In Vitro synthesis of, Methyl Salicylate & Methyl Benzoate, by Nicotiana Suaveolens via BSMT
Pedroso, Farah L.
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Many plants like those of the Solanaceae family produce an array of volatiles, airborne chemicals emitted from planta tissue, through a methylating pathway. In which methyl group is donated and transferred from S-adenosyl-Lmethionnine, SAM, to carboxylic acid-bearing substrates like salicylic or benzoic acid (Zubita et. al., 2003). It is believed that volatiles have an array of effects on the environment. They are thought of being responsible for the attraction of pollinators, defense from herbivory and also of having anti-microbial functions. The enzymes responsible for transferring the methyl group in many of the Solanaceae members are salicylic acid methyl transferase (SAMT) and benzoic acid methyl transferase (BSMT). This two are part of the SABATH gene family, in which all its members produce volatiles in planta through a methylating pathway using SAM as its donor (Effmert, 2005). Nicotiana Suaveolens, Australian tobacco is a BSMT -type plant that produces methyl benzoate in large amounts due to its high affinity for benzoic acid. It has been found in previous studies that N. Suaveolens produces a 4-fold greater amount of methyl benzoate than methyl salicylate (Pott, 2004). The purpose of this study was to isolate methyl benzoate in vitro from the BSMT gene of the N. Suaveolens' sequence. The second part of the study was to create a single amino acid mutation in amino acid position 150, changing the amino acid from Histidine to Methionine. This change will make the affinity for salicylic acid higher and the production of methyl salicylate will also increase, in order words the gene will be changed from BSMT to SAMT. Showing that the main difference between these two types of plants is the existence of 1 amino acid or another in position 150, with SAMT -type plants having Methionine and BSMT -type plants having Histidine. The main results from this study were tested with the use of protein assays and GC-MS. The results suggested that in vitro N. Suaveolens WT-BSMT was able to methylate both substrates, as expected, but it had a higher affinity for salicylic acid over benzoic acid; thereby having a 6-fold higher production of methyl salicylate. These results are very contradicting to what has been reported previously by a very important study, which suggested that there would be a 4-fold higher amount of methyl benzoate produced over methyl salicylate. The BSMT genes that were mutated to perform as SAMT genes had zero yields.
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