In Vitro synthesis of, Methyl Salicylate & Methyl Benzoate, by Nicotiana Suaveolens via BSMT
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Authors
Pedroso, Farah L.
Issue Date
2007
Type
Thesis
Language
en_US
Keywords
Alternative Title
Abstract
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.
Description
v, 34 p.
Citation
Publisher
Kalamazoo College
License
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