Table 3.
Steroid substrate ranges of four KshA homologues of R. rhodochrous DSM43269a
| Steroid substrate | KshA1 |
KshA3 |
KshA4c |
KshA5 |
||||
|---|---|---|---|---|---|---|---|---|
| Enzyme activity | Rel. activity (%) | Enzyme activity | Rel. activity (%) | Enzyme activity | Rel. activity (%) | Enzyme activity | Rel. activity (%) | |
| 4-Androstene-3,17-dione | 2.6 × 102 ± 10 | 100 | 1.7 × 102 ± 22 | 100 | 2.8 × 102 ± 20 | 100 | 1.5 × 102 ± 30 | 100 |
| 1,4-Androstadiene-3,17-dione | 7.5 × 102 ± 90 | 288 | 3.3 × 102 ± 49 | 194 | 2.5 × 102 ± 20 | 89 | 73 ± 11 | 49 |
| 4-Androstene-17β-ol-3-one | 3.3 × 103 ± 70 | 127 | 1.6 × 102 ± 40 | 94 | 2.8 × 102 ± 29 | 101 | 1.7 × 102 ± 24 | 113 |
| 4-Pregnene-3,20-dione | 1.1 × 103 ± 94 | 423 | 1.9 × 102 ± 33 | 112 | 2.7 × 102 ± 38 | 99 | 1.0 × 102 ± 12 | 67 |
| 19-Nor-4-androstene-3,17-dione | 63 ± 7 | 24 | 55 ± 10 | 32 | 2.2 × 102 ± 25 | 78 | 1.6 × 102 ± 25 | 107 |
| 1-(5α)-Androstene-3,17-dione | 59 ± 5 | 23 | 48 ± 5 | 28 | 19 × 102 ± 20 | 68 | 14 × 102 ± 32 | 93 |
| 5α-Androstane-3,17-dione | 23 ± 13 | 9 | ND | ND | 1.8 × 102 ± 19 | 64 | 12 × 102 ± 26 | 80 |
| 5β-Androstane-3,17-dione | 50 ± 7 | 19 | ND | ND | 1.6 × 102 ± 22 | 58 | 1.4 × 102 ± 27 | 93 |
| 5α-Androstane-17β-ol-3-one (stanolon) | ND | ND | ND | ND | ND | ND | 1.4 × 102 ± 27 | 93 |
| 11β-Hydrocortisone | ND | ND | ND | ND | ND | ND | 1.4 × 102 ± 29 | 93 |
| 4-Cholestene-3-oneb | 34 ± 5 | 13 | 39 ± 5 | 23 | ND | ND | 39 ± 5 | 26 |
| 23,24-Bis-nor cholesta-4-ene-22-oic acid | 1.3 × 103 ± 1.2 × 102 | 500 | 2.1 × 102 ± 33 | 124 | —d | 23 | 1.2 × 102 ± 17 | 80 |
| 23,24-Bis-nor-cholesta-1,4-diene-22-oic acid | 1.1 × 103 ± 1.3 × 102 | 423 | 3.0 × 102 ± 44 | 176 | —d | 22 | 64 ± 6 | 43 |
Initial enzyme activities with a 200 μM steroid substrate concentration were calculated in nmol min−1 mg−1 of purified KSH enzyme. The relative activities are expressed as percentages compared to activities with 4-androstane-3,17-dione, which was set at 100%. Standard errors of the means (n = 3) are also reported. ND, no detectable initial activity. No KSH activity was observed with the following compounds: 5-cholestene-3β-ol (cholesterol), 5α-androstane-17-one, 3α-hydroxy-5α-pregnane-20-one, 3β-hydroxy-5α-androstane-17-one, 9α-hydroxy-4-androstene-3,17-dione (negative control).
Due to its limited solubility, this steroid was used at 25 μM. (The lower concentration was also used for 5-cholestene-3β-ol and 3α-hydroxy-5α-pregnane-20-one [data are not shown].)
Data were obtained from reference 24.
Measurements were performed with enzyme batches with higher activities (initial activity on 4-androstane-3,17-dione of 512 ± 62 nmol min−1 mg−1 of purified KSH enzyme) than those described in reference 24 due to the slightly optimized expression and purification methods used in our study. However, the relative activities remained constant. For chemical structures of the tested steroids, see Fig. S1 in the supplemental material.