Untersuchungen von Methyltransferasen aus Linum-Arten

Lignans are widely distributed throughout the plant kingdom and play a role in the defense against herbivores and microorganisms. These compounds are of interest due to their distinct biological activity. An example for a medically used lignan is the aryltetralin lignan podophyllotoxin (PTOX) that has cytotoxic properties. It is applied against warts but also serves in the production of the PTOX derivatives etoposide and teniposide. These drugs are utilized in cancer therapy. The phosphate derivative of etoposide (Etopophos®) is also used because it shows a better solubility. PTOX is extracted from Podophyllum hexandrum and P. peltatum (Berberidaceae). The biosynthetic pathway of aryltetralin lignans can be divided into the early and late steps. The early steps have been completely elucidated, whereas from the late steps only few enzymes and reactions are known. An enzyme class which especially is of interest are methyltransferases. These transfer a methyl group from a methyl group donor to the substrate. The experiments described in this work were conducted with suspension cultures of Linum nodiflorum, L. album and L. flavum belonging to the family Linaceae. These Linum species accumulate aryltetralin lignans. To start the investigations, seeds from Linum nodiflorum were germinated and callus and suspension cultures were established from these shoots in different media. Suspension cultures from the other plants already existed. The established cultures in CB2 and B5 medium produced less lignans than those in MSLi medium. Regarding the two cultures in MSLi medium, Ln1 mainly produced deoxypodophyllotoxin (DOP) but also 6-methoxypodophyllotoxin (MPTOX). Ln2 accumulated PTOX as principal lignan apart from MPTOX and DOP. Additionally, experiments were performed with the elicitor methyl jasmonate to observe the possible enhancement of the lignan amount in suspension cultures Ln1 and Ln2. The results did not show the desired effects on the lignan content. Suspension culture Ln1 was examined in a culture characterization over a period of 14 days. Data were collected concerning the medium (sugar content, pH value, conductivity), cell parameters (fresh and dry weight), lignan and protein content, phenylalanine ammonia-lyase activity and the gene expression of various methyltransferases. Besides the investigations on the suspension cultures, a biochemical characterization of different methyltransferases was also conducted. Coniferyl alcohol 9-O-methyltransferase catalyzes the methylation of an aliphatic hydroxy group. Wolters et al., (2013) produced mutants of this enzyme to identify the reaction mechanism. The task was to determine the Km value and maximal reaction rate of the mutants C271A and C271S. The Km value for C271A was about 8.91 µM and for C271S 2.58 µM. The second enzyme from Linum nodiflorum that was characterized was a putative caffeic acid O-methyltransferase (LnCOMT). It was shown that it catalyzes the reaction from caffeic acid to ferulic acid. Yet, the activity was too low to determine any biochemical parameters. In the process of identifying enzymes involved in lignan biosynthesis, the focus was on methyltransferases. Degenerate primer from conserved regions were used in PCR and two partial sequences of methyltransferases from Linum album and Linum nodiflorum were received. The methyltransferase from Linum album (LaCOMT) showed a high activity with caffeic acid but also a lower activity with isoferulic acid. The optimal temperature with caffeic acid was 40°C and the optimal pH was 6. The calculated Km value was 90.75 µM for caffeic acid and for SAM 17.35 µM. The comparison of LaCOMT and LnCOMT showed an identity of 95.5% (nucleotide sequence), but a considerable difference in their activity was detected. After successfully obtaining the full-length sequence of the methyltransferase LnMT3 from Linum nodiflorum, two possible start codons were identified. For this reason, two methyltransferases (LnMT3 and LnMT3s) were isolated, cloned and expressed. Both enzymes were tested to check their activity, and both converted the same substrates. A clarification of the actual start codon was not possible. The utilized substrates were the flavonoids hesperetin, quercetin, chrysin and to a lower extent luteolin-7-glucoside. Regarding hesperetin a methylation of the 3’-hydroxy group was proven. Tests could not be performed with all substrates from the lignan biosynthesis due to their unavailability, though future tests can show whether LnMT3/LnMT3s are involved in this biosynthesis.