Progress 09/01/05 to 08/31/10
Outputs
OUTPUTS: Over the course of this 5-year PECASE grant, we completed our aims and began new research that build on successful completion of this project. Aim 1. Structure/function analysis of OASS. This aim was completed during the year one of the proposal. Subsequently, we expanded our work to examine how OASS-related proteins in the beta-substituted alanine synthase (BSAS) family evolved distinct specificities. During the last year, we determined the three-dimensional structure of soybean cyanoalanine synthase (CAS), which detoxifies cyanide produced by ethylene synthesis. This work complements mutagenesis studies that define the determinants of chemical specificity between CAS and OASS. This work also suggests how the active site of BSAS enzymes are modified to yield chemodiversity. Building from our understanding of how OASS functions, in collaboration with Dr. Krishnan (USDA and U. Missouri), we successfully overexpressed OASS in soybean to increase cysteine content in seed to meet recommended levels required for the optimal growth of monogastric animals. Aim 2. Role of the OASS beta8A-beta9A loop. Experiments using OASS mutants targeted to this loop demonstrate the importance of this loop protein-protein interaction. Given the success of Aim 3, we placed priority on analyzing the structural and energetic basis of complex formation. Aim 3. Formation of the Cysteine Regulatory Complex (CRC). Association of OASS and SAT to form the CRC plays a role in regulating sulfur assimilation and cysteine biosynthesis. The x-ray crystal structure of OASS bound with a peptide corresponding to the C-terminal ten residues of SAT was determined. Formation of the CRC was examined using biophysical methods. The stability of the CRC derives from rapid association and extremely slow dissociation of OASS with SAT, and requires the C-terminus of SAT for the interaction. Kinetic analysis shows that CRC formation enhances SAT activity and releases SAT from substrate inhibition and feedback inhibition by cysteine, the final product of the biosynthesis pathway. Cysteine inhibits SAT and the CSC with Ki values of 2 and 70 uM, respectively. These results suggest a new model for the architecture of the CRC and additional control mechanisms for biochemically controlling plant cysteine biosynthesis. Based on previous work and our results, we suggest that OASS acts as an enzyme chaperone of SAT in the CRC. Recently, we determined the three-dimensional structure of soybean SAT. These studies provide insight on CRC formation and how these interactions stabilize SAT activity to prevent its cold-denaturation and inactivation. Because cysteine synthesis increases in response to cold stress in plants, formation of the CRC to maintain SAT activity and synthesis of cysteine may play an key physiological function. New work. USDA funding has also been used to explore other aspects of thiol metabolism and its regulation in plants. We have expanded our studies to examine key regulatory enzymes in methionine synthesis and in understanding how signal transduction systems control sulfur metabolism through the action of 14-3-3 proteins. PARTICIPANTS: Eric R. Bonner, Ph.D. Postdoctoral Research Associate (current position: Scientist, Akermin, Inc., St. Louis, MO) Julie A. Francois, Ph.D., Postdoctoral Research Associate (current position: Scientist, Sigma-Aldrich, St. Louis, MO) Sangaralingam Kumaran, Ph.D.,Postdoctoral Research Associate (current position: Assitant Professor, National Institute of Microbial Technology, Chandigarh, India) Hankuil Yi, Ph.D., Postdoctoral Research Associate Rebecca E. Cahoon, Research Associate III (current position: Lab Manager, University of Nebraska, Lincoln, NE) Sarah M. Knapke, Undergraduate Lab Assistant (current position: Scientist, Monsanto, St. Louis, MO) Amy C. Schroeder, Undergraduate Lab Assistant (current position: NSF Graduate Fellow, BMB Program, UC-Davis) Kiani Arkus, Undergraduate Lab Assistant (current position: NIH Graduate Fellow, MCM Program, Duke) Leia Wachsstock, Undergraduate Lab Assistant Jeremy Bleeke, Undergraduate Lab Assistant Matthew Juergens, Undergraduate Lab Assistant TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: This proposal was originally written as a 3-year NRI grant but was converted into a 5-year PECASE award. Our efforts on the original aims were successful and lead to new research directions and opportunities, as described in the summary.
