Generation of Product by Incubation of Recombinant Enzyme

Since many of the uroporphyrinogen III methyltransferases display substrate inhibition, uroporphyrinogen III is normally incubated with the enzyme at a final concentration of 5 pM, with SAM at a concentration of 50 pM (3). The high concentra-

tion of SAM helps to overcome inhibition with S-adenosyl-L-homocysteine. The reaction should be undertaken at pH 8.0 in 50 mM Tris-HCl buffer at either 30° or 37°C. As precorrin-2 is so unstable, we recommend that a high concentration of the uroporphyrinogen methyltransferase be used in the reaction at a concentration of about 50 pg/mL. This ensures a rapid synthesis of precorrin-2, which can be monitored visually since the solution turns a bright yellow color. In fact, precorrin-2 has a broad absorption maximum around 350 to 400 nm. The newly synthesized precorrin-2 can be separated from the other components of the incubation mixture by ion exchange chromatography. After mixing in a few milliliters of ion exchange resin such as DEAE Sephacel, the solution is slowly stirred for about 1 minute. Once the resin has settled, the majority of the supernatant can be decanted, and the resin slurry can be trans ferred to a small plastic column. The resin is washed with buffer, and buffer containing 250 mM NaCl, to remove the more loosely bound proteins, and the precorrin-2 is elut-ed in buffer containing 2 M NaCl. Precor-rin-2 is highly unstable with a tendency to form mono- and dilactones. The compound is difficult to store and should be used immediately. The uroporphyrinogen methyltransferases are very susceptible to feedback inhibition by S-adenosyl-L-homo-cysteine, and therefore, to achieve high yields of precorrin-2 (in excess of 90%), a high concentration of enzyme and SAM are required in the incubation mixture.

Sirohydrochlorin can be synthesized from precorrin-2 by the inclusion of either CysG or Met8p together with NAD+ to the above incubation (25). These enzymes are purified in the same manner as described for the CobA (above) from the appropriate strains shown in Table 1. The

Figure 6. Spectra of precorrin-2, sirohydrochlorin, and cobalt-sirohydrochlorin. The spectrum of precorrin-2 (large dashed line) has a broad absorption maximum around 350 to 400 nm. The spectrum of sirohydrochlorin (filled line) has a more defined absorption maximum at 378 nm, while cobalt sirohydrochlorin (dashed line) has defined maxima at 410 and 595 nm.

Figure 6. Spectra of precorrin-2, sirohydrochlorin, and cobalt-sirohydrochlorin. The spectrum of precorrin-2 (large dashed line) has a broad absorption maximum around 350 to 400 nm. The spectrum of sirohydrochlorin (filled line) has a more defined absorption maximum at 378 nm, while cobalt sirohydrochlorin (dashed line) has defined maxima at 410 and 595 nm.

CysG or Met8p should be added at a concentration of 50 pg/mL with NAD+ at 25 pM. Sirohydrochlorin is characterized by the appearance of a new absorption maximum at 378 nm (Figure 6).

Siroheme can be synthesized by the inclusion of ferrous iron with the incubation. However, this reaction is difficult to follow, and a clearer spectral difference can be obtained by the use of cobalt, which produces a spectrum with absorption maxima at 410 and 595 nm (Figure 6). The metal ions should be added to a concentration no higher than 10 pM, otherwise the enzymes become inactivated.

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