Rubisco: Maladapted or Misunderstood

Mk, Morell; Paul, Kalanethee; Kane, HJ; Andrews, T. John

doi:10.1071/bt9920431

Cited by 42 publications

(30 citation statements)

References 24 publications

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“…Whether overexpressing the GroES-GroEL chaperonins can improve the yield of Rubisco-fusion complexes produced in E. coli, as it can for cyanobacterial L 8 S 8 Rubisco (23, 37), remains to be examined. Curiously the incorporation of successive histidine residues appeared to hamper proper folding and/or assembly of the c S60H 4 L and c S40LH 6 peptides. As well, the fusion peptides containing the 40-or 60-amino linker sequences appeared more adept to functional assembly than the fusion peptides containing the 20-amino acid linker (Table 1).…”

Section: Discussionmentioning

confidence: 99%

“…As the primary port of entry of inorganic carbon into the biosphere, it is surprising that plant Rubisco is not very efficient, particularly at limiting CO 2 concentrations where the rate of catalytic turnover is less than one-thousandth that of many other plant enzymes (4). The tendency of Rubiscos to confuse CO 2 with the more abundant atmospheric gas, O 2 , encumbers photosynthesis in higher plants with both a requirement to invest large amounts of protein in Rubisco and also a requirement for an energy-intensive photorespiratory metabolism to recycle the oxygenated waste product.…”

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confidence: 99%

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Linked Rubisco Subunits Can Assemble into Functional Oligomers without Impeding Catalytic Performance

Whitney

Sharwood

2007

Journal of Biological Chemistry

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Although transgenic manipulation in higher plants of the catalytic large subunit (L) of the photosynthetic CO 2 -fixing enzyme ribulose 1,5-bisphospahte carboxylase/oxygenase (Rubisco) is now possible, the manipulation of its cognate small subunit (S) is frustrated by the nuclear location of its multiple gene copies. To examine whether L and S can be engineered simultaneously by fusing them together, the subunits from Synechococcus PCC6301 Rubisco were tethered together by different linker sequences, producing variant fusion peptides. In Escherichia coli the variant PCC6301 LS fusions assembled into catalytically functional octameric ([LS] 8 ) and hexadecameric ([[LS] 8 ] 2 ) quaternary structures that excluded the integration of co-expressed unfused S. Assembly of the LS fusions into Rubisco complexes was impaired 50 -90% relative to the assembly of unlinked L and S into L 8 S 8 enzyme. Assembly in E. coli was not emulated using tobacco SL fusions that accumulated entirely as insoluble protein. Catalytic measurements showed the CO 2 /O 2 specificity, carboxylation rate, and Michaelis constants for CO 2 and ribulose 1,5-bisphosphate for the cyanobacterial Rubisco complexes comprising fusions where the S was linked to the N terminus of L closely matched those of the wild-type L 8 S 8 enzyme. In contrast, the substrate affinities and carboxylation rate of the Rubisco complexes comprising fusions where L was fused to the N terminus of S or a six-histidine tag was appended to the C terminus of L were compromised. Overall this work provides a framework for implementing an alternative strategy for exploring simultaneous engineering of modified, or foreign, Rubisco L and S subunits in higher plant plastids.The catalytic efficiency of the photosynthetic CO 2 -fixing enzyme D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) has a central role in determining how efficiently plants use their resources of water, fertilizer nutrient, and light (1-3). As the primary port of entry of inorganic carbon into the biosphere, it is surprising that plant Rubisco is not very efficient, particularly at limiting CO 2 concentrations where the rate of catalytic turnover is less than one-thousandth that of many other plant enzymes (4). The tendency of Rubiscos to confuse CO 2 with the more abundant atmospheric gas, O 2 , encumbers photosynthesis in higher plants with both a requirement to invest large amounts of protein in Rubisco and also a requirement for an energy-intensive photorespiratory metabolism to recycle the oxygenated waste product. However, higher plant Rubisco is not the pinnacle of evolution as more efficient and specific forms are found in nature, particularly in a number of red algae (5). The benefits of replacing the Rubisco in C 3 crops with these natural variants have been modeled and show substantial improvements that come at no additional energy or resource cost (1, 2, 6). Against this background, it is no surprise efforts are continuing into understanding Rubisco's catalytic mechanism, how it ha...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Linked Rubisco Subunits Can Assemble into Functional Oligomers without Impeding Catalytic Performance

Whitney

Sharwood

2007

Journal of Biological Chemistry

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“…However, the many examples of algal and bacterial Rubiscos that lack fallover inhibition (27,43,51,52) suggest additional significance. Like the Val-335 mutant, fallover-free Rubiscos generally have lower CO 2 /O 2 specificity and lower catalytic effectiveness (k cat /K m for carboxylation), than the higher plant enzyme (53). They also have differences in the sequence of loop 6 and its environs.…”

Section: Slow Inhibition During Catalysis Is Less Severe and Differenmentioning

confidence: 99%

The Relationship between Side Reactions and Slow Inhibition of Ribulose-bisphosphate Carboxylase Revealed by a Loop 6 Mutant of the Tobacco Enzyme

Pearce

Andrews

2003

Journal of Biological Chemistry

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The first directed mutant of a higher plant ribulosebisphosphate carboxylase/oxygenase (Rubisco), constructed by chloroplast transformation, is catalytically impaired but still able to support the plant's photosynthesis and growth (Whitney, S. M., von Caemmerer, S., Hudson, G. S., and Andrews, T. J. (1999) Plant Physiol. 121, 579 -588). This mutant enzyme has a Leu to Val substitution at residue 335 in the flexible loop 6 of the large subunit, which closes over the substrate during catalysis. Its active site was intact, as judged by its barely impaired competency in the initial enolization step of the reaction sequence, and its ability to bind tightly the intermediate analog, 2-carboxy-D-arabinitol-1,5-bisphosphate. Prompted by observations that the mutant enzyme displayed much less slow inhibition during catalysis in vitro than the wild type, its tendency to catalyze side reactions and its response to the slow inhibitor D-xylulose-1,5-bisphosphate were studied. The lessening in slow inhibition was not caused by reduced production of inhibitory side products. Except for pyruvate production, these reactions were strongly enhanced by the mutation, as was the ability to catalyze the carboxylation of D-xylulose-1,5-bisphosphate. Rather, reduced inhibition was the result of lessened sensitivity to these inhibitors. The slow isomerization phase that characterizes inhibition of the wild-type enzyme by D-xylulose-1,5-bisphosphate was completely eliminated by the mutation, and the mutant was more adept than the wild type in catalyzing the benzylic acid-type rearrangement of D-glycero-2,3-pentodiulose-1,5-bisphosphate (produced by oxidation of the substrate, D-ribulose-1,5-bisphosphate). These observations are consistent with increased flexibility of loop 6 induced by the mutation, and they reveal the underlying mechanisms by which the side reactions cause slow inhibition.Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, 1 EC 4.1.1.39) catalyzes the carboxylation and oxygenation of ribulose-P 2 in photosynthetic CO 2 fixation and photorespiration (1-5). Despite excellent crystal structures of various liganded and unliganded Rubiscos from various sources (6) and intensive mutagenic study of algal (3) and bacterial (7) Rubiscos, progress toward understanding the catalytic mechanism has been hampered by an inability to express the dominant higher plant form