Impacts
Understanding cysteine biosynthesis will open future research directions for improving nutritional sulfur sources by altering amino acid content. Corn, soybean, and rice contain low levels of one or more essential amino acids, including cysteine and methionine. Since animals obtain these nutrients from their diets, feeds are supplemented with amino acids. For example, soybean provides a protein source in human diet and is a high-protein feed component in livestock and poultry production, but the poultry and swine industries spend $100 million annually to augment feeds with sulfur-containing amino acids to promote growth and development of animals consuming grain-soybean feed. Therefore, developing soybean cultivars with high sulfur content may have a significant and positive impact on the livestock and poultry industry. Plant breeding and genetic technology have increased total protein content in soybean, but have not been used to enhance methionine or cysteine levels. Understanding the sulfur assimilatory pathway in soybean is a prerequisite for improving sulfur-containing amino acid content. An adequate supply of cysteine and methionine in developing seeds may facilitate accumulation of sulfur-rich proteins to a level sufficient to meet the nutritional requirements of livestock and poultry. Using information of the cysteine biosynthetic enzymes to engineer their activity for increasing levels of cysteine (and potentially methionine) offers the potential for enhancing amino acid content in crops and improving their nutritional value.
Publications
- 2. Phartiyal, P., Kim, W.S., Cahoon, R.E., Jez, J.M., and Krishnan, H.B. 2006. Soybean ATP sulfurylase, a homodimeric enzyme involved in sulfur assimilation, is abundantly expressed in roots and induced by cold treatment. Archives of Biochemistry and Biophysics, 450: 20-9.
- 3. Francois, J.A., Kumaran, S., and Jez, J.M. 2006. Structural basis for interaction of O-acetylserine sulfhydrylase and serine acetyltransferase in the Arabidopsis cysteine synthase complex. The Plant Cell, 18: 3647-55.
- 4. Kumaran, S. and Jez, J.M. 2007. Thermodynamics of the interaction between O-acetylserine sulfhydrylase and the C-terminus of serine acetyltransferase. Biochemistry, 46: 5586-94.
- 5. Phartiyal, P., Kim, W.S., Cahoon, R.E., Jez, J.M., and Krishnan, H.B. 2008. The role of 5-adenylylsulfate reductase in the sulfur assimilation pathway of soybean: molecular cloning, gene expression, and kinetic characterization. Phytochemistry, 69: 356-64.
- 6. Kumaran, S., Francois, J.A., Krishnan, H.B., and Jez, J.M. 2008. Regulatory protein-protein interactions in primary metabolism: the case of the cysteine synthase complex In Sulfur Assimilation and Abiotic Stress in Plants (NA Khan, RP Singh, Eds), pp. 97-109, Springer-Verlag, NY.
- 7. Jez, J.M. and Fukagawa, N.K. 2008. Plant sulfur compounds and human health In Sulfur: A Missing Link Between Soils, Crops, and Nutrition (Jez JM, Ed.) pp. 281-292, ASA-CSSA-SSSA Publishing, Madison, WI.
- 8. Jez, J.M. and Krishnan, H.B. 2009. Sulfur assimilation and cysteine biosynthesis in soybean seeds: towards engineering sulfur amino acid content In Modification of Seed Composition to Promote Health and Nutrition (Krishnan HB, Ed.), pp. 249-262, ASA-CSSA-SSSA Publishing, Madison, WI.
- 9. Kumaran, S., Yi, H., Krishnan, H.B., and Jez, J.M. 2009. Assembly of the cysteine synthase complex and the regulatory role of protein-protein interactions. Journal of Biological Chemistry, 284: 10268-75.
- 10. Schroeder, A.C., Zhu, C., Yanamadala, S.R., Cahoon, R.E., Arkus, K.A.J., Wachsstock, L., Bleeke, J., Krishnan, H.B., and Jez, J.M. 2010. Threonine-insensitive homoserine dehydrogenase from soybean: genomic organization, kinetic characterization, and in vivo activity. Journal of Biological Chemistry, 285: 827-34.
- 11. Yi, H., Galant, A., Ravilious, G.E., Preuss, M.L., and Jez, J.M. 2010. Sensing sulfur conditions: simple to complex biochemical regulatory mechanisms in plant thiol metabolism. Molecular Plant, 3: 269-79.
