Figure 3
Mutations of the active site clamp affect the regio and stereo specificity of CspLOX2. (A) The conformation of the arachidonic acid (20:4n-6) substrate crystallized in the coral 8R-LOX (grey sticks) fits quite well into the predicted channel of CspLOX2, which is shown from the entry part (left) and from the bottom part (right). The double bonds that belong to the reacting pentadiene are shown in dark red. The residues shown in blue belong to the active site clamp with Ala300 and Ile296 closer to the n-2 which corresponds to position C9 on linoleic acid (18:2n-6) and with Leu258, Leu304, Leu502 and Leu506 closer to the n-2 position (C13 on linoleic acid). (B) The residues shown in (A) were exchanged by smaller residues and the product distribution was analyzed by HPLC. Shown are mean values and error bars show standard deviation. Additionally to the distribution of the positional isomers 9-HPODE, 11-HPODE and 13-HPODE, the chirality of each product is indicated. The relative activities shown on top were determined by measuring the consumption of oxygen. All experiments were carried out in triplicates.
