mini-lipoxygenase-1 (miniLOX)” that retains the catalytically active iron, but in a more accessible position2. In this study, we investigated by near-UV-circular dichroism and fluorescence spectroscopies the structural and functional effects of iron removal, reconstitution and vicariation in miniLOX. Moreover, we report the kinetic analysis and the membrane binding ability of the apo- and metal-substituted forms of miniLOX, using fluorescence resonance energy transfer and monolamellar vesicles. Taken together, these data demonstrate an unprecedented structural role of iron, which is involved not only in the catalytic activity but also in the membrane binding ability of lipoxygenase-1.1. Dainese E. Sabatucci A. van Zadelhoff G. Angelucci C. B. Vachette P., Veldink G. Finazzi Agrò A. and Maccarrone M. (2005). J. Mol. Biol. 349, 143-152.2. Maccarrone M. Salucci M. L. van Zadelhoff G. Malatesta F. Veldink, G. Vliegenthart J. F. and Finazzi Agrò A. (2001). Biochemistry 40, 6819-6827.[...]I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