- 12. Yi, H., Ravilious, G., Galant, A., Krishnan, H.B., and Jez, J.M. 2010. Thiol metabolism in soybean: sulfur to homoglutathione. Amino Acids, 39: 963-78.
- 13. Shin, R., Jez, J.M., Basra, A., Zhang, B., and Schachtman, D.P. 2010. 14-3-3 Proteins fine-tune plant nutrient metabolism. FEBS Letters, in press.
- 14. Kim, W.S., Chronis, D., Juergens, M., Schroeder, A.C., Jez, J.M., and Krishnan, H.M. 2011. Transgenic soybean plants overexpressing O-acetylserine sulfhydrylase accumulate enhanced levels of cysteine and Bowman-Birk protease inhibitor in seeds. Plant Biotechnology Journal, submitted.
- 15. Yi, H., Juergens, M., and Jez, J.M. 2011. Molecular basis for cyanide detoxification and evolution of enzyme activity in the beta-substituted alanine synthase enzyme family. in preparation.
- 16. Yi, H. and Jez, J.M. 2011. Structure of soybean serine acetyltransferase and isoform interactions in formation of the cysteine regulatory complex. in preparation.
- 1. Bonner, E.R., Cahoon, R.E., Knapke, S.M., and Jez, J.M. 2005. Molecular basis of plant cysteine biosynthesis: structural and functional analysis of O-acetylserine sulfhydrylase from Arabidopsis thaliana. Journal of Biological Chemistry, 280: 38803-13.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: During the fourth year of this project, we continued to dissect the interaction between O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT) in the cysteine synthase complex and to further examine thiol metabolism into methionine synthesis. Progress made during the fourth year of the project is summarized below by aim. Aim 1. Structure/function analysis of OASS. We have expanded our analysis of OASS-like enzymes in plants to examine a related enzyme, β-cyanoalanine synthase (CAS), involved in the detoxification of cyanide produced by ethylene biosynthesis. CAS shares high sequence similarity with OASS. Using structural and sequence comparisons, we have identified a subset of amino acids that differ in the active site of each enzyme. We have generated point mutations in both CAS and OASS to examine the effect of these changes on enzyme activity. None of the single mutations interconvert enzymatic activity. Future work: we will incorporate the complete set of changes in each protein to test if substrate specifity and reaction chemistry can be interconverted between CAS and OASS. Aim 2. Role of the OASS β8A-β9A loop. Work on this aim is completed. Aim 3: Cysteine synthase complex (CSC) formation. We have obtained crystals of soybean SAT alone and in complex with substrates and products. These are now the focus of structural studies. In addition, we are exploring the role of OASS as an enzyme chaperone to stabilize SAT as part of the CSC. Interaction of these two proteins is important to prevent cold-denaturation of SAT and inactivation of its activity. In vitro experiments have demonstrated the critical role of complex formation in maintaining SAT activity. Importantly, because cysteine synthesis increases in response to cold stress in soybean and other plants, formation of the CSC to maintain SAT activity and synthesis of cysteine may play an key physiological function. Future work: Our goals for the next year are to complete crystallographic studies and to probe the role of CSC formation as a response to cold stress in soybean (and other plants). New work. Aspartate kinase (AK) and homoserine dehydrogenase (HSD) function as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback inhibited by threonine. To investigate the role of HSD, we cloned the cDNA and gene encoding the monofunctional HSD (GmHSD) from soybean. Initial velocity and product inhibition studies support an ordered bi bi kinetic mechanism in which nicotinamide cofactor binds first and leaves last in the reaction sequence. Threonine inhibition of GmHSD occurs at concentrations more than 1000-fold above physiological levels. This is in contrast to the two AK-HSD isoforms in soybean that are sensitive to threonine inhibition. GmHSD is not inhibited by other aspartate-derived amino acids. The ratio of threonine-resistant to threonine-sensitive HSD activity in soybean tissues varies and likely reflects different demands for amino acid biosynthesis. Threonine-resistant HSD offers a useful biotechnology tool for manipulating the aspartate amino acid pathway for improved nutritional content. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Understanding cysteine biosynthesis will open future research directions for improving nutritional sulfur sources by altering amino acid content. Corn, soybean, and rice contain low levels of one or more essential amino acids, including cysteine and methionine. Since animals obtain these nutrients from their diets, feeds are supplemented with amino acids. For example, soybean provides a protein source in human diet and is a high-protein feed component in livestock and poultry production, but the poultry and swine industries spend $100 million annually to augment feeds with sulfur-containing amino acids to promote growth and development of animals consuming grain-soybean feed. Therefore, developing soybean cultivars with high sulfur content may have a significant and positive impact on the livestock and poultry industry. Plant breeding and genetic technology have increased total protein content in soybean, but have not been used to enhance methionine or cysteine levels. Understanding the sulfur assimilatory pathway in soybean is a prerequisite for improving sulfur-containing amino acid content. An adequate supply of cysteine and methionine in developing seeds may facilitate accumulation of sulfur-rich proteins to a level sufficient to meet the nutritional requirements of livestock and poultry. Using information of the cysteine biosynthetic enzymes to engineer their activity for increasing levels of cysteine (and potentially methionine) offers the potential for enhancing amino acid content in crops and improving their nutritional value.
