Preparative-scale enzymic synthesis of <scp>d</scp>-[14C]ribulose 1,5-bisphosphate

Kuehn, Glenn D.; Hsu, T C

doi:10.1042/bj1750909

Cited by 45 publications

(20 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The substrate specificity (⍀) values were measured with purified enzymes (25-250 g) in 400-l assays performed in 1.5-ml vials under saturating (1.23 mM) O 2 concentrations (23). [1-3 H]RuBP used in these assays was synthesized and purified as described elsewhere (30). Rates of Rubisco activation were measured with unactivated Rubisco enzymes using a modified procedure adapted from elsewhere (31).…”

Section: Methodsmentioning

confidence: 99%

Structure-Function Studies with the Unique Hexameric Form II Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) from Rhodopseudomonas palustris

Satagopan

Chan²,

Perry³

et al. 2014

Journal of Biological Chemistry

103

View full text Add to dashboard Cite

Background: Rubisco fixes atmospheric CO 2 to organic carbon and sustains life on earth. Results: A form II Rubisco structure has been solved, and functional analysis was conducted with divergent residues. Conclusion: The unique structure combined with functional analysis can help better understand and improve Rubisco catalysis. Significance: This is the first high resolution structure of an activated transition-state analog (CABP)-bound form II Rubisco.

show abstract

Section: Methodsmentioning

confidence: 99%

Structure-Function Studies with the Unique Hexameric Form II Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) from Rhodopseudomonas palustris

Satagopan

Chan²,

Perry³

et al. 2014

Journal of Biological Chemistry

103

View full text Add to dashboard Cite

show abstract

“…RuBP carboxylase and oxygenase activities were measured by the incorporation of acid-stable 14 C from NaH 14 CO 3 (17). (33,35).…”

Section: Methodsmentioning

confidence: 99%

Phylogenetic engineering at an interface between large and small subunits imparts land-plant kinetic properties to algal Rubisco

Spreitzer

Peddi

Satagopan

2005

Proc. Natl. Acad. Sci. U.S.A.

115

107

View full text Add to dashboard Cite

Ribulose-1,5-bisphosphate carboxylase͞oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO 2 fixation and, thus, limits agricultural productivity. However, Rubisco enzymes from different species have different catalytic constants. If the structural basis for such differences were known, a rationale could be developed for genetically engineering an improved enzyme. Residues at the bottom of the large-subunit ␣͞␤-barrel active site of Rubisco from the green alga Chlamydomonas reinhardtii (methyl-Cys-256, Lys-258, and Ile-265) were previously changed through directed mutagenesis and chloroplast transformation to residues characteristic of land-plant Rubisco (Phe-256, Arg-258, and Val-265). The resultant enzyme has decreases in carboxylation efficiency and CO 2͞O2 specificity, despite the fact that land-plant Rubisco has greater specificity than the Chlamydomonas enzyme. Because the residues are close to a variable loop between ␤-strands A and B of the small subunit that can also affect catalysis, additional substitutions were created at this interface. When largesubunit Val-221 and Val-235 were changed to land-plant Cys-221 and Ile-235, they complemented the original substitutions and returned CO 2͞O2 specificity to the normal level. Further substitution with the shorter ␤A-␤B loop of the spinach small subunit caused a 12-17% increase in specificity. The enhanced CO 2͞O2 specificity of the mutant enzyme is lower than that of the spinach enzyme, but the carboxylation and oxygenation kinetic constants are nearly indistinguishable from those of spinach and substantially different from those of Chlamydomonas Rubisco. Thus, this interface between large and small subunits, far from the active site, contributes significantly to the differences in catalytic properties between algal and land-plant Rubisco enzymes.catalysis ͉ Chlamydomonas ͉ chloroplast ͉ photosynthesis ͉ ribulosebisphosphate carboxylase͞oxygenase C O 2 and O 2 compete at the active site of ribulose-1,5-bisphosphate (RuBP) carboxylase͞oxygenase [Ribulose-1,5-bisphosphate carboxylase͞oxygenase (Rubisco), Enzyme Commission 4.1.1.39] for either the carboxylation or oxygenation of RuBP (reviewed in refs. 1-3). Whereas carboxylation is responsible for the accumulation of carbon in the biosphere, oxygenation is a nonessential reaction that leads to the loss of fixed carbon via the photorespiratory pathway. The ratio of the catalytic efficiencies (V max ͞K m ) of carboxylation (V c ͞K c ) and oxygenation (V o ͞K o ) defines the CO 2 ͞O 2 -specificity kinetic constant ⍀ (4), which is determined by the differential stabilization of the carboxylation and oxygenation transition states for the rate-limiting partial reactions (5). However, net CO 2 fixation is determined by the difference between the velocities of carboxylation and oxygenation at the CO 2 and O 2 concentrations that occur in vivo (4, 6). Because of its pivotal role in catalyzing the rate-limiting step of photosynthesis, genetic engineering of Rubisco aimed at increasing net CO 2 fixa...

