NICOTIANAMINE BIOSYNTHESIS IN PLANTS

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dc.contributor.advisorBorthakur, Dulal
dc.contributor.authorCarrillo, James Thomas
dc.contributor.departmentMolecular Biosciences and Bioengineering
dc.date.accessioned2024-10-09T23:46:17Z
dc.date.available2024-10-09T23:46:17Z
dc.date.issued2024
dc.description.degreePh.D.
dc.identifier.urihttps://hdl.handle.net/10125/108710
dc.subjectMolecular biology
dc.subjectBiochemistry
dc.subjectBioengineering
dc.titleNICOTIANAMINE BIOSYNTHESIS IN PLANTS
dc.typeThesis
dcterms.abstractPlants rely upon a small chemical ligand, nicotianamine (NA), to chelate metallic ions during transport throughout tissues and across cell membranes. The production of this essential compound is achieved by three enzymes: S-adenosylmethionine (SAM) synthetase, NA synthase and 5’-methylthioadenosine (MTA) nucleosidase. The characterization of each of these enzymes from any plant species is lacking or absent, therefore the coding sequence for these genes were isolated from local hardwood species and characterized in vitro. The cDNA encoding for SAM-synthetase, NA synthase and MTA nucleosidase was isolated from giant leucaena (Leucaena leucocephala subsp. glabrata) root tissue mRNA. Transcriptome data and/or 5’-RLM-RACE were used to obtain the full transcript sequence. The complete coding sequences were cloned into a T7-expression vector and expressed in Escherichia coli. The giant leucaena SAM-synthetase displayed optimal enzyme activity at pH 10.0, 55 oC, and 200 mM KCl. In addition to thermophilic activity, giant leucaena SAM-synthetase remains highly active in solutions containing up to 4 M KCl and accepts Na+ to some extent as a substitute for K+, a known required cofactor for SAM-synthetases. The enzyme followed Michaelis–Menten kinetics (Km = 1.82 mM, Kcat = 1.17 s-1, Vmax 243.9 µM. min-1) and was not inhibited by spermidine, spermine or nicotianamine. Plant SAM-synthetases have not been characterized previously and the activity of the giant leucaena SAM-synthetase differed greatly from that of nonplant SAM-synthetases. Therefore, a second plant SAM-synthetase from Acacia koa was investigated. The activity of the Acacia koa SAM-synthetase resembles that from the giant leucaena (Km = 1.44 mM, Kcat = 1.29 s-1, Vmax 170 µM. min-1) and exhibited similar alkali-thermophilic properties. Four single-point mutation variants of the Acacia koa SAM-synthetase were produced, each with varying degrees of reduced reaction rate, greater sensitivity to product inhibition and loss of thermophilic properties. Although an enhanced mutant was not produced, the addition of organic solvent was shown to have a beneficial effect on enzyme activity. Feedback inhibition was reduced in SAM-synthetase enzyme assays containing 25% acetonitrile, methanol or dimethylformamide and total SAM production improved by 30-65%. Transcriptome sequence data and 5’-RLM-RACE methods identified the complete NA synthase coding sequence, which was cloned for heterologous expression and in vitro assays. Soluble expression was highly repressed, however heat-shocking and high-NaCl media improved recombinant enzyme production approximately 6-fold. Feedback inhibition by MTA is known to severely limit the in vitro activity of SAM dependent enzymes, however this work demonstrates comparable effects produced by substrate inhibition. Producing enantiomerically pure substrate in situ, using a recombinant SAM-synthetase, enabled 5-fold faster NA synthase activity (Vmax = 1.25 µM. h-1) and 2-fold net nicotianamine production than when commercially purified substrate, a racemate, is provided. Sequence and structural analyses suggest residues involved in azetidine ring formation, and other aspects of the mechanism are explored. The complete coding sequence of a MTA nucleosidase was obtained from the giant leucaena transcriptome and cloned into pet39b, thereby expressing a fusion protein secreted into the E. coli periplasm. The crude periplasmic extract was tested for MTA-nucleosidase activity and when combined with SAM-synthetase and NA-synthase, further improved the reaction rate 22-fold (Vmax = 27.5 µM. h-1) and net NA production 3-fold. In vitro NA production is optimal in solutions containing all three enzymes (SAM-synthetase, NA-synthase, and MTA-nucleosidase), however the reaction becomes fully inhibited upon producing 58.5 µM NA. In addition to the in vitro characterization of recombinant enzymes, the in plantae transcription of NA synthase, SAM synthetase and 17 other genes was quantified in giant leucaena seedlings. In response to high-iron fertilization, real-time PCR performed on root and foliar tissue showed overall upregulation of most genes. Notable genes affected include glutathione synthase (20-fold increase in leaf), ferric chelate reductase (15-fold increase in root), mimosinase (20-fold increase in leaf) and nicotianamine synthase (30-fold increase in root). In response to iron fertilization, mimosine metabolism was shown to be increased by metal induced stress; that is oxidative stress and not iron itself. Furthermore, the gene expression profile of leaf tissue indicated that plants with high mimosinase activity were less affected by iron and experienced less oxidative stress. Therefore, mimosine metabolism may broadly affect iron-regulated processes and enable the hyperaccumulation of iron or other metals.
dcterms.extent150 pages
dcterms.languageen
dcterms.publisherUniversity of Hawai'i at Manoa
dcterms.rightsAll UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
dcterms.typeText
local.identifier.alturihttp://dissertations.umi.com/hawii:12332

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