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

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 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.

Impact of Embedded Endocannabinoids and Their Oxygenation by Lipoxygenase on Membrane Properties

“…DPPC large unilamellar vesicles (LUVs) were prepared at a final concentration of 1 mM, in the absence or presence of 0.1 mM AEA, 2-AG, or AA (lipid/DPPC molar ratio of 1:10), by standard hydration and extrusion methods in 50 mM Tris/HCl buffer, pH 7.6. 30 For the measurements in the presence of calcium ions, 0.1 mM EGTA and 0.3 mM CaCl 2 were added to the buffer. Small unilamellar vesicles (SUVs) for FTIR measurements were prepared by the same procedure in the same buffer at a final concentration of 55 mM, in the absence or presence of 5 mM AEA (lipid/DPPC molar ratio of 1:10).…”

“…The binding isotherms were built as already reported. 30 FTIR Measurements. FTIR measurements were performed using a Perkin-Elmer Spectrum ONE (Perkin-Elmer, Waltham, MA, USA) FTIR spectrometer, with a demountable temperature-controlled transmission cell for liquid samples (Specac Ltd. Buckingham, Slough, UK) and equipped with two CaF 2 windows.…”

“…Kinetic parameters of the reaction catalyzed by LOX-1, i.e., apparent Michaelis−Menten constant (K M ), maximum velocity (V max ), catalytic constant (K cat ), and catalytic efficiency (K cat /K M ), were determined as previously described. 30 Statistical Analysis. Data reported in this article are the means (±SD) of at least three independent determinations, each performed in triplicate.…”

“…It has been demonstrated that the binding of LOX-1 to lipid membranes can be stabilized by Ca 2+31,42 and that this ion is able to increase the amount of membrane-bound enzyme. 30 Therefore, we repeated our measurements after the addition of 100 μM Ca 2+ and failed to detect any variation in the T m of the DPPC liposomes (data not shown).…”

“…The increase of Trp quenching was analyzed as a function of lipid concentration, in the absence or presence of calcium ions in solution, corroborating the evidence that LOX-1 interaction with membranes is strengthened by this divalent cation. 30 FRET curves clearly showed a hyperbolic shape ( Figure 3) and could be fitted according to a standard binding curve with a halfsaturation binding constant ([L] 1/2 ) of 39.2 ± 3.7 μM for pure DPPC membranes (Table 3).…”