Kinetics of Bis‐Allylic Hydroperoxide Synthesis in the Iron‐Containing Lipoxygenase 2 from Cyanothece and the Effects of Manganese Substitution

Abstract

Lipoxygenases (LOX) catalyze the regio- and stereospecific insertion of dioxygen into polyunsaturated fatty acids. While the catalytic metal of LOX is typically a non-heme iron, some fungal LOX contain manganese as catalytic metal (MnLOX). In general, LOX insert dioxygen at C9 or C13 of linoleic acid leading to the formation of conjugated hydroperoxides. MnLOX (EC 1.13.11.45), however, catalyze the oxygen insertion also at C11, resulting in bis-allylic hydroperoxides. Interestingly, the iron-containing CspLOX2 (EC 1.13.11.B6) from Cyanothece PCC8801 also produces bis-allylic hydroperoxides. What role the catalytic metal plays and how this unusual reaction is catalyzed by either MnLOX or CspLOX2 is not understood. Our findings suggest that only iron is the catalytically active metal in CspLOX2. The enzyme loses its catalytic activity almost completely when iron is substituted with manganese, suggesting that the catalytic metal is not interchangeable. Using kinetic and spectroscopic approaches, we further found that first a mixture of bis-allylic and conjugated hydroperoxy products is formed. This is followed by the isomerization of the bis-allylic product to conjugated products at a slower rate. These results suggest that MnLOX and CspLOX2 share a very similar reaction mechanism and that LOX with a Fe or Mn cofactor have the potential to form bis-allylic products. Therefore, steric factors are probably responsible for this unusual specificity. As CspLOX2 is the LOX with the highest proportion of the bis-allylic product known so far, it will be an ideal candidate for further structural analysis to understand the molecular basis of the formation of bis-allylic hydroperoxides.

Lipoxygenase 2 from Cyanothece sp. controls dioxygen insertion by steric shielding and substrate fixation

“…CspLOX2 is the LOX with the highest relative amount of bis-allylic product reported so far 13 , hence it is a suitable candidate to study the molecular basis of the dioxygen positioning at the bis-allylic position. We therefore crystallized the enzyme and determined its structure to 1.8 Å resolution (PDB code 5MED).…”

“…1). Interestingly, even when the iron cofactor is replaced by manganese 13 , the coordination geometry is not affected (PDB code 5MEG, Supplemental Fig. 2.

Figure 1Structure of CspLOX2.

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“…These LOX are mainly fungal enzymes that contain manganese in the active site 9–11 , but also the iron-containing enzyme CspLOX2 from the cyanobacterium Cyanothece PCC8801 forms (11 R )-hydroperoxy octadecadienoic acid (11-HPODE) as major product 12, 13 . Even though the first enzyme with bis-allylic products was reported in 1998 9 and the first structures of a manganese-containing LOX were recently published 14, 15 , the underlying structural factors responsible for this unusual reaction could not yet be determined.…”

Crystal structure of linoleate 13R-manganese lipoxygenase in complex with an adhesion protein

“…This also occurs nonenzymatically by Mn 3+ in methanol, but not by Fe 3+ (42,43). FeLOX catalyze -fragmentation at an insignificant rate with one exception, the 9R-LOX of Cyanothece (CspLOX2) (18,44). Enzymatic -fragmentation likely requires positioning of the hydroperoxide group near the catalytic center and steric factors are therefore of obvious importance (42,43).…”

“…In addition, the metal ligands may adjust the redox potentials of protein-bound iron and manganese to support -fragmentation and lipoxygenation. The redox potentials of Mn 2+ /Mn 3+ and Fe 2+ /Fe 3+ differ by a factor of two, and Mn-substituted FeLOX lose their catalytic activities (44). How do Mnand FeLOX adjust redox potentials to support lipoxygenation and -fragmentation?…”