Table 3.
Product specificities of different PpAOS1 and PpHPL variants
| Substrate | Enzyme-variant | ω-oxo fatty-acids (= HPL activity) | Ketols (= AOS activity) | Cyclopentenone (= AO cyclization) |
|---|---|---|---|---|
| 9-HPOD |
HPL Wt |
++++ |
+ |
n.d. |
| |
AOS1 Wt |
+ |
++++ |
n.d. |
| |
AOS1 F93L |
++++ |
+ |
n.d. |
| 9-HPOT(n-3) |
HPL Wt |
++++ |
+ |
n.d. |
| |
HPL F151L |
++++ |
+ |
n.d. |
| |
HPL A169S |
++++ |
+ |
n.d. |
| |
HPL F151L, A169S |
++++ |
+ |
n.d. |
| |
AOS1 Wt |
+ |
++++ |
n.d. |
| |
AOS1 F93L |
++++ |
- |
n.d. |
| 13-HPOD |
HPL Wt |
++++ |
+ |
n.d. |
| |
HPL F151L |
++++ |
+ |
n.d. |
| |
HPL A169S |
++++ |
+ |
n.d. |
| |
HPL F151L, A169S |
++++ |
+ |
n.d. |
| |
AOS1 Wt |
+ |
++++ |
n.d. |
| |
AOS1 F93L |
++++ |
+ |
n.d. |
| 13-HPOT(n-3) |
HPL Wt |
++++ |
+ |
n.d. |
| |
HPL F151L |
++++ |
+ |
n.d. |
| |
HPL A169S |
++++ |
+ |
n.d. |
| |
HPL F151L, A169S |
++++ |
+ |
n.d. |
| |
AOS1 Wt |
+ |
+++ |
+ |
| AOS1 F93L | ++ | ++ | + |
Affinity purified enzymes were incubated with [1-14C]-labeled hydroperoxy fatty acids for approx. 30 min. After extraction products were analyzed by RP-HPLC that was coupled to a radio-detector and quantified by integration of the respective peak area. For simplicity the relative amounts of each product is indicated by the number of “+”. AOS, allene oxide synthase; HPL, hydroperoxide lyase; Wt, wild type; n.d., not determined. The data represent between 2 and 5 independent experiments.