Progress 11/01/01 to 10/31/05
Outputs
Rubisco catalyzes the rate determining step of photosynthesis, but it has a carboxylation kcat of only a few per second and is competitively inhibited by O2. Oxygenation of RuBP generates phosphoglycolate, which is the first intermediate in the nonessential photorespiratory pathway that leads to the loss of CO2. These properties make Rubisco an obvious target for genetic engineering aimed at improving agricultural productivity. Our research has relied on various genetic approaches for elucidating the structural basis for Rubisco catalytic efficiency. The green alga Chlamydomonas reinhardtii is the model organism of choice because photosynthesis-deficient mutants can be maintained with acetate as an alternative carbon source. The chloroplast large-subunit gene and family of nuclear small-subunit genes can also be eliminated or replaced by directed mutagenesis/transformation or by appropriate genetic crosses. Various large-subunit mutants have been recovered via mutant
screening and genetic selection or via directed mutagenesis and chloroplast transformation. Analysis of many of these mutant enzymes has shown that changes relatively far from the active site can influence CO2/O2 specificity. In collaboration with Dr. Inger Andersson (Swedish Agricultural University, Uppsala), X-ray crystal structures have now been solved for many of the mutant, revertant, and suppressor Rubisco enzymes. These structures may reveal the mechanisms by which distant alterations affect active-site catalysis. Rubisco catalysis also depends on a conserved set of large-subunit active-site residues, but catalytic efficiency and CO2/O2 specificity vary among Rubisco enzymes from diverse species. Because more than 2,000 large-subunit gene sequences are now available, phylogenetic/bioinformatic approaches are being employed to define the structural basis for this catalytic diversity. When 500 large-subunit sequences from flowering plants were compared with the Chlamydomonas
sequence, only 34 residues were found to be unique to Chlamydomonas. However, Chlamydomonas Rubisco has a higher kcat for carboxylation but lower CO2/O2 specificity than the land-plant enzymes. When directed mutagenesis and chloroplast transformation were used to change three Chlamydomonas residues to those characteristic of land plants at the bottom of the active site, substantial decreases in carboxylation catalytic efficiency and CO2/O2 specificity were observed. Two additional phylogenetic substitutions have recently been found to increase the CO2/O2 specificity of the triple-mutant enzyme back to the wild-type level. These five residues surround a small-subunit loop. When the Chlamydomonas loop was replaced with the spinach loop and then combined with the five large-subunit phylogenetic substitutions, the resultant enzyme had kinetic properties similar to those of the spinach enzyme. Thus, this region at the interface between large and small subunits is, at least in part,
responsible for catalytic diversity, and warrants further engineering aimed at improving Rubisco.
Impacts
As global population increases, so does world hunger. Because Rubisco catalyzes the rate-limiting step of photosynthesis, our attempts to understand the structural basis for catalysis can identify potential targets for beneficial engineering. Future genetic engineering of Rubisco may result in substantial increases in crop-plant productivity.
Publications
- Karkehabadi, S., Taylor, T. C., Spreitzer, R. J., and Andersson, I. (2005) Altered intersubunit interactions in crystal structures of catalytically-compromised ribulose-1,5-bisphosphate carboxylase/oxygenase. Biochemistry 44, 113-120.
- Spreitzer, R. J., Peddi, S. R., and Satagopan, S. (2005) Phylogenetic engineering at an interface between large and small subunits imparts land-plant kinetic properties to algal ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc. Natl. Acad. Sci. U. S. A. 102, 17225-17230.
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Progress 11/01/03 to 10/31/04
Outputs
Rubisco catalyzes the rate determining step of photosynthesis, but it has a carboxylation kcat of only a few per second and is competitively inhibited by O2. Oxygenation of RuBP generates phosphoglycolate, which is the first intermediate in the nonessential photorespiratory pathway that leads to the loss of CO2. These properties make Rubisco an obvious target for genetic engineering aimed at improving agricultural productivity. The green alga Chlamydomonas reinhardtii is the model organism of choice because photosynthesis-deficient mutants can be maintained with acetate as an alternative carbon source. The chloroplast large-subunit gene and family of nuclear small-subunit genes can also be eliminated or replaced by directed mutagenesis/transformation or by appropriate genetic crosses. Rubisco catalysis depends on a conserved set of large-subunit active-site residues, but catalytic efficiency and CO2/O2 specificity vary among Rubisco enzymes from diverse species.
Because more than 2,000 rbcL sequences are now available, phylogenetic/bioinformatic approaches are being employed to define the structural basis for this catalytic diversity. When 500 large-subunit sequences from flowering plants were compared with the Chlamydomonas sequence, only 34 residues were found to be unique to Chlamydomonas. However, Chlamydomonas Rubisco has a higher kcat for carboxylation but lower CO2/O2 specificity than the land-plant enzymes. When directed mutagenesis and chloroplast transformation were used to change three Chlamydomonas residues to those characteristic of land plants at the bottom of the a/b-barrel active site, substantial decreases in carboxylation catalytic efficiency and CO2/O2 specificity were observed. Because land-plant Rubisco enzymes have greater CO2/O2 specificity than the Chlamydomonas enzyme, one must assume that this group of residues is complemented by other residues in land plants, and these residues must be different from those of
Chlamydomonas. In fact, two additional phylogenetic substitutions have recently been found to increase the CO2/O2 specificity of the triple-mutant enzyme back to the wild-type level. These five residues surround the small-subunit bA-bB loop. When the Chlamydomonas loop was replaced with the spinach loop and then combined with the five large-subunit phylogenetic substitutions, the resultant enzyme had kinetic properties similar to those of the spinach enzyme. Thus, this region at the interface between large and small subunits is, at least in part, responsible for catalytic diversity, and warrants further engineering aimed at improving Rubisco.
