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

Coffa and Brash. 10.1073/pnas.0406727101.

Supporting Information

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Supporting Figure 8
Supporting Text
Supporting Figure 9

Supporting Figure 8

Fig. 8. Reversed-phase high-pressure liquid chromatography (RP-HPLC) and chiral HPLC analysis of products formed from [1-¹⁴C]arachidonic acid (AA) by WT and Ala-416Gly mutant human 15-LOX-2. (A) RP-HPLC analysis of WT. (B) RP-HPLC analysis of Ala416Gly mutant. (C) Chiral HPLC analysis of 15-hydroxyeicosatetraenoic acid (HETE) methyl ester from WT. (D) Chiral HPLC analysis of 15-HETE-Me from Ala416Gly mutant. (E) Chiral HPLC analysis of 11-HETE-Me from Ala416Gly mutant. [1-¹⁴C]AA (100 m M) was incubated with aliquots of His-tagged WT and mutant human 15-LOX-2 as described in Experimental Procedures. Products were analyzed by using a Waters Symmetry C₁₈ 5-m m column (0.46 × 25 cm) eluted at a flow rate of 1 ml/min with methanol/water/acetic acid (80:20:0.01, by volume) for 22 min and finally with methanol to elute unreacted AA. Chiral HPLC analyses were carried out as described in the legend to Fig. 2.

Supporting Text

Metabolism of 1-Palmitoyl-2-arachidonoyl Phosphatidylcholine (C₁₆/AA-PC) by Coral 8R-LOX. According to our current understanding of the orientation of substrate binding in R and S lipoxygenases, two of the enzymes we studied here should bind the substrate with a tail-first entry into the active site. These two enzymes are the 15S-LOX and the 8R-LOX. The other two, 8S-LOX and 12R-LOX, should bind carboxyl end first. Tail-first entry should be compatible with the ability to metabolize fatty acids esterified in phospholipids. This ability is a well known property of 15S-LOX enzymes, and in Results, we used the metabolism of C₁₆/AA-PC to study product formation in the 15-LOX-2 Ala416Gly mutant. Previously we had not been successful in demonstrating metabolism of C₁₆/AA-PC by the 8R-LOX domain of the coral fusion protein. This result is despite the fact that a different 8R-LOX from coral (1) readily converts C₁₆/AA-PC to its 8R-hydroperoxyeicosatetraenoic acid (HPETE) derivative (unpublished observations). Activity of the 8R-LOX domain is strongly stimulated by calcium (2), and we reasoned that the tendency of calcium to precipitate phospholipids could be counterproductive. In the absence of calcium, we could detect weak activity of the 8R-LOX domain in metabolizing C₁₆/AA-PC. In these experiments, the level of activity was sufficiently low so that the chiral purity of the products was diluted by a low level of nonenzymic oxygenation. Nonetheless, the WT 8R-LOX was found to convert C₁₆/AA-PC to the 8R-HPETE PC ester (82% 8R), whereas the Gly427Ala mutant formed the 12-HPETE derivative, 83% 12S in chirality. The results are compatible only with a tail-first orientation of the arachidonate moiety in the active site, both for the WT 8R-LOX and the Gly427Ala mutant enzyme.

1. Brash, A. R., Boeglin, W. E., Chang, M. S. & Shieh, B.-H. (1996) J. Biol. Chem. 271, 20949-20957.

2. Boutaud, O. & Brash, A. R. (1999) J. Biol. Chem. 274, 33764-33770.

Supporting Figure 9

Fig. 9. Straight-phase (SP)-HPLC and chiral HPLC analysis of products formed by WT and mutant human 12R-LOX. (A) SP-HPLC analysis of WT. (B) SP-HPLC analysis of Gly441Ala mutant. (C) Chiral HPLC analysis of 12-HETE-Me from WT. (D) Chiral HPLC analysis of 12-HETE-Me from Gly441Ala mutant. (E) Chiral HPLC analysis of 8-HETE from Gly441Ala mutant. [1-¹⁴C]AA (100 mM) was incubated with aliquots of WT and mutant human 12R-LOX as described in Experimental Procedures. SP-HPLC and chiral HPLC analyses were carried out as described in the legend to Fig. 2.