The goal of this project is to improve sulfur amino acid-enriched soybean using genetic engineering, while maintaining agronomicly desirable characteristics. The research results should aid the understanding of sulfur assimilation in plants and provide a means to increase the accumulation of sulfur-containing amino acids by manipulating rate-limiting enzymes in the biochemical pathway.
In this project, the researchers propose to employ biochemical, structural and molecular approaches to manipulate the expression of ATP sulfurylase in transgenic soybean plants with the goal of increasing seed cysteine and methionine levels. The specific objectives are: (1) to generate transgenic soybean plants overexpressing ATP sulfurylase; and (2) to elucidate the biochemical regulation of soybean ATP sulfurylase.
Methods
To increase the biosynthesis of cysteine and methionine, the researchers will overexpress ATP sulfurylase, the entry point enzyme of sulfur assimilation, in soybean to provide increased metabolic flux through the pathway. In addition, they will determine the three-dimensional structure of this enzyme to understand its metabolic regulation; this information is essential for engineering a deregulated version of ATP sulfurylase for expression in soybean seeds.
In the second stage of the project, the researchers will cross the ATP sulfurylase overexpressing soybean with soybean plants expressing the methionine-rich d-zein protein. Since antibodies specific for the transgenic d-zein protein and the His-tagged ATP sulfurylase are available, rapid selection of progeny carrying both traits will be possible. Analysis of succeeding generations will continue until they obtain plants homozygous for both traits. Biochemical analysis of homozygous plants accumulating both methionine-rich d-zein protein and the His-tagged ATP sulfurylase will then be performed.
Generating transgenic soybean plants overexpressing ATP sulfurylase.
The first research goal will be to generate a transgenic soybean plant overexpressing soybean ATP sulfurylase. To prepare constructs for soybean transformation, the coding region of soybean ATP sulfurylase (ATPS) preceded by the chloroplast transit peptide (CTP) sequence will be amplified from the cDNA clone. To distinguish the introduced ATP sulfurylase in transgenic soybean from the native enzyme the researchers will introduce a hexahistidine tag at the C-terminus of the enzyme. Using His-tag antibodies, they will monitor accumulation of the introduced ATP sulfurylase protein.
Elucidating the biochemical regulation of soybean ATP sulfurylase
The ATP sulfurylase from soybean and other plants differs from the enzyme from other species in its domain structure and oligomeric state. The plant enzyme lacks the regulatory APS kinase domain found in the mammalian, fungal, and yeast homologs, suggesting the regulation of the plant ATP sulfurylase occurs in a different manner. The goal of this component of research is to determine the three-dimensional structure of the soybean enzyme using x-ray crystallography. Moreover, the investigators will also examine the effect of small molecules known to regulate the non-plant enzymes on the soybean enzyme's activity. These studies will provide a structural understanding of how the plant enzyme functions and is regulated as the entry point into sulfur assimilation and its regulation. In addition, detailed knowledge of the enzyme's architecture will be the basis for future protein engineering experiments to deregulate its activity.
The initial efforts to solve the structure of the enzyme will focus on molecular replacement approaches using a homology model generated from already determined crystal structures as a search model (Ullrich et al., 2001; Benyon et al., 2001; MacRae et al., 2001). If necessary, the structure will be determined using multiple isomorphous replacement with anomalous scattering (MIRAS) or MAD phasing. Researchers will screen for heavy atom derivatives in house and with synchrotron access can perform MAD experiments using the heavy atom derivatives. Heavy atom analysis and refinement will be accomplished with SHARP (de la Fourtelle & Bricogne, 1997). Heavy atom positions derived from MIRAS or MAD data will be located by inspection of difference Patterson maps (Bruker software suite) or in an automated fashion through SOLVE (Terwilliger & Berendzen, 1999). Additional derivative data sets will be analyzed by difference Fourier analysis. They will improve and extend initial phase sets with the program DM (CCP4, 1994) and with solvent flipping implemented in SOLOMON (Abrahams & Leslie, 1996). Iterative rounds of model building with O (Jones et al., 1994) and refinement in CNS (Brunger et al., 1998) will be carried out. The three-dimensional structures of soybean ATP sulfurylase complexed with ligands will be solved by difference Fourier analysis or molecular replacement.
The enzymatic activity of soybean ATP sulfurylase will be tested in the presence of small molecule effectors (3'-phosphoadenosine-5'-phosphosulfate (PAPS), thiosulfate, and chlorate) using a spectrophotometric assay (Renosto et al., 1993). [Earlier studies influenced the choice of compounds.] PAPS binds to the APS kinase domain to inhibit ATP sulfurylase activity in mammals, yeast, and fungi (Ullrich et al., 2001; Benyon et al., 2001; MacRae et al., 2001). Although the plant ATP sulfurylase lacks the regulatory domain, PAPS may bind at a different site on the enzyme. Chlorate binds to the yeast enzyme at an effector site adjacent to the active site, stabilizing a conformation of the enzyme that prevents product release (Ullrich & Huber, 2001). If this also occurs in the plant enzyme, site-directed mutagenesis of the site may eliminate inhibition by this compound.
Commercialization
After the transformations and initial crosses are completed, approximately five generations will be necessary to produce plants homogeneous for both traits. Utilizing the Sears greenhouse facility at the University of Missouri-Columbia, this process will require approximately three years. Seed bearing the desired characteristics will be conveyed to soybean breeders for incorporation into current lines under the established regulations and protocols. IMBA can expect economic benefits from this research soon after release of a soybean cultivar, which is expected to take about five years from the beginning of the research project.
For a full research proposal, contact one of the Principal Investigators.
