Dissertation
IDENTIFICATION AND CHARACTERIZATION OF THE MISSING PHOSPHATASE ON THE RIBOFLAVIN BIOSYNTHESIS PATHWAY IN ARABIDOPSIS THALIANA
Doctor of Philosophy (PhD), Washington State University
01/2016
Handle:
https://hdl.handle.net/2376/111527
Abstract
As the direct precursor of cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), riboflavin is required in all living organisms. The pathway for synthesis of riboflavin in plants has been identified, and most of the enzymes involved have been cloned and biochemically characterized with one exception, which is the phosphatase catalyzing the hydrolysis of 5-amino-6-ribitylamino-2,4(1H,3H) pyrimidinedione 5'-phosphate (ARPP). Lack of such knowledge is an important problem, preventing further understanding of the riboflavin biosynthesis in plants and other organisms.
In order to fill this gap in knowledge, the main goal of my dissertation is to identify and characterize the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana. Starting with a previously identified FMN hydrolase AtcpFHy1 (At1g79790), seven more plastidial phosphatases belonging to the HAD superfamily were found. These candidates for the missing ARPP phosphatase of the riboflavin biosynthesis pathway were cloned and purified. Their phosphatase activities on the substrate ARPP were tested, and enzymes encoded by genes At1g79790 (AtcpFHy1), At4g11570 and At4g25840 were found capable of hydrolyzing ARPP in vitro. AtcpFHy1 was therefore renamed AtcpFHy/PyrP1; At4g11570 and At4g25840 were named AtPyrP2 and AtPyrP3, respectively. Subcellular localization of AtPyrP2 and AtPyrP3 was studied, and it was found that AtPyrP2 is a plastidial enzyme, while AtPyrP3 is localized in mitochondria. Since the entire riboflavin biosynthesis resides in chloroplast, AtcpFHy/PyrP1 and AtPyrP2 were further characterized with respect to Michaelis-Menten kinetics, substrate specificity, pH and temperature optima, and molecular weight. The physiological roles of these two HAD phosphatases in riboflavin production were investigated. T-DNA insertional knockout of AtcpFHy/PyrP1 did not affect the flavin profile of the transgenic plants, whereas down-regulation of AtPyrP2 resulted in a decreased accumulation of riboflavin, FMN, and FAD.
Our results thus indicate that AtPyrP2 is the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis. This identification of the last missing enzyme on this pathway closes a long-standing gap in understanding of the riboflavin biosynthesis pathway in plants.
Metrics
7 File views/ downloads
17 Record Views
Details
- Title
- IDENTIFICATION AND CHARACTERIZATION OF THE MISSING PHOSPHATASE ON THE RIBOFLAVIN BIOSYNTHESIS PATHWAY IN ARABIDOPSIS THALIANA
- Creators
- Na Sa
- Contributors
- Sanja Roje (Advisor)Asaph Cousins (Committee Member)Helmut Kirchhoff (Committee Member)Bernd Markus Lange (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Program in Molecular Plant Sciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 102
- Identifiers
- 99900581725501842
- Language
- English
- Resource Type
- Dissertation