Impacts
As global population increases, so does world hunger. Because Rubisco catalyzes the rate-limiting step of photosynthesis, our attempts to genetically engineer this enzyme may result in substantial increase in crop-plant productivity.
Publications
- Du, Y.C., Peddi, S.R. and Spreitzer, R.J. 2003. Assessment of structural and functional divergence far from the large subunit active site of ribulose-1,5-bisphosphate carboxylase/ oxygenase. J. Biol. Chem. 278:49401-49405.
- Satagopan, S. and Spreitzer, R.J. 2004. Substitutions at the Asp-473 latch residue of Chlamydomonas ribulosebisphosphate carboxylase/oxygenase cause decreases in carboxylation efficiency and CO2/O2 specificity. J. Biol. Chem. 279:14240-14244.
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Progress 11/01/02 to 10/31/03
Outputs
To gain a deeper understanding of the structure-function relationships of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), as a requisite step for its eventual genetic engineering aimed at improving plant productivity, genetic methods have been employed in the green alga Chlamydomonas reinhardtii. With the recent elimination of the small-subunit gene family, Chlamydomonas has now become the only organism in which both nuclear-encoded small subunits and chloroplast-encoded large subunits of the Rubisco holoenzyme can be manipulated by both classical and molecular genetic approaches. Rubisco enzymes from divergent species differ with respect to catalytic efficiency and CO2/O2 specificity despite conservation of three-dimensional structure and active-site residues. A deeper understanding of the structural basis for these differences may provide a rationale for engineering an improved enzyme, thereby leading to an increase in photosynthetic CO2 fixation and
agricultural productivity. By comparing 500 active-site large-subunit sequences from flowering plants with that of Chlamydomonas, a small number of residues were found to differ in regions previously shown by mutant screening to influence CO2/O2 specificity. When directed mutagenesis and chloroplast transformation were used to change Chlamydomonas Met-42 and Cys-53 to land-plant Val-42 and Ala-53 in the large-subunit N-terminal domain, little or no change in Rubisco catalytic properties was observed. However, changing Chlamydomonas methyl-Cys-256, Lys-258, and Ile-265 to land-plant Phe-256, Arg-258, and Val-265 at the bottom of the alpha/beta-barrel active site caused a 10% decrease in CO2/O2 specificity largely due to an 85% decrease in carboxylation catalytic efficiency (Vmax/Km). Because land-plant Rubisco enzymes have greater CO2/O2 specificity than the Chlamydomonas enzyme, this group of residues must be complemented by other residues that differ between Chlamydomonas and land
plants. The Rubisco X-ray crystal structures indicate that these residues may reside in a variable loop of the nuclear-encoded small subunit, more than 20 angstrom away from the active site.
Impacts
As global population increases, so does world hunger. Because Rubisco catalyzes the rate-limiting step of photosynthesis, our attempts to genetically engineer this enzyme may result in substantial increase in crop-plant productivity.
Publications
- Esquivel, M.G., Anwaruzzaman, M. and Spreitzer, R.J. 2002. Deletion of nine carboxy-terminal residues of the Rubisco small subunit decreases thermal stability but does not eliminate function. FEBS Lett. 520:73-76.
- Spreitzer, R.J. 2003. Role of the Rubisco small subunit. Arch. Biochem. Biophys. 414:141-149.
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Progress 10/01/01 to 09/30/02
Outputs
Two large-subunit regions farthest from the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been investigated by directed mutagenesis and chloroplast transformation. One of these regions was previously defined by pseudoreversion of a photosynthesis-deficient G54D null mutant. A G54V substitution in the large-subunit N-terminal domain causes a decrease in CO2/O2 specificity. The other was defined by intragenic suppression in which either an A222T or V262L substitution complements the low CO2/O2 specificity and thermal instability of an L290F mutant enzyme. At the restrictive temperature of 35C, the L290F mutant strain completely lacks photosynthesis due to the loss of Rubisco holoenzyme. Residues 222, 262, and 290 reside at the bottom of the a/b-barrel, more than 20 angstroms away from the active-site residues. Changing two Chlamydomonas residues to those characteristic of land plants in the large-subunit N-terminal domain had little
effect on catalysis whereas three at the bottom of the a/b-barrel domain caused a dramatic decrease in CO2/O2 specificity. These latter residues must be complemented by other residues in land-plant Rubisco that differ from those characteristic of Chlamydomonas Rubisco. Examination of the Chlamydomonas Rubisco X-ray crystal structure indicates that these other residues may reside in the Rubisco small subunit.
Impacts
To gain a deeper understanding of the structure-function relationships of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), as a requisite step for its eventual genetic engineering aimed at improving plant productivity, genetic methods have been employed in the green alga Chlamydomonas reinhardtii. As global population increases, so does world hunger. Because Rubisco catalyzes the rate-limiting step of photosynthesis, our attempts to genetically engineer this enzyme may result in substantial increase in crop-plant productivity.
Publications
- Taylor, T. C., Backlund, A., Bjorhall, K., Spreitzer, R. J. and Andersson, I. 2001. First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii. J. Biol. Chem. 276:48159-48164.
- Spreitzer, R. J. and Salvucci, M. E. 2002. Rubisco: Structure, regulatory interactions, and possibilities for a better enzyme. Annu. Rev. Plant Biol. 53:449-475.
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