Publications
- Yi H, Galant A, Ravilious GE, Preuss ML, Jez JM (2009) Sensing sulfur conditions: simple to complex biochemical regulatory mechanisms in plant thiol metabolism. Molecular Plant (in press)
- Yi H, Ravilious GE, Galant A, Krishnan HB, Jez JM (2009) Thiol metabolism in soybean: sulfur to homoglutathione. Amino Acids (in press)
- Jez JM, Krishnan HB (2009) Sulfur assimilation and cysteine biosynthesis in soybean seeds: towards engineering sulfur amino acid content In Modification of Seed Composition to Promote Health and Nutrition (Krishnan HB, Ed.), pp. 249-262, ASA-CSSA-SSSA Publishing, Madison, WI
- Kumaran S, Yi H, Krishnan HB, Jez JM (2009) Assembly of the cysteine synthase complex and the regulatory role of protein-protein interactions. J. Biol. Chem. 284, 10268-75
- Schroeder AC, Zhu C, Yanamadala SR, Cahoon RE, Arkus KAJ, Wachsstock L, Bleeke J, Krishnan HB, Jez JM (2009) Threonine-insensitive homoserine dehydrogenase from soybean: genomic organization, kinetic characterization, and in vivo activity. J. Biol. Chem. (in press)
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: During the third year of this project, we continued to dissect the structural and energetic basis of the interaction between O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT) in the cysteine synthase complex. Progress made during the third year of the project is summarized below by aim. Aim 1. Structure/function analysis of OASS. Although work on this aim was completed during the first year of the project, we are now examining the evolution of OASS-like enzymes in plants. Future work: In plants, the detoxification of cyanide produced by ethylene biosynthesis is catalyzed by β-cyanoalanine synthase (CAS). CAS shares high sequence similarity with OASS and presumably uses a similar catalytic mechanism. We are now exploring how CAS and OASS perform there respective reactions. Aim 2. Role of the OASS β8A-β9A loop. Examination of plant and bacterial OASS sequences suggest that the highly conserved β8A-β9A surface loop may be important for interaction with SAT. Initial protein-protein interaction experiments using AtOASS mutants targeted to this loop support this hypothesis. In the year 2, we generated mutants of the surface loop; however, as the progress in the third aim is more important for understanding the foundation of complex formation, we concentrated our efforts on that work during year 3. Future work: We plan to use the generated mutants for interaction analysis with SAT. Aim 3: Cysteine synthase complex formation. Previously, we elucidated elements of the structural and energetic basis for interactions in the cysteine synthase complex using a protein-peptide model (Francois et al., 2006; Kumaran et al., 2007 & 2008). During Year 3, we have moved this analysis to examine the interaction of SAT and OASS using intact proteins. The cysteine synthase complex (CSC) functions as a multienzyme complex that responds to changes in sulfur levels resulting from metabolic stresses in plants and bacteria. We have examined the oligomerization and energetics of formation of the soybean CSC using analytical ultracentrifugation, isothermal titration calorimetry, and surface plasmon resonance. Biophysical examination of CSC oligomerization indicates that this macromolecular assembly from soybean consists of a single SAT trimer and three OASS dimers. Under physiological conditions, the stability of the CSC derives from tight binding resulting from rapid association and extremely slow dissociation of the assembly, as determined by isothermal titration calorimetry and surface plasmon resonance. Addition of multiple OASS dimers to the SAT trimer displays negative cooperatively and requires the C-terminus of SAT for interaction with OASS. Steady-state kinetic analysis shows that the functional consequences of CSC formation include increased SAT activity and the release of feedback inhibition by cysteine, the final product of the pathway. These results suggest a new model for the architecture of this regulatory complex and a possible physiological function of the assembly in modulating cysteine biosynthesis. Future work: We are now shifting to structural elucidation of the CSC by x-ray crystallography. PARTICIPANTS: To date, this project has trained two postdocs (S. Kumaran & H. Yi) and two undergraduate students (M. Juergens & A. Schroeder). TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Understanding cysteine