TABLE 1.
Relative activities of the CbiH60 wild type and C402A and C443A mutants
Cobalt-precorrin-3 or cobalt-factor III was used as the substrate (5 μm) and incubated with 10 μm of CbiH60, 500 μm SAM, and 1 mm DTT in a 2.5-ml reaction. The reactions were left overnight at 37 °C, stopped with 1% (v/v) acetic acid, and purified by reverse phase chromatography. Bound tetrapyrroles were eluted in 2 ml of ethanol, freeze-dried, and resuspended in 200 μl of 1% (v/v) acetic acid. 50 μl was analyzed by LC-MS, with cobalt-precorrin-4 (CP4) and cobalt-factor IV (CFIV) eluting at 17 and 18 min, respectively. The products are represented as high (++) or low (+), relative to the CbiH60 wild-type reaction. Low product formation indicates a turnover of less than 10%, relative to the CbiH60 wild-type reaction.
| Substrate | CbiH60 | CP4 | CFIV |
|---|---|---|---|
| Cobalt-precorrin-3 | − | − | − |
| Wild-type | + | ++ | |
| C402A | + | ++ | |
| C443A | + | ++ | |
| Cobalt-factor III | − | − | − |
| Wild-type | + | ++ | |
| C402A | − | + | |
| C443A | − | + |