Investigating Plant One-carbon Metabolism Through Engineering and Enzyme Characterization
One-carbon (C1) metabolism is essential to all organisms. In plants, C1 units are required for the synthesis of proteins, nucleic acids, pantothenate, and many methylated molecules. Due to the low abundance and lability of the C1-substituted folates, S-adenosylmethionine (AdoMet), and S-adenosyl-homocysteine (AdoHcy), and the presence of multiple isoforms for enzymes interconverting the C1 units, plant C1 metabolism has been under investigated.Plant C1 metabolism consists of folate-mediated reactions, folate-independent reactions, the activated methyl cycle, and the related S-methylmethionine (SMM) cycle. To better understand this area of metabolism, recruitment of C1 moiety to the C1 metabolism through serine hydroxymethyltransferase (SHMT) and provision of C1 moiety to methionine biosynthesis involving 5,10-methylenetetrahydrofolate reductase (MTHFR) have been studied in this dissertation project.SHMT catalyzes the reversible interconversion of serine and tetrahydrofolate (H4PteGlun) to glycine (Gly) and 5,10-methylenetetrahydrofolate (5,10-CH2-H4PteGlun). SHMT activity has been found in the cytosol, plastids, and mitochondria in plants. Mitochondrial SHMT (mSHMT) from plants plays an important role in catabolizing Gly and regenerating Ser during photorespiration. Two isoforms of mSHMT from Arabidopsis thaliana have been cloned, expressed, and purified. Those two isoforms have also been proven active through enzyme assays.Methionine (Met) is a sulfur-containing amino acid, required as a building block of proteins and a precursor of many essential metabolites. This essential amino acid cannot be synthesized de novo by mammals and needs to be obtained from the diet. C1 metabolism contributes to the synthesis of Met by providing the methyl group from 5-methyltetrahydrofolate (5-CH3-H4PteGlun) for methylation of homocysteine (Hcy) to Met. Due to the absence of MTHFR, which catalyzes the reduction of 5,10-CH2-H4PteGlun to 5-CH3-H4PteGlun, synthesis of Met is hypothesized to be limited in plastids by the availability of the methyl group donor, 5-CH3-H4PteGlun. Therefore, Escherichia coli (E. coli) MTHFR has been overexpressed in plastids of tobacco plants in an attempt to elevate Met content in these organelles. Transgenic plants have accumulated up to 4-fold more soluble Met than the wild type together with increased levels of various other amino acids. Total folate content has also been increased up to 4 fold. However, these transgenic plants barely photosynthesize and have severe growth defects, which could be partially rescued by growth under elevated CO2 concentration.