The following are current highlights from the research focus areas in the Leustek Group
Sulfur is an essential nutrient for all organisms including plants. The biological role of sulfur traces back to the initial events in the origin of life, which might have arisen as catalytic reactions on iron sulfide surfaces under anaerobic, hydrothermal conditions. The aerobic atmosphere of the modern Earth ensures that sulfur exists predominantly in the +6 valance state in the form of sulfate. But sulfur in its other oxidation states exist in anaerobic or volcanic environments and within living cells. Plants and microorganisms reduce sulfate, changing the valance to –2 (sulfide) through the process of assimilative reduction. The process is assimilative because sulfide is used exclusively for the synthesis of cysteine, methionine, and other metabolites. On Earth sulfur is in constant flux between oxidized and reduced states through the action of living organisms and chemical processes. The cycle is termed the bio-geochemical sulfur cycle. Although the Leustek Group has worked on many aspects of sulfate assimilation in plants the recent work has focused on the biochemical properties of a key enzyme of the sulfate assimilation pathway known as 5'-adenylylsulfate (APS) reductase. The most recent work, carried out in collaboration with Drs. David Knaff and Sung-Kun Kim of Texas Tech University, has been a comparative analysis of the enzymes from Pseudomonas aeruginosa and from the plants Arabidopsis thaliana and Enteromorpha intestinalis. APS reductases from these species contain an FeS center. The plant enzymes differ from the Pseudomonas enzyme in that they contain a carboxyl terminal extension that function as a thioredoxin or glutaredoxin. The catalytic mechanism or the function of the FeS centers are not known, but likely involve dithiol/difulfide chemistry.
An important question when considering how plants coordinate the synthesis of amino acids with the demand is whether amino acids are transported throughout the plant body. For some amino acids that are involved in transporting nitrogen, such as glutamine, the answer is clearly, yes. However, for many of other amino acids nothing is known about inter-organ translocation. To address this question the Leustek Group has been analyzing amino acid biosynthesis mutants of Arabidopsis thaliana. One class of mutants in the genes for His biosynthesis have an embryo lethal phenotype, meaning, that homozygous mutants abort at an early stage in embryo development. This result suggests that Arabidopsis embryos may not obtain His from the maternal source, rather, they must synthesize His on their own. Further studies aim to characterize the physiological and molecular response of mutant embryos to His starvation.
Met is synthesized from three components. The carbon skeleton is derived from Asp as are the amino acids Lys, Thr and Ile. The sulfur moiety is derived from Cys. The methyl group is derived from methyltetrahydrofolate. Any or all of these pathways could be involved in the control of Met synthesis. The aim of Leustek's research has been to define the points for control of Met synthesis. This work, along with that of other researchers, has implicated three control points. Regulation of cystathionine gamma-synthase (CGS) and threonine synthase (TS) activities directly control Met synthesis, whereas the regulation of aspartate kinase (AK) indirectly controls Met synthesis by regulating the production of homoserine. The three control mechanisms can be illustrated from the results of metabolic engineering experiments and from chemically treated plants. The effect of CGS on Met synthesis is shown by the affect of repressing CGS expression using an antisense RNA technique. The plants are unable to complete their life cycle unless Met is supplied to them. Overexpression of CGS results in the accumulation of soluble Met, which is readily observed as resistance to ethionine, a toxic analog of Met.
Although lysine biosynthesis in plants is known to occur by way of a pathway that utilizes diaminopimelic acid (DAP) as a central intermediate the available evidence suggested that none of the known DAP pathway variants found in nature occurs in plants. The Leustek group, in collaboration with Dr. Charles Gilvarg of Princeton University, set out to determine the Lys biosynthesis pathway using the model Arabidopsis thaliana. They discovered that plants utilizes a novel transaminase that specifically catalyzes the inter-conversion of tetrahydrodipicolinate and L,L-diaminopimelate, a reaction requiring 3 enzymes in the DAP pathway variant found in Escherichia coli. A similar enzyme was discovered in cyanobacteria, indicating the conservation of Lys biosynthesis between plants and the ancestor of chloroplasts. Moreover, these results demonstrate that the lysine biosynthetic pathway in plants and cyanobacteria is distinct from the pathways that have so far been defined in microorganisms.
Almost nothing is known about the properties of plant aminoacyl-tRNA synthetases. The Leustek group is focusing on these enzymes from a variety of plant species in order to determine whether they are the basis for resistance to pathogens. Although the project is still in its earliest stages, it has already produced significant results that will be the topic of future research papers.
Thomas Leustek would like to gratefully acknowledge the various funding agencies that have supported his work since 1992. The public agencies include the National Science Foundation, the US Department of Agriculture, the National Institutes of Health, and the Office of Naval Research. Private groups have also provided funding including Pioneer Hi-Bred International Inc., Nucycle Inc, and Integrated BioPharma.
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Last updated:
01/04/2006
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