Dissertation
Characterization of the plastidial serine hydroxymethyl-transferase from Arabidopsis thaliana
Washington State University
Doctor of Philosophy (PhD), Washington State University
05/2010
DOI:
https://doi.org/10.7273/000006002
Abstract
One-carbon (C1) metabolism is crucial to all living organisms. Tetrahydrofolate (H4PteGlun) -bound C1 units are needed in biosynthesis of purines, thymidylate, methionine, and N-formylmethionine, in the methylation of DNA, proteins, and phospholipids, and in biosynthesis of important plant secondary metabolites such as lignin, alkaloids, and betaines. Folates can only be synthesized de novo by plants and prokaryotes. Folate deficiency in humans has been linked to cardiovascular disease and cancer. To fight the problem of folate deficiency, wheat products have been fortified with synthetic folic acid. Dietary folate intake could also be increased by consuming folate-enriched plants; this could potentially be accomplished by engineering C1 metabolism. Serine hydroxymethyltransferase (SHMT) catalyzes reversible conversion of H4PteGlun and serine to 5,10-CH2-H4PteGlun and glycine and is one of the three enzymes that derivatize folates with C1 units. Understanding of SHMT features such as subcellular localization and kinetic properties for polyglutamylated H4PteGlun is necessary groundwork for plant C1 engineering. However, such knowledge is still lacking although SHMTs from several plant species have been studied in the past. Sequencing of the Arabidopsis genome revealed seven SHMTs predicted to be localized in mitochondria, plastids, the cytosol, and the nuclei. As a part of the effort to investigate all seven SHMTs in Arabidopsis, this study presents the first report on the characterization of a putative plastid SHMT (AtSHMT3). The localization of AtSHMT3 in plastids was verified with GFP fusions. Additionally, the presence of SHMT and 10-formyltetrahydrofolate synthetase in plastids of pea and barley was demonstrated. In order to assay SHMT at low concentrations of H4PteGlun, we developed a sensitive HPLC-based fluorometric assay. By using this HPLC assay, we obtained kinetic parameters for H4PteGlun=1-8 showing that AtSHMT3 has increased substrate affinity and catalytic efficiency as n grows from 1 to 8. This work also showed that both 5-CH3-H4PteGlu1&5 and 5-HCO-H4PteGlu1&5 inhibit AtSHMT3-catalyzed hydroxymethyl-group transfer from serine to H4PteGlu6 with the pentaglutamylated inhibitors being more effective. Calculations revealed that these pentaglutamylated inhibitors could lead to little reduction in AtSHMT3 activity under estimated folate concentrations in plastids.
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Details
- Title
- Characterization of the plastidial serine hydroxymethyl-transferase from Arabidopsis thaliana
- Creators
- Yi Zhang
- Contributors
- Sanja Roje (Chair)Andrew McCubbin (Committee Member) - Washington State University, School of Biological SciencesJohn A Browse (Committee Member) - Washington State University, Institute of Biological ChemistryMICHAEL L. KAHN (Committee Member) - Washington State University, Institute of Biological Chemistry
- Awarding Institution
- Washington State University
- Academic Unit
- Program in Molecular Plant Sciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Publisher
- Washington State University
- Number of pages
- 98
- Identifiers
- 99901055026201842
- Language
- English
- Resource Type
- Dissertation