Strictosidine synthetase

Strictosidine synthetase catalyzes the stereospecific condensation of trypt-amine and the iridoid glucoside secologanin to form strictosidine. The product is the precursor of the monoterpenoid-derived indole and quinoline alkaloids. 

Strictosidine, tryptamine, and codeine (internal standard) were separated on a LiChrosorb RP-8 Select B column (4 mm X 250 mm, 7 /xm) at a flow rate of 1 mL/min. The mobile phase contained 7 mM sodium dodecyl sulfate and 25 mM sodium phosphate (pH 6.2) in 32% methanol (v/v). The effluent was monitored at 280 nm. Quantitation was based on standard curves for tryptamine and strictosidine. 

The reaction mixture contained in a final volume of 0.1 mL, 0.1 M sodium phosphate buffer (pH 6.8), 1.0 mM tryptamine, 5 mM secologanin, and 3 mM dithiothreitol. To inhibit glucosidase activity, 100 mM D(+)-gluconic acid-5-D-gluconolactone was included. The incubation was started by addition of 10 /xL of enzyme. After 30 minutes of incubation at 30°C, the reaction was stopped by addition of 0.1 mL of 5% trichloroacetic acid. Before centrifugation, 25 /xL of 8 mM codeine hydrochloride was added as the internal standard. HPLC analysis was performed on 4 /xL aliquots. With enzyme purified to a specific activity of 710 pkat per milligram of protein, the reaction was linear with time for 1 hour and with protein up to at least 50 /xg of protein during a 20-minute incubation period. 

Two enzymes catalyze the last two steps of tetracycline biosynthesis Anhy-drotetracycline oxygenase produces dehydrotetracycline, and tetracycline dehydrogenase converts dehydrotetracycline to tetracycline. Usage of a diode- 

Anhydrotetracycline, dehydrotetracycline, and tetracycline were separated at 40°C on a Separon ODS glass-pack column (1 mm X 150 mm, 5 /urn). Mobile phase A was a 20 80 mixture of 20 mM EDTA (pH 6.4) and dimethyl-formamide. Solvent B was methanol. After 0.5 minute on solvent A, a 0.5-minute linear gradient from 0 to 50% B was started, with the higher concentration held for 2 minutes. This was followed by a return to the starting conditions with a 0.5-minute gradient. After a 1.5-minute delay, the next sample was injected. A diode-array detector was used, with peak areas integrated at 440, 400, and 360 nm for anhydrotetracycline, dehydrotetracycline, and tetracycline, respectively. 

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Figure 9.151 Determination of strictosidine synthetase activity by HPLC. Codeine (a), tryptamine (b), and strictosidine (c) were separated on a 4.0 (i.d.) X 250 mm LiChrosorb RP-8 Select B column at a flow rate of 1.0 mL/min. Incubation was for 30 minutes at 30°C with enzyme from Catharanthus roseus after ammonium sulfate precipitation (35-50% saturation) and gel filtration on Sephadex G-25, in the presence of 100 mM fi-D-gluconolactone. Injection volume was 8 pL and the UV detector was set at 0.02 AUFS. (From Pennings et al., 1989.)...

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