Characterization of rosmarinic acid synthase from cell cultures of Coleus blumei

Abstract

Rosmarinic acid synthase (RAS) catalyses the transesterification reaction of CoA-activated cinnamic acids, e.g. 4-coumaroyl- or caffeoyl-CoA, and hydroxyphenyllactic acids, e.g. 4-hydroxy- or 3,4-dihydroxyphenyllactic acid, during the biosynthesis of rosmarinic acid. The enzyme isolated from suspension cultures of Coleus blumei is soluble and has an optimal pH of 7.0–7.5. Michaelis-Menten kinetics were observed with respect to the substrates 4-coumaroyl- and caffeoyl-CoA with K m -values of 20 and 33 μM, respectively. Only R(+)-stereoisomers of the hydroxyphenyllactic acid are accepted by RAS. For 4-hydroxy- and 3,4-dihydroxyphenyllactic acid the K m -values were 0.17 and 0.37 mM, respectively. The RAS reaction was inhibited by 4-hydroxymercuribenzoate, the S(−)-stereoisomer of 3,4-dihydroxyphenyllactic acid, hydroxyphenylpyruvates and rosmarinic acid, whereas cinnamic acids were not inhibitory. Coenzyme A showed a non-competitive inhibition. The reverse reaction of RAS, the splitting of rosmarinic acid with help of coenzyme A into caffeoyl-CoA and 3,4-dihydroxyphenyllactate, was readily detected. The kinetic data for this reaction are K m -values of 310 μM for coenzyme A and 15 μM for rosmarinic acid.