Abstract.A biosynthetic pathway for rosmarinic acid is proposed. This pathway is deduced from studies of the enzymes detectable in preparations from suspension cells of Coleus blumei. Phenylalanine is transformed to 4-coumaroyl-CoA by the enzymes of the general phenylpropanoid pathway: phenylalanine ammonia-lyase (EC 4.3.1.5), cinnamic acid 4-hydroxylase (EC 1.14.13.11) and hydroxycinnamic acid:CoA ligase (EC 6.2.1.12). Tyrosine is metabolized to 4-hydroxyphenyllactate by tyrosine aminotransferase (EC 2.6.1.5) and hydroxyphenylpyruvate reductase. The ester can be formed from 4-coumaroyl-CoA and 4-hydroxyphenyllactate by the catalytic activity of rosmarinic acid synthase with concomitant release ofCoA. Microsomal hydroxylase activities introduce the hydroxyl groups at positions 3 and 3' of the aromatic rings of the ester 4-coumaroyl-4'-hydroxyphenyllactate giving rise to rosmarinic acid.

Suspension cultures of Coleus blumei accumulate very high amounts of rosmarinic acid, an ester of caffeic acid and 3,4-dihydroxyphenyllactate, in medium with elevated sucrose concentrations. Since the synthesis of this high level of rosmarinic acid occurs in only five days of the culture period, the activities of the enzymes involved in the biosynthesis are very high. Therefore all the enzymes necessary for the formation of rosmarinic acid from the precursors phenylalanine and tyrosine could be isolated from cell cultures of Coleus blumei: phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, hydroxycinnamoyl:CoA ligase, tyrosine aminotransferase, hydroxyphenylpyruvate reductase, rosmarinic acid synthase and two microsomal 3-and 3'-hydroxylases. The main characteristics of these enzymes of the proposed biosynthetic pathway of rosmarinic acid will be described.

The amount of chlorophyll (Fig. 1) and hepatotoxin (Fig. 2) in cell samples rose during three weeks. After that the decay of the cells began and toxins started to leak into the water phase. After 60 days only traces of toxin were present in cell samples. Biological decomposition of the toxin was not detected. The total amount of toxin (cells and water) did not differ in the inoculated and in the axenic samples. Results support the assumption that hepatotoxic cyanobacteria in water supplies are a potential health risk. References

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NADH 190iM NADPH 95M position 3 of the phenolic ring occurs only at the ester stage of RA formation. HPPR is competitively inhibited by RA and pyruvate. Caffeic acid, p-coumaric acid, and cinnamic acid do not affect the enzyme activity, p-coumaroyl-CoA inhibits HPPR. The back reaction, i.e. the oxidation of pHPL to the corresponding pyruvate, takes place only to a small extent.