A novel role for iron in modulating the activity and membrane-binding ability of a trimmed soybean lipoxygenase-1.
Lipoxygenases (LOXs) are iron-containing enzymes that play critical roles in plants and animals. As yet, metal atom extraction, reconstitution, and substitution have not been successfully applied to soybean LOX-1 [Glycine max (L.) Merrill], a prototype member of the LOX family that is widely used in structural and kinetic studies. Here, tryptic digestion of native LOX-1, used as a control, allowed us to isolate the 60-kDa C-terminal region (termed miniLOX), that retains the catalytically active iron in a more accessible position. Then, iron was removed to obtain an unprecedented apo-miniLOX, which was reconstituted and substituted with different metal ions. These forms of miniLOX were characterized vs. native LOX-1 by kinetic analysis, near UV circular dichroism, steady-state fluorescence, and fluorescence resonance energy transfer. MiniLOX showed a 2-fold increase in the membrane-binding affinity compared with native LOX-1 and a remarkable 4-fold increase compared with apo-miniLOX (K(d)=9.2+/-1.0, 17.9+/-2.0, and 45.4+/-4.3 microM, respectively). Furthermore, miniLOX reconstituted with Fe(II) or Fe(III) partially recovered its membrane-binding ability (K(d)=21.4+/-2.4 and 18.9+/-5.5 microM, respectively), overall supporting a novel noncatalytic role for iron in the LOX family.
Dainese
Enrico
E
Department of Biomedical Sciences, University of Teramo, Teramo, Italy.
Angelucci
Clotilde B
CB
Sabatucci
Annalaura
A
De Filippis
Vincenzo
V
Mei
Giampiero
G
Maccarrone
Mauro
M
D001051:Apoenzymes; D001665:Binding Sites; D002942:Circular Dichroism; D007501:Iron / Q000502:physiology*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism*; D008084:Lipoxygenase / Q000378:metabolism*; D008670:Metals / Q000032:analysis / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology*; D013054:Spectrophotometry, Atomic; D013379:Substrate Specificity
D001051:Apoenzymes; D008081:Liposomes; D008670:Metals; D007501:Iron; C101084:lipoxygenase L-1; D008084:Lipoxygenase