Acridonbiosynthese in Ruta graveolens L.: Molekularbiologie und Biochemie der Anthranilat N-Methyltransferase

Acridones represent a special type of alkaloids as they were identified so far mainly from genera belonging to the Rutaceae family. There is only little information about their biosynthesis and benefit to the plant. Ruta graveolens L., the common rue, is a small evergreen shrub originating from the mediterrainien region. It was used as a traditional medicinal herb and spice already in the ancient world. The accumulation of acridones renders Ruta graveolens an excellent model plant for investigating their biosynthesis in vitro; this task was greatly simplified by introducing elicitor-inducible Ruta cell suspension cultures. The Anthranilate N-methyltransferase (ANMT) is catalysing an important step in acridone biosynthesis by channeling anthranilate from primary into secondary metabolism; however, the isolation of the enzyme failed so far (Maier et al., 1995; Burga, 2005). A protein band of approximately 40 kDa could be partially purified from elicitor-induced Ruta graveolens cell cultures. After peptide microsequencing, degenerated primers were designed, which were successfully applied for amplification of a full length cDNA in combination with RACE techniques. The cDNA exhibits an open reading frame of 1095 bp, coding for a polypeptide sequence of 365 amino acids with a calculated mass of 40059 Da and a predicted isoelectric point (pI) of 5,72. After recombinant expression in E. coli the enzyme showed narrow substrate specificity with Km-values of 7,1 µM and 3,3 µM for anthranilate and S-adenosyl-L-methionine (SAM), respectively. No other tested substrates were accepted. Most interestingly, anthraniloyl-CoA was not converted; this provides evidence that in Ruta graveolens the first step in acridone biosynthesis is the N-methylation of anthranilate yielding N-methylanthranilate, followed by an activation to the corresponding CoA-ester. Acridones without the N-methylation pattern might be demethylated later in the biosynthetic pathway. Biochemical characterization showed an optimal enzyme activity in sodium-glycinate-buffer pH 7.5 at 37 °C. Enzyme activity is inhibited by metal ions, especially Fe2+, Fe3+, Cu2+ and Zn2+. No influence of Mg2+-ions was detected, which clusters the ANMT into class II of SAM-dependent plant methyltransferases (MTs). This is confirmed by the molecular mass of 40 kDa for the denatured protein on SDS-PAGE and 80 ± 5 kDa for the native active protein on a calibrated gel permeation chromatography column. Enzyme activity can thus be assigned to a homodimer, as described for most SAM-dependant plant MTs. For further characterization, expression studies in Ruta cell cultures and plants were conducted. Both systems showed a constitutive expression of the ANMT, which emphazises the function of the acridone alkaloids as phytoanticipins. The cell culture showed a slight increase in protein amount and enzyme activity upon induction with yeast elicitor. In intact plants, the highest abundance of RgANMT transcripts was found in flowers and roots, with lower amounts in leaves and stems. These findings coincide with the main locations of acridone alkaloid biosynthesis, so that no translocation of acridones from roots into other plant tissues seems to be required as described for other alkaloid types in other plant genera (cf. Junghanns et al., 1998). Phylogenetic analysis revealed a surprisingly high similarity of the ANMT polypeptide to the group of O-methyltransferases (OMTs), which points to an evolutionary relation of the ANMT to this MT class. The successful isolation and characterization of the ANMT represents an important step not only for the understanding of acridone biosynthesis. It also provides an important tool for studies on the regulation of acridone biosynthesis or anthranilate metabolism, respectively.