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The enzymes that take part in rosmarinic acid biosynthesis could be identified and characterized in cell cultures of Coleus blumei and several other species of the Lamiaceae and Boraginaceae. But there is still little known about the regulation of the biosynthetic pathway on genomic level. This work aimed to provide a detailed investigation of the biosynthesis of rosmarinic acid in a diploid suspension culture of Melissa officinalis L. for future investigations into these regulatory mechanisms. For this purpose a thorough characterization of the cell culture was necessary. Additionally a detailed characterization of the enzymes involved in the rosmarinic acid biosynthesis both in the crude extract and after cloning and heterologous expression in E. coli was performed.
Rosmarinic acid, the main component of the so called “Labiatengerbstoffe”, is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid that can be found wide spread in the plant kingdom. In plants, rosmarinic acid is supposed to act as a preformed constitutively accumulated defence compound. The biosynthesis starts with the amino acids L-phenylalanine and L-tyrosine. After enzymatic conversion to 4-coumaric acid and 4-hydroxyphenyllactic acid they are esterified by rosmarinic acid synthase, an enzyme belonging to the superfamily of BAHD acyltransferases. Finally the reaction product is converted to rosmarinic acid by 3- and 3’-hydroxylases.
During short culture periods suspension cultures of Melissa officinalis are able to accumulate rosmarinic acid up to 7 % of the dry weight in medium with 4 % sucrose. After elicitation with methyl jasmonate a further increase is possible.
The transformation of L-phenylalanine to CoA-esters of hydroxycinnamic acid is catalyzed by the enzymes of the general phenylpropanoid pathway: phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (C4H) and 4-coumaric acid CoA-ligase (4CL). In addition to the characterization of the native enzymes a successful cloning and characterization of a PAL- and a 4CL-isoform took place. Southern blot analysis of both enzymes revealed two gene copies of each enzyme in the genome. According to the mRNA expression profiles the cloned isoforms may be involved in rosmarinic acid biosynthesis. By using the full-length primers it was also possible to determine the genomic sequence of both enzymes. The genomic sequence of PAL contains a characteristic intron that can be found in a conserved position in the genomic sequences of all known PAL-sequences of angiosperms. In contrast to this the genomic sequence of the cloned 4CL is free of introns.
L-Tyrosine is transformed to 4-hydroxyphenylpyruvate catalyzed by tyrosine aminotransferase (TAT). The kinetic parameter of the TAT in the crude extract of suspension cultures of lemon balm correlated well with the data determined for the enzyme in other plants (Coleus blumei, Anchusa officinalis), mammalians (rat), or protozoa (Trypanosoma cruzi). Using the method of RACE-PCR a partial cDNA could be identified that showed high similarity to other TAT sequences.
The reduction of 4-hydroxyphenylpyruvic acid to the corresponding lactate is catalyzed by hydroxyphenylpyruvate reductase (HPPR). The substrate preference of the native enzyme is comparable, both in the crude extract of Coleus blumei and Melissa officinalis suspension cultures: 4-hydroxyphenylpyruvic acid (5 µmol/l) is preferred over 3,4-dihydroxyphenylpyruvic acid (28 µmol/l) and the apparent Km-value for NADPH (58 µmol/l) is lower than the one for NADH (90 µmol/l). It is still unknown whether HPPR is a specific enzyme of rosmarinic acid biosynthesis. It could also be possible that the reac-tion is catalyzed by an enzyme of the photorespiratory pathway, namely cytosolic hydroxypyruvate reductase (HPR).
Rosmarinic acid synthase (RAS) catalyzes the esterification of the coenzyme A activated (hydroxy-) cinnamic acid with the reaction product of HPPR. Up to now only four RAS-cDNAs could be cloned and characterized after heterologous expression in E. coli. The cDNAs were isolated from two different Coleus species, Plectranthus fruticosus and Melissa officinalis. As acceptor substrates they accept only 4-hydroxyphenyllactic acid, and 3,4-dihydroxyphenyllactic acid, but neither quinic acid nor shikimic acid. Because of this high substrate specificity RAS is supposed to be a specific enzyme of this biosynthetic pathway. According to the results of Southern blot analysis there is only one copy of RAS in the genome of lemon balm.