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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.22, #3)

Analysis of the aspartic acid metabolic pathway using mutant genes by R. A. Azevedo (pp. 217-230).

Amino acid metabolism is a fundamental process for plant growth and development. Although a considerable amount of information is available, little is known about the genetic control of enzymatic steps or regulation of several pathways. Much of the information about biochemical pathways has arisen from the use of mutants lacking key enzymes. Although mutants were largely used already in the 60's, by bacterial and fungal geneticists, it took plant research a long time to catch up. The advance in this area was rapid in the 80's, which was followed in the 90's by the development of techniques of plant transformation. In this review we present an overview of the aspartic acid metabolic pathway, the key regulatory enzymes and the mutants and transgenic plants produced for lysine and threonine metabolism. We also discuss and propose a new study of high-lysine mutants.

Keywords: Keywords: Aspartate kinase; Aspartic acid; Lysine; Lysine 2-oxoglutarate reductase; Methionine; Threonine

Molecular and biochemical analysis of serine acetyltransferase and cysteine synthase towards sulfur metabolic engineering in plants by M. Noji; K. Saito (pp. 231-243).

Serine acetyltransferase (SATase) and cysteine synthase (O-acetylserine (thiol)-lyase) (CSase) are committed in the final step of cysteine biosynthesis. Six cDNA clones encoding SATase have been isolated from several plants, e.g. watermelon, spinach, Chinese chive and Arabidopsis thaliana. Feedback-inhibition pattern and subcellular localization of plant SATases were evaluated. Two types of SATase that differ in their sensitivity to the feedback inhibition by l-cysteine were found in plants. In Arabidopsis, cytosolic SATase was inhibited by l-cysteine at a physiological concentration in an allosteric manner, but the plastidic and mitochondrial forms were not subjected to this feedback regulation. These results suggest that the regulation of cysteine biosynthesis through feedback inhibition may differ depending on the subcellular compartment. The allosteric domain responsible for l-cysteine inhibition was characterized, using several SATase mutants. The single change of amino acid residue, glycine-277 to cysteine, in the C-terminal region of watermelon SATase caused a significant decrease of the feedback-inhibition sensitivity of watermelon SATase. We made the transgenic Arabidopsis overexpressing point-mutated watermelon SATase gene whose product was not inhibited by l-cysteine. The contents of OAS, cysteine, and glutathione in transgenic Arabidopsis were significantly increased as compared to the wild-type Arabidopsis. Transgenic tobacco (Nicotiana tabacum) (F₁) plants with enhanced CSase activities both in the cytosol and in the chloroplasts were generated by cross-fertilization of two transgenic tobacco expressing either cytosolic CSase or chloroplastic CSase. Upon fumigation with 0.1 μL L⁻¹ sulfur dioxide, both the cysteine and glutathione contents in leaves of F₁ plants were increased significantly, but not in leaves of non-transformed control plants. These results indicated that both SATase and CSase play important roles in cysteine biosynthesis and its regulation in plants.

Keywords: Keywords: Amino acids; Cysteine biosynthesis; Serine acetyltransferase; Cysteine synthase

Molecular and biochemical analysis of the enzymes of cysteine biosynthesis in the plant Arabidopsis thaliana by R. Hell; R. Jost; O. Berkowitz; M. Wirtz (pp. 245-257).

Among the amino acids produced by plants cysteine plays a special role as a mediator between assimilatory sulfate reduction and provision of reduced sulfur for cell metabolism. Part of this characteristic feature is the presence of cysteine synthesis in plastids, mitochondria and cytosol. Plants are the major source of reduced sulfur for human and animal nutrition. Cysteine biosynthesis deserves special attention, since reduced sulfur is channelled from cysteine into many sulfur-containing compounds in food and feed. Recent investigations are reviewed that focus on structure and regulation of cysteine synthesis in the model plant Arabidopsis thaliana. These data indicate that cysteine synthesis is not just an intermediate reaction step but that it is part of a regulatory network that mediates between inorganic sulfur supply and the demand for reduced sulfur during plant growth and in response to environmental changes.

Keywords: Keywords: Sulfur amino acids; Plant biotechnology; Metabolic regulation

Engineering of cysteine and methionine biosynthesis in potato by V. Nikiforova; S. Kempa; M. Zeh; S. Maimann; O. Kreft; A. P. Casazza; K. Riedel; E. Tauberger; R. Hoefgen; H. Hesse (pp. 259-278).

Methionine and cysteine, two amino acids containing reduced sulfur, are not only an important substrate of protein biosynthesis but are also precursors of various other metabolites such as glutathione, phytochelatines, S-adenosylmethionine, ethylene, polyamines, biotin, and are involved as methyl group donor in numerous cellular processes. While methionine is an essential amino acid due to an inability of monogastric animals and human beings to synthesise this metabolite, animals are still able to convert methionine consumed with their diet into cysteine. Thus, a balanced diet containing both amino acids is necessary to provide a nutritionally favourable food or feed source. Because the concentrations of methionine and cysteine are often low in edible plant sources, e.g. potato, considerable efforts in plant breeding and research have been and are still performed to understand the physiological, biochemical, and molecular mechanisms that contribute to their synthesis, transport, and accumulation in plants. During the last decade molecular tools have enabled the isolation of most of the genes involved in cysteine and methionine biosynthesis, and the efficient plant transformation technology has allowed the creation of transgenic plants that are altered in the activity of individual genes. The physiological analysis of these transgenic plants has contributed considerably to our current understanding of how amino acids are synthesised. We focused our analysis on potato (Solanum tuberosum cv. Désirée) as this plant provides a clear separation of source and sink tissues and, for applied purposes, already constitutes a crop plant. From the data presented here and in previous work we conclude that threonine synthase and not cystathionine gamma-synthase as expected from studies of Arabidopsis constitutes the main regulatory control point of methionine synthesis in potato. This article aims to cover the current knowledge in the area of molecular genetics of sulfur-containing amino acid biosynthesis and will provide new data for methionine biosynthesis in solanaceous plants such as potato.

Keywords: Keywords: Cysteine biosynthesis; Methionine biosynthesis; Cystathionine gamma-synthase; Cystathionine beta-lyase; Methionine synthase; Serine actetyltransferase; Threonine synthase; Nutritional quality; Potato; Transgenic plants

Biosynthesis and metabolic engineering of glucosinolates by M. D. Mikkelsen; B. L. Petersen; C. E. Olsen; B. A. Halkier (pp. 279-295).

Glucosinolates are amino acid-derived natural plant products found throughout the Capparales order. Glucosinolates and their degradation products have a wide range of biological activities, e.g. in plant defense as deterrents against insect and fungi. The conversion of amino acids to aldoximes is a key step in glucosinolate biosynthesis. This step is catalyzed by cytochromes P450 from the CYP79 family. The post-aldoxime enzymes in the glucosinolate pathway have high substrate-specificity for the functional group and low substrate-specificity for the side chain. Therefore, we have been able to metabolically engineer new glucosinolate profiles into Arabidopsis by altering the levels of endogenous CYP79s and by introducing exogenous CYP79s. The approach has great potential for design of metabolically engineered plants with improved pest resistance and increased nutritional value.

Keywords: Keywords: Glucosinolates; Metabolic engineering; CYP79s; Aldoximes; Arabidopsis; CYP83s

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.22, #3)

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.22, #3)