Table 1.
Mutational analysis of the AtSOT18 enzyme.
| Activity in pkatal mg−1 | ||||
|---|---|---|---|---|
| AtSOT18 | 3 MTP | 8MTO | Sinigrin | I3M |
| Wild-type* | 1624 ± 122 | 1618 ± 272 | 879 ± 410 | 501 ± 46 |
| Lys93Ala | N.D. | N.D. | N.D. | — |
| Thr96Ala | 122 ± 19 | 536 ± 73 | N.D. | — |
| Thr97Ala | N.D. | N.D. | N.D. | — |
| Tyr130Ala | N.D. | N.D. | N.D. | — |
| His155Ala | N.D. | N.D. | N.D. | — |
| Pro136Ala | 1726 ± 264 | — | — | 473 ± 18 |
Selected amino acids in the catalytic centre were mutated to alanine. The activity was tested with short-chained aliphatic 3 MTP, long-chained aliphatic Gl 8MTO, co-crystallized sinigrin and indolic Gl I3M. The 150 µL assays contained 80 mM Tris/HCl, pH 8.0, 9.2 mM MgCl2, 60 µM of the respective ds-Gl substrates, 1 µg purified protein, and 60 µM PAPS. The reactions were started by the addition of PAPS, incubated for 20 min at 37 °C, and stopped by incubation at 95 °C for 10 min. The formation of the respective sulphated product was analysed by HPLC at 229 nm. The specific activities are given in pkatal mg−1. N.D., not detectable; (—), not tested.