Table 2.
rDl3βHSD, rCt3KSI, and rAtSt5βR_F342A_F153A or rAtSt5βR_R64D_F342A_F153A were combined (1:1:1)
| Enzymes combined | Cosubstrate (mM) | After incubation | ||
|---|---|---|---|---|
| Progesterone |
5β‐Pregnane‐ 3,20‐dione |
5β‐Pregnane−3β‐ol−20‐one | ||
|
3βHSD 3KSI AtSt5βR_DMCb |
0.4 NAD+ | 25.8 ± 2.6 | n.d.a | n.d. |
|
3βHSD 3KSI AtSt5βR_DMC |
0.4 NAD+ 0.4 NADH/H+ | 17.2 ± 0.7 | n.d. | 0.1 ± 0.03 |
|
3βHSD 3KSI AtSt5βR_DMC |
0.8 NAD+ | 31.4 ± 0.3 | n.d. | 0.2 ± 0.07 |
|
3βHSD 3KSI AtSt5βR_DMC |
0.4 NAD+ | 31.4 ± 0.3 | n.d. | 0.2 ± 0.07 |
|
3βHSD 3KSI AtSt5βR_DMc |
0.4 NAD+ 0.4 NADPH/H+ |
5.0 ± 0.04 | 8.5 ± 0.3 | 67.5 ± 1.2 |
Pregnenolone (0.3 mM) was added, and the assays were incubated for 2 hr at 40°C and then analyzed by GC‐MS. Amounts of substrate and products were combined and set to equal 100%.
a
n.d.: not detectable.
b
AtSt5βR_DMC:AtSt5βR_R64D_F342A_F153A.
c
AtSt5βR_DM: AtSt5βR_F342A_F153A.