Plant Metabolic Pathways and Regulatory Networks for Aromatic Amino Acids and Hormones
Başlık:
Plant Metabolic Pathways and Regulatory Networks for Aromatic Amino Acids and Hormones
Yazar:
Holland, Cynthia K., author. (orcid)0000-0001-6099-7998
ISBN:
9780438094352
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (323 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-11(E), Section: B.
Advisors: Joseph M. Jez Committee members: Barbara N. Kunkel; Lucia C. Strader; Richard D. Vierstra; Timothy A. Wencewicz.
Özet:
Unlike humans and other metazoans, organisms such as fungi, bacteria, and plants have retained the enzymatic machinery necessary to synthesize their aromatic amino acids de novo. Chorismate, the final product of the shikimate pathway, is the precursor to the three aromatic amino acids--- tryptophan, tyrosine, and phenylalanine--- and is upstream of a number of plant growth hormones, including auxins and benzoates. Phenylalanine and tyrosine both stem from the precursor prephenate, which is formed from chorismate by chorismate mutase, and use dehydrogenases, aminotransferases, and dehydratases in their biosynthetic pathways. Although aromatic amino acid biosynthesis is important for protein synthesis, secondary metabolism, and human health, much of what is known about plant aromatic amino acid biosynthesis and its regulation has been inferred from microbial investigations of the pathway.
L-Tyrosine is essential for protein synthesis and is a precursor of numerous specialized metabolites crucial for plant and human health. Tyrosine can be synthesized via two alternative routes by a key regulatory TyrA family enzyme, prephenate or arogenate dehydrogenase (PDH/TyrAp or ADH/TyrAa), representing a unique divergence of primary metabolic pathways. The molecular foundation underlying the evolution of the alternative Tyr pathway is currently unknown. Here we characterized recently-diverged plant PDH and ADHs, obtained the x-ray crystal structure of soybean PDH, and identified a single amino acid residue that defines TyrA substrate specificity and regulation. Structures of mutated PDHs co-crystallized with Tyr indicate that substitutions of Asn222 confers ADH activity and Tyr-sensitivity. Subsequent mutagenesis of the corresponding residue in divergent plant ADHs introduced PDH activity and relaxed Tyr sensitivity, highlighting the critical role of this residue in TyrA substrate specificity that underlies the evolution of alternative Tyr biosynthetic pathways in plants.
The three-dimensional structure of soybean PDH1 allowed for the identification of both the cofactor- and ligand-binding sites. Here, we present steady-state kinetic analysis of twenty site-directed active site mutants of the soybean (Glycine max) PDH compared to wild-type. Molecular docking of the substrate, prephenate, into the active site of the enzyme reveals its potential interactions with the active site residues and makes a case for the importance of each residues in substrate recognition and/or catalysis, most likely through transition state stabilization. Overall, these results suggest that the active site of the enzyme is highly sensitive to any changes, as even subtle alterations substantially reduced the catalytic efficiency of the enzyme.
Chorismate mutase catalyzes the branch point reaction of phenylalanine and tyrosine biosynthesis to generate prephenate. In Arabidopsis thaliana , there are two plastid-localized chorismate mutases that are allosterically regulated (AtCM1 and AtCM3) and one cytosolic isoform (AtCM2) that is unregulated. Previous analysis of plant chorismate mutases suggested that the enzymes from early plants (i.e., bryophytes/moss, lycophytes, and basal angiosperms) formed a clade distinct from the isoforms found in flowering plants; however, no biochemical information on these enzymes is available. To understand the evolution of allosteric regulation in plant chorismate mutases, we analyzed a basal lineage of plant enzymes homologous to AtCM1 based on sequence similarity. The chorismate mutases from the moss/bryophyte Physcomitrella patens (PpCM1 and PpCM2), the lycophyte Selaginella moellendorffii (SmCM), and the basal angiosperm Amborella trichopoda (AmtCM1 and AmtCM2) were characterized biochemically. Tryptophan was a positive effector for each of the five enzymes examined. Histidine was a weak positive effector for PpCM1 and AmtCM1. Neither tyrosine nor phenylalanine altered the activity of SmCM; however, tyrosine was a negative regulator of the other four enzymes. Phenylalanine down-regulates both moss enzymes and AmtCM2. The 2.0 A x-ray crystal structure of PpCM1 in complex with the tryptophan identified the allosteric effector site and reveals structural differences between the R- (more active) and T-state (less active) forms of plant chorismate mutases. Molecular insight into the basal plant chorismate mutases guides our understanding of the evolution of allosteric regulation in these enzymes.
Plants make tyrosine and phenylalanine by a different pathway from many microbes, which requires prephenate aminotransferase (PAT) as the key enzyme. PAT produces arogenate, the unique and immediate precursor for both tyrosine and phenylalanine in plants, and also has aspartate aminotransferase (AAT) activity. The molecular mechanisms governing the substrate specificity and activation or inhibition of PAT are currently unknown. (Abstract shortened by ProQuest.).
Notlar:
School code: 0252
Mevcut:*
Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(695046.1) | 695046-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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