Methyltransferases from Ruta graveolens L.: Molecular Biology and Biochemistry
The common rue, Ruta graveolens L., is an aromatic plant. It contains acridone alkaloids, furoquinolines, coumarins, and numerous volatile compounds with antimicrobial and allelopathic activity. The starting point of this work was the biosynthesis of acridone alkaloids in Ruta graveolens L., that a...
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|Summary:||The common rue, Ruta graveolens L., is an aromatic plant. It contains acridone alkaloids, furoquinolines, coumarins, and numerous volatile compounds with antimicrobial and allelopathic activity.
The starting point of this work was the biosynthesis of acridone alkaloids in Ruta graveolens L., that are restricted to the family of Rutaceae. The N-methylation of anthranilate by the anthranilate N-methyltransferase represents the first step in acridone alkaloid biosynthesis by which the anthranilic acid is removed from the primary metabolism.
The anthranilate N-methyltransferase coding cDNA had to be isolated from R. graveolens cell culture material and the recombinant enzyme expressed and characterized. An appropriated method to clone an unknown protein is to isolate it from native tissue followed by microsequencing of the resulted peptides. Specific primers designed from the determined peptides would then be used for the cDNA amplification.
In this work, 500 g wet weight of Ruta graveolens R-20 cells grown in suspension culture, were employed for a six-step purification strategy leading to a 450 times purification fold. The gel electrophoresis showed a polypeptide band with an apparent molecular weight of 42-43 kDa. Radioactive mesurements with [Methyl-14C]-SAM showed a high specific activity.
The microsequencing of the protein, performed by Dr. Peter Hunziker (Biochemistry Department, University of Zürich), resulted in only five short peptide fragments with no homology with other known methyltransferases. Degenerated oligonucleotide primers, designed based on the peptide fragments, produced a PCR amplicon with no homology to methyltransferases.
As the purification did not bring the desired result, degenerated oligonucleotide primers were designed based on the conserved motives (SAM binding site) in O-methyltransferases (OMT). With this method, two full length cDNAs were isolated, R-23 and R-27. R-23 was coding for a 366 amino acid protein with a calculated molecular weight of 40 kDa. R-27 was coding for a 41.6 kDa protein of 374 amino acid residues. These genes were cloned in pQE-60 vector and expressed in E. coli M-15. The protein R-23 had 79% similarity with caffeic acid O-methyltransferases and preserved all the amino acids important for methylation. R-27 showed all the important methyltransferases conserved motifs and had 50% similarity with a putative methyltransferase from Prunus dulcis respectively orcinol and chavicol O-methyltrasnferases from Rosa hybrida. Both bacterial overexpressed proteins were tested for substrate specificity. R-23 coded enzyme did not methylate any of the tested substrates, including caffeic acid. R-27 did methylate 3,5-dimethoxyphenol and other methoxylated phenols.
The protein R-27 was purified in four chromatographic steps and characterized. The enzyme showed narrow substrate specificity. The highest affinity was with 3,5-dimethoxyphenol (Km 20.1 µM) with an optimum pH of 7.5. Other accepted substrates were: 3-methoxyphenol, guaiacol, 3,4-dimethoxyphenol and 3,5-dihydroxyanisole. The reaction was independent on cations. The optimum temperature was around 36˚C. On Coomassie stained SDS gel, the purified protein showed a characteristic band of 42 kDa. The calculated molecular weight from a calibrated size exclusion chromatographie was 84 kDa, suggesting that the native enzyme is a homodimer.
The reaction product of 3,5-dimethoxyphenol methylation, 1,3,5-trimethoxybenzene is an important component of the scent of roses. Therefore, is reasonable to postulate that the product of the methylation described in this work is a volatile component of the scent of R. graveolens. Based on its substrate specificity, the protein was designated as 3,5-dimethoxyphenol O-methyltransferase.
It could be shown that in the presence of Zn2+ the protein efficiently methylate DTT. The product was identified by LC-MS as monomethylthioether. Therefore, 3,5-dimethoxyphenol O-methyltransferase, in addition to the OMT activity, had a thiolmethyltransferase (TMT) activity. Kinetic analysis of 3,5-dimethoxyphenol methylation in the presence of various Zn2+/DTT concentrations showed a competitive DTT binding with an affinity of Ki = 52.0 µM. These results indicate that the OMT and TMT take place in the same active site of the enzyme. Altogether, these results suggest that the substrate specificity of plant O-methyltransferases II and their function in vivo is probably much wider as assumed.|
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