A single active site residue directs oxygenation stereospecificity in lipoxygenases: Stereocontrol is linked to the position of oxygenation

Abstract

Lipoxygenases are a class of dioxygenases that form hydroperoxy fatty acids with distinct positional and stereo configurations. Several amino acid residues influencing regiospecificity have been identified, whereas the basis of stereocontrol is not understood. We have now identified a single residue in the lipoxygenase catalytic domain that is important for stereocontrol; it is conserved as an Ala in S lipoxygenases and a Gly in R lipoxygenases. Our results with mutation of the conserved Ala to Gly in two S lipoxygenases (mouse 8S-LOX and human 15-LOX-2) and the corresponding Gly-Ala substitution in two R lipoxygenases (human 12R-LOX and coral 8R-LOX) reveal that the basis for R or S stereocontrol also involves a switch in the position of oxygenation on the substrate. After the initial hydrogen abstraction, antarafacial oxygenation at one end or the other of the activated pair of double bonds (pentadiene) gives, for example, 8S or 12R product. The Ala residue promotes oxygenation on the reactive pentadiene at the end deep in the substrate binding pocket and S stereochemistry of the product hydroperoxide, and a Gly residue promotes oxygenation at the proximal end of the reactive pentadiene resulting in R stereochemistry. A model of lipoxygenase reaction specificity is proposed in which product regiochemistry and stereochemistry are determined by fixed relationships between substrate orientation, hydrogen abstraction, and the Gly or Ala residue we have identified. Lipoxygenases are a class of nonheme iron oxygenases that catalyze the conversion of arachidonic acid (AA) and other polyunsaturated fatty acids to their hydroperoxy derivatives (1, 2). The products are involved in a series of biological events such as inflammation (3, 4), cell development, and differentiation (5, 6). Lipoxygenase isoforms of differing regiospecificity and stereospecificity are widespread in both the animal (7,8) and plant kingdoms (9-11).Lipoxygenase regiospecificity has been studied extensively, and there is now experimental evidence to support the control of substrate binding as critical to positional specificity (12). Both the depth of substrate entry into the catalytic domain of the protein and the head-to-tail orientation of the substrate are known to be important. The difference in positional specificity of 12S-LOX and 15S-LOX, for example, is due to a ''frame shift'' of the substrate, where the position of oxygenation is determined by how deeply the substrate enters into the active site (13-15). Positional specificity is also determined by the substrate orientation, whereby different oxygenation products are formed if the substrate enters the active site with the carboxylic or the methyl end first (e.g., 8S-LOX, 15S-LOX) (16)(17)(18)(19). All these changes have been demonstrated with S lipoxygenases, and in these experiments the oxygenation remained S-specific with the change in positional specificity. The mechanism controlling the R or S stereospecificity in lipoxygenases is less understood, and although a model has been propose...

On the Role of Molecular Oxygen in Lipoxygenase Activation

“…This will not be unexpected because this residue lies within the active site and may be directly involved in the substrate/intermediate/product binding (18,48). Consistent with this possibility, the two epoxyallylic hydroperoxide products from 13S-HPODE are in a ratio of 42:58 (cf.…”

“…To explain this effect of the A451G mutation, we have considered two possibilities not mutually exclusive. In the first possibility, this mutation opens up space either directly (18) or indirectly (48), thereby allowing O 2 to react with the epoxyallylic radical intermediate in the enzyme active site. Consistent with this possibility is the finding by Cristea and Oliw (49) that the opposite mutation in the fungal manganese-LOX, G316A, produces the opposite effect, i.e.…”

“…15S-HPETE or 13S-HPODE was synthesized by reacting soybean LOX-1 with arachidonic acid or linoleic acid at pH 9 followed by SP-HPLC purification (24). [ 18 O]13S-HPODE was synthesized under 18 O 2 atmosphere (25). Nordihydroguaiaretic acid (NDGA) was purchased from Cayman Chemical Co. (Ann Arbor, MI).…”

Inhibition of arachidonic acid metabolism and its implication on cell proliferation and tumour-angiogenesis

“…The amino acid residues crucial for iron binding and enzyme activity were determined to include His367, His550, His372 and Glu376 [132]. Site-directed mutagenesis studies have identified the critical residue for enzyme activity and control of stereochemistry of oxygenation to be Ala404, which is located between the iron binding site and the likely entrance to the substrate binding channel [133]. LOX substrate is believed to bind to the protein through π-electron, charged and hydrophobic interactions.…”

Crystal Structure of a Lipoxygenase in Complex with Substrate

“…This determines which side of the selected pentadiene is accessible to the catalytic iron. In addition to the mode of fatty acid binding, the direction of oxygen attack across the face of the pentadiene determines the R or S chirality of the products (18). As an example, 8R and 12S products have the hydroperoxide moiety on the same side of the carbon chain and the 8S/12R-hydroperoxides are on the opposite side.…”