biosynthesis will open future research directions for improving nutritional sulfur sources by altering amino acid content. Corn, soybean, and rice contain low levels of one or more essential amino acids, including cysteine and methionine. Since animals obtain these nutrients from their diets, feeds are supplemented with amino acids. For example, soybean provides a protein source in human diet and is a high-protein feed component in livestock and poultry production, but the poultry and swine industries spend $100 million annually to augment feeds with sulfur-containing amino acids to promote growth and development of animals consuming grain-soybean feed. Therefore, developing soybean cultivars with high sulfur content may have a significant and positive impact on the livestock and poultry industry. Plant breeding and genetic technology have increased total protein content in soybean, but have not been used to enhance methionine or cysteine levels. Understanding the sulfur assimilatory pathway in soybean is a prerequisite for improving sulfur-containing amino acid content. An adequate supply of cysteine and methionine in developing seeds may facilitate accumulation of sulfur-rich proteins to a level sufficient to meet the nutritional requirements of livestock and poultry. Using information of the cysteine biosynthetic enzymes to engineer their activity for increasing levels of cysteine (and potentially methionine) offers the potential for enhancing amino acid content in crops and improving their nutritional value.
Publications
- Jez JM, Fukagawa NK (2008) Plant sulfur compounds and human health In Sulfur: A Missing Link Between Soils, Crops, and Nutrition (Jez JM, Ed.) pp. 281-292, ASA-CSSA-SSSA Publishing, Madison, WI
- Jez JM, Krishnan HB (2008) Sulfur assimilation and cysteine biosynthesis in soybean seeds: towards engineering sulfur amino acid content In Modification of Seed Composition to Promote Health and Nutrition (Krishnan HB, Ed.) ASA-CSSA-SSSA Publishing, Madison, WI (in press)
- Phartiyal P, Kim WS, Cahoon RE, Jez JM, Krishnan HB (2008) The role of 5'-adenylylsulfate reductase in the sulfur assimilation pathway of soybean: molecular cloning, gene expression, and kinetic characterization. Phytochemistry 69, 356-364
- Kumaran S, Francois JA, Krishnan HB, Jez JM (2008) Regulatory protein-protein interactions in primary metabolism: the case of the cysteine synthase complex In Sulfur Assimilation and Abiotic Stress in Plants (NA Khan, S Singh, S Umar, Eds.) pp. 97-109, Springer-Verlag, NY
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Progress 09/01/06 to 08/31/07
Outputs
Progress Report During the second year of this project, we began to dissect the structural and energetic basis of the interaction between O-aceylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT) in the cysteine synthase complex. Progress made during the second year of the project is summarized below by aim. Aim 1. Structure/function analysis of OASS. This aim was completed during the first year of the project. Aim 2. Role of the OASS b8A-b9A loop. Examination of plant and bacterial OASS sequences suggest that the highly conserved b8A-b9A surface loop may be important for interaction with SAT. Initial protein-protein interaction experiments using AtOASS mutants targeted to this loop support this hypothesis. In the past year, we have generated mutants of the surface loop; however, as the progress in the third aim is more important for understanding the foundation of complex formation, we concentrated our efforts on that work. Future work: We plan to use the
generated mutants for interaction analysis with SAT. Aim 3: Cysteine synthase complex formation. Previously, we elucidated elements of the structural basis for interactions in the cysteine synthase complex by determining the crystal structure of Arabidopsis OASS bound with a peptide corresponding to the C-terminal ten residues of Arabidopsis SAT (C10) (Francois et al., 2006). Subsequent work in the past year examined the detailed thermodynamic basis of this interation (Kumaran et al., 2007 & 2008). Analysis of the interaction between OASS and the C10 peptide using fluorescence spectroscopy and isothermal titration calorimetry (ITC) suggest that the C-terminus of SAT provides the major contribution to total binding energy in the cysteine synthase complex. The C10 peptide binds to the AtOASS homodimer in a 2:1 complex. Interaction between AtOASS and the C10 peptide is tight (Kd = 5-100 nM) over a range of temperatures (10-35 C) and NaCl concentrations (0.02-1.3 M). AtOASS binding of the
