Small Oligomers of Ribulose-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Are Required for Biological Activity

Keown, J.R.; Griffin, Michael D. W.; Mertens, Haydyn D. T.; Pearce, F. Grant

doi:10.1074/jbc.m113.466383

Cited by 34 publications

(45 citation statements)

References 58 publications

(86 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In this work, Rca hydrolytic activity was determined at a subunit concentration of 5 M, and in published works, the activity was measured utilizing 2 M Rca or less. A spike in ATPase activity has been reported to occur at 1-2 M subunit concentration, followed by a downward trend at somewhat higher concentrations (30). In support of this notion, preliminary data in our laboratory indicate a substantial reduction in turnover rate with increasing protein concentration.…”

Section: Discussionsupporting

confidence: 79%

“…Previous reports have shown that the minimal oligomeric unit for ATP hydrolysis consists of about three subunits (30,32). It is not surprising that the estimated stoichiometry for ATP hydrolysis is smaller than that for Rubisco reactivation, because in AAA ϩ proteins, active sites functional in hydrolysis are formed at the molecular interface of two subunits.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, a subunit concentration of 5 M (0.2 mg/ml) or larger was shown to provide a weight-averaged sedimentation coefficient consistent with a hexamer (29). However, these experiments also demonstrated that tobacco ␤-Rca remained highly polydisperse over the entire concentration range tested (30). The stable formation of a hexameric species could only be demonstrated for the tobacco ␤-Rca-R294V mutant (31,32).…”

mentioning

confidence: 83%

See 2 more Smart Citations

Regulation of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase

Hazra

Henderson

Liles

et al. 2015

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: Rubisco activase (Rca) maintains carbon fixation, coordinates photosynthetic pathways, and regulates darklight transitions. Results: Positive cooperativity of ATP hydrolysis is afforded by ADP binding to oligomeric assemblies in the Rca-Mg-ATP-Mg state. Conclusion: Magnesium-mediated allosteric regulation involves the coexistence of ATP-and ADP-bound states. Significance: In dark-adapted chloroplasts, wasteful ATP hydrolysis may be prevented by low magnesium.

show abstract

Section: Discussionsupporting

confidence: 79%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 83%

See 1 more Smart Citation

Regulation of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase

Hazra

Henderson

Liles

et al. 2015

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Although species-specific differences in Rca assembly remain poorly understood, it appears likely that significant differences in K d values exist. For example, SAXS data reported for tobacco apo-β-Rca at 2.4 μM were consistent with hexameric assemblies, 32 whereas cotton β-Rca-AC appears to require concentrations of ∼30 μM to form substantial amounts of hexamers. Below 500 nM, our data indicate that the β-Rca-AC monomer is the primary species in the presence of both ATPγS and ADP.…”

Section: ■ Discussionmentioning

confidence: 80%

ATP and Magnesium Promote Cotton Short-Form Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Hexamer Formation at Low Micromolar Concentrations

et al. 2014

View full text Add to dashboard Cite

We report a fluorescence correlation spectroscopy (FCS) study of the assembly pathway of the AAA+ protein ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a ring-forming ATPase responsible for activation of inhibited Rubisco complexes for biological carbon fixation. A thermodynamic characterization of simultaneously populated oligomeric states appears critical in understanding Rca structure and function. Using cotton β-Rca, we demonstrate that apparent diffusion coefficients vary as a function of concentration, nucleotide, and cation. Using manual fitting procedures, we provide estimates for the equilibrium constants for the stepwise assembly and find that in the presence of ATPγS, the Kd for hexamerization is 10-fold lower than with ADP (∼0.1 vs ∼1 μM). Hexamer fractions peak at 30 μM and dominate at 8-70 μM Rca, where they comprise 60-80% of subunits with ATPγS, compared with just 30-40% with ADP. Dimer fractions peak at 1-4 μM Rca, where they comprise 15-18% with ATPγS and 26-28% with ADP. At 30 μM Rca, large aggregates begin to form that comprise ∼10% of total protein with ATPγS and ∼25% with ADP. FCS data collected on the catalytically impaired WalkerB-D173N variant in the presence of ATP provided strong support for these results. Titration with free magnesium ions lead to the disaggregation of larger complexes in favor of hexameric forms, suggesting that a second magnesium binding site with a Kd value of 1-3 mM mediates critical subunit contacts. We propose that closed-ring toroidal hexameric forms are stabilized by binding of Mg·ATP plus Mg2+, whereas Mg·ADP promotes continuous assembly to supramolecular aggregates such as spirals.

show abstract

“…All Rca isoforms presented with the concentration-dependent polydisperse oligomeric state typical for green-type Rca when analyzed by size exclusion chromatography (Supplemental Fig. S2; Blayney et al, 2011;Keown et al, 2013).…”

Section: Rubisco Activase From Agave Is Highly Functional At Activatimentioning

confidence: 99%

In Vitro Characterization of Thermostable CAM Rubisco Activase Reveals a Rubisco Interacting Surface Loop

Shivhare

Mueller‐Cajar

2017

Plant Physiol.

View full text Add to dashboard Cite

To maintain metabolic flux through the Calvin-Benson-Bassham cycle in higher plants, dead-end inhibited complexes of Rubisco must constantly be engaged and remodeled by the molecular chaperone Rubisco activase (Rca). In C3 plants, the thermolability of Rca is responsible for the deactivation of Rubisco and reduction of photosynthesis at moderately elevated temperatures. We reasoned that crassulacean acid metabolism (CAM) plants must possess thermostable Rca to support Calvin-Benson-Bassham cycle flux during the day when stomata are closed. A comparative biochemical characterization of rice (Oryza sativa) and Agave tequilana Rca isoforms demonstrated that the CAM Rca isoforms are approximately10°C more thermostable than the C3 isoforms. Agave Rca also possessed a much higher in vitro biochemical activity, even at low assay temperatures. Mixtures of rice and agave Rca form functional hetero-oligomers in vitro, but only the rice isoforms denature at nonpermissive temperatures. The high thermostability and activity of agave Rca mapped to the N-terminal 244 residues. A Glu-217-Gln amino acid substitution was found to confer high Rca activity to rice Rca. Further mutational analysis suggested that Glu-217 restricts the flexibility of the a4-b4 surface loop that interacts with Rubisco via Lys-216. CAM plants thus promise to be a source of highly functional, thermostable Rca candidates for thermal fortification of crop photosynthesis. Careful characterization of their properties will likely reveal further protein-protein interaction motifs to enrich our mechanistic model of Rca function.

show abstract

Small Oligomers of Ribulose-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Are Required for Biological Activity

Cited by 34 publications

References 58 publications

Regulation of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase

Regulation of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase

ATP and Magnesium Promote Cotton Short-Form Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Hexamer Formation at Low Micromolar Concentrations

In Vitro Characterization of Thermostable CAM Rubisco Activase Reveals a Rubisco Interacting Surface Loop

Contact Info

Product

Resources

About