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Title: Studies of pantothenate biosynthesis in Arabidopsis in vivo and in vitro
Author: Coxon, M. M.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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With a view to developing inhibitors, two enzymes of the Arabidopsis pantothenate pathway (KPHMT2 and PS) were expressed in E. coli, as MBP fusion proteins purified and used to perform kinetic studies to identify their characteristics. KPHMT2 showed an apparent Km, Vmax and kcat of 0.534 ± 0.089 mM, 0.802 ± 0.032 nmoles/min and 2.79 min/1 respectively for α-KIVA whilst the enzyme appeared to be inhibited by its co-factor, 5,10-methylene tetrahydrofolate, at levels above approximately 25μM. In addition to its potential as a target for herbicides and anti-microbials the pathway is also of commercial interest for the production of pantothenate for use as a supplement in animal feed and for addition to cosmetics. By constitutive expression of the genes of the E. coli pathway, Arabidopsis lines with higher levels of pantothenate than WT were obtained. Expression of E. coli panC resulted in increased levels of the vitamin in some lines, as did the expression of panD, however, expression of panB showed no increase. The β-alanine levels of the transgenic lines ere also assayed and showed hugely elevated levels in the lines expressing panD.  To determine if expression of the E. coli genes or the accumulation of pantothenate effected the expression of the endogenous genes, RT-PCR was performed and showed no significant difference in the expression of endogenous panB2 or panC in lines expressing E. coli panB, panC or panD. Expression of the genes of the pathway was also studied using promoter:GUS fusions, RT-PCR, western blot analysis and enzyme assays. The results showed expression of the genes throughout the plant suggesting de novo pantothenate biosynthesis with panB1 and panB2 promoter activity showing up-regulation in flowers and siliques, tissues known to require large amounts of pantothenate.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available