C10 peptide displays negative cooperativity at higher temperature. ITC studies reveal compensating changes in enthalpy and entropy of binding that also depend on temperature. The enthalpy of interaction has a significant temperature dependence (deltaCp = -401 cal mol-1 K-1). The heat capacity change and salt-dependence studies suggest that hydrophobic interactions drive formation of the AtOASS-C10 peptide complex. Temperature may have a potential regulatory effect on complex formation that correlates to in vivo activity changes in cysteine synthesis. Future work: Although these studies defined the role of the SAT C-terminus in complex formation, understanding the interaction between each full-length protein is a priority. Efforts in year 3 will focus on examining the thermodynamics of the interaction between soybean SAT and OASS.
Impacts
Understanding cysteine biosynthesis will open future research directions for improving nutritional sulfur sources by altering amino acid content. Corn, soybean, and rice contain low levels of one or more essential amino acids, including cysteine and methionine. Since animals obtain these nutrients from their diets, feeds are supplemented with amino acids. For example, soybean provides a protein source in human diet and is a high-protein feed component in livestock and poultry production, but the poultry and swine industries spend $100 million annually to augment feeds with sulfur-containing amino acids to promote growth and development of animals consuming grain-soybean feed. Therefore, developing soybean cultivars with high sulfur content may have a significant and positive impact on the livestock and poultry industry. Plant breeding and genetic technology have increased total protein content in soybean, but have not been used to enhance methionine or cysteine levels.
Understanding the sulfur assimilatory pathway in soybean is a prerequisite for improving sulfur-containing amino acid content. An adequate supply of cysteine and methionine in developing seeds may facilitate accumulation of sulfur-rich proteins to a level sufficient to meet the nutritional requirements of livestock and poultry. Using information of the cysteine biosynthetic enzymes to engineer their activity for increasing levels of cysteine (and potentially methionine) offers the potential for enhancing amino acid content in crops and improving their nutritional value.
Publications
- Phartiyal P, Kim WS, Cahoon RE, Jez JM, Krishnan HB (2007) The role of 5'-adenylylsulfate reductase in the sulfur assimilation pathway of soybean: molecular cloning, gene expression, and kinetic characterization. Phytochemistry (in press)
- Kumaran S, Francois JA, Krishnan HB, Jez JM (2008) Regulatory protein-protein interactions in primary metabolism: the case of the cysteine synthase complex in Sulfur Assimilation and Abiotic Stress in Plants (NA Khan, RP Singh, Omar, eds). Springer-Verlag, NY (in press)
- Jez JM (2008) Plant sulfur compounds and human health In Sulfur: A Missing Link Between Soils, Crops, and Nutrition (Jez JM, Ed.) (in press)
- Francois JA, Kumaran S, Jez JM (2006) Structural basis for interaction of O- acetylserine sulfhydrylase and serine acetyltransferase in the Arabidopsis cysteine synthase complex. Plant Cell 18: 3647-3655
- Kumaran S, Jez JM (2007) Thermodynamics of the interaction between O-acetylserine sulfhydrylase and the C-terminus of serine acetyltransferase. Biochemistry 46: 5586-5594
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Progress 09/01/05 to 08/31/06
Outputs
Progress made during the preceding year is summarized by aim. Aim 1. Structure/function analysis of AtOASS. Overall, the planned work was completed (Bonner et al., 2005). The structures of AtOASS and the AtOASS K46A mutant with pyridoxal phosphate (PLP) and methionine linked as an external aldimine were determined. Although the plant and bacterial OASS share a conserved set of amino acids for PLP binding, the structure of AtOASS reveals differences from the bacterial enzyme in the positioning of an active site loop formed by residues 74-78 when methionine is bound. Site-directed mutagenesis, kinetic analysis, and ligand binding titrations probed the functional roles of active site residues. These experiments indicate that Asn77 and Gln147 are key amino acids for O-acetylserine binding and that Thr74 and Ser75 are involved in sulfur incorporation into cysteine. Aim 2. Role of the OASS beta8A-beta9A loop. Examination of plant and bacterial OASS sequences suggest that
