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 apominiLOX (Kd=9.2+/-1.0, 17.9+/-2.0, and 45.4+/-4.3 M, respectively). Furthermore, miniLOX reconstituted with Fe(II) or Fe(III) partially recovered its membrane-binding ability (Kd=21.4+/-2.4 and 18.9+/-5.5 M, respectively), overall supporting a novel noncatalytic role for iron in the LOX family.

A novel role for iron in modulating the activity and membrane-binding ability of a trimmed soybean lipoxygenase-1

MACCARRONE M
2010-01-01

Abstract

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 apominiLOX (Kd=9.2+/-1.0, 17.9+/-2.0, and 45.4+/-4.3 M, respectively). Furthermore, miniLOX reconstituted with Fe(II) or Fe(III) partially recovered its membrane-binding ability (Kd=21.4+/-2.4 and 18.9+/-5.5 M, respectively), overall supporting a novel noncatalytic role for iron in the LOX family.
File in questo prodotto:
Non ci sono file associati a questo prodotto.
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11697/156049
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 24
  • ???jsp.display-item.citation.isi??? 23
social impact