show abstract

“…⍀ of purified and activated Rubisco (20 g/reaction) was determined by assaying carboxylase and oxygenase activities simultaneously with [1-3 H]RuBP (7.2 Ci/mol) and NaH 14 CO 3 (0.5 Ci/mol) in 30-min reactions at 25°C (35,36). [1-3 H]RuBP and phosphoglycolate phosphatase were synthesized/purified by standard methods (35,37). To measure Rubisco thermal stability (6,20), purified enzyme (10 g/ml) was incubated in 10 mM NaHCO 3 , 10 mM MgCl 2 , 1 mM dithiothreitol, and 50 mM Bicine, pH 8.0, at various temperatures for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Suppressor Mutations in the Chloroplast-encoded Large Subunit Improve the Thermal Stability of Wild-type Ribulose-1,5-bisphosphate Carboxylase/Oxygenase

Spreitzer

2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

A temperature-conditional, photosynthesis-deficient mutant of the green alga Chlamydomonas reinhardtii, previously recovered by genetic screening, results from a leucine 290 to phenylalanine (L290F) substitution in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39). Rubisco purified from mutant cells grown at 25°C has a reduction in CO 2 /O 2 specificity and is inactivated at lower temperatures than those that inactivate the wild-type enzyme. Second-site alanine 222 to threonine (A222T) or valine 262 to leucine (V262L) substitutions were previously isolated via genetic selection for photosynthetic ability at the 35°C restrictive temperature. These intragenic suppressors improve the CO 2 /O 2 specificity and thermal stability of L290F Rubisco in vivo and in vitro. In the present study, directed mutagenesis and chloroplast transformation were used to create the A222T and V262L substitutions in an otherwise wild-type enzyme. Although neither substitution improves the CO 2 /O 2 specificity above the wild-type value, both improve the thermal stability of wild-type Rubisco in vitro. Based on the x-ray crystal structure of spinach Rubisco, large subunit residues 222, 262, and 290 are far from the active site. They surround a loop of residues in the nuclear-encoded small subunit. Interactions at this subunit interface may substantially contribute to the thermal stability of the Rubisco holoenzyme.Genetic screening for conditional lethal mutants identifies only those relatively few amino acid substitutions that are critical for protein structure or function (reviewed in Refs. 1 and 2). One such mutant of the green alga Chlamydomonas reinhardtii results from an L290F substitution in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, 1 EC 4.1.1.39) (3, 4). This mutant contains enough Rubisco holoenzyme at 25°C to grow photoautotrophically but lacks holoenzyme and requires acetate for growth at the 35°C restrictive temperature (3). Because the mutant enzyme subunits are synthesized and assembled into holoenzyme at apparently normal rates (4), the reduced level of Rubisco must result from increased degradation of an unstable holoenzyme in vivo (4 -6). Rubisco purified from mutant cells grown at 25°C has decreases in catalytic efficiency, CO 2 /O 2 specificity, and holoenzyme thermal stability (4 -6) despite the fact that residue 290 resides at the bottom of the ␣/␤-barrel active-site domain, relatively far from the active-site residues (7). In an attempt to understand the nature of this distant effect, photosynthesis-competent revertants were selected from the L290F mutant strain at 35°C (5, 8). Either an A222T or V262L large subunit substitution was found to complement the original mutant substitution (8). Comparison of enzymes purified from cells grown at 25°C revealed that the A222T and V262L substitutions increased the CO 2 /O 2 specificity of the L290F enzyme back to the wild-type level (8). Residues 222 and 262 ...

show abstract

Preparative-scale enzymic synthesis of d-[14C]ribulose 1,5-bisphosphate

Cited by 45 publications

References 11 publications

Structure-Function Studies with the Unique Hexameric Form II Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) from Rhodopseudomonas palustris

Structure-Function Studies with the Unique Hexameric Form II Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) from Rhodopseudomonas palustris

Phylogenetic engineering at an interface between large and small subunits imparts land-plant kinetic properties to algal Rubisco

Suppressor Mutations in the Chloroplast-encoded Large Subunit Improve the Thermal Stability of Wild-type Ribulose-1,5-bisphosphate Carboxylase/Oxygenase

Contact Info

Product

Resources

About