the highly conserved beta8A-beta9A surface loop may be important for interaction with SAT. Initial protein-protein interaction experiments using AtOASS mutants targeted to this loop support this hypothesis. Future plans: During the next year, we will focus on generating GmOASS mutants and analyzing their effect on GmSAT binding by calorimetry. Aim 3: CS complex formation. To elucidate the structural basis of protein-protein interactions in the CS complex, we determined the 2.8 A resolution crystal structure of AtOASS bound with a peptide corresponding to the C-terminal ten residues of AtSAT (C10). Interactions with key active site residues (Thr74, Ser75, and Gln147) lock the C10 peptide in the binding site. Binding of the C10 peptide blocks access to the OASS active site; this explains how OASS is down-regulated upon formation of the CS complex. Comparison with the bacterial OASS suggests that structural plasticity in the active site allows for the binding of SAT C-termini with
dissimilar sequences at structurally similar active sites. Analysis of the effect of active site mutations by calorimetry demonstrates that these residues are important for binding of the C10 peptide and that changes at these positions disrupt communication between active sites in the homodimeric enzyme. In addition, we demonstrate that the C-terminal isoleucine of the C10 peptide is required for molecular recognition by AtOASS. These results provide new insights into the molecular mechanism underlying formation of the cysteine synthase complex and provide a structural basis for the biochemical regulation of cysteine biosynthesis in plants. This work has been submitted for publication (Francois et al., 2006). Future plans: Although these interaction studies offer insight into formation of the cysteine synthase complex, additional studies are needed. Efforts in the next year will focus on examining the thermodynamics of the protein-peptide interaction and on establishing calorimetry
studies using soybean SAT and OASS.
Impacts
Understanding cysteine biosynthesis will open future research directions for improving nutritional sulfur sources by altering amino acid content. Corn, soybean, and rice contain low levels of one or more essential amino acids, including cysteine and methionine. Since animals obtain these nutrients from their diets, feeds are supplemented with amino acids. For example, soybean provides a protein source in human diet and is a high-protein feed component in livestock and poultry production, but the poultry and swine industries spend $100 million annually to augment feeds with sulfur-containing amino acids to promote growth and development of animals consuming grain-soybean feed. Therefore, developing soybean cultivars with high sulfur content may have a significant and positive impact on the livestock and poultry industry. Plant breeding and genetic technology have increased total protein content in soybean, but have not been used to enhance methionine or cysteine
levels. Understanding the sulfur assimilatory pathway in soybean is a prerequisite for improving sulfur-containing amino acid content. An adequate supply of cysteine and methionine in developing seeds may facilitate accumulation of sulfur-rich proteins to a level sufficient to meet the nutritional requirements of livestock and poultry. Using information of the cysteine biosynthetic enzymes to engineer their activity for increasing levels of cysteine (and potentially methionine) offers the potential for enhancing amino acid content in crops and improving their nutritional value.
Publications
- Bonner ER, Cahoon RE, Knapke SM, Jez JM (2005) Molecular basis of plant cysteine biosynthesis: structural and functional analysis of O-acetylserine sulfhydrylase from Arabidopsis thaliana. J. Biol. Chem. 280, 38803-38813
- Phartiyal P, Kim WS, Cahoon RE, Jez JM, Krishnan HB (2006) Soybean ATP sulfurylase, a homodimeric enzyme involved in sulfur assimilation, is abundantly expressed in roots and induced by cold treatment. Arch. Biochem. Biophys. 450, 20-29
- Francois JA, Kumaran S, Jez JM (2007) Structural basis for interaction of O-acetylserine sulfhydrylase and serine acetyltransferase in the plant cysteine synthase complex. Plant Cell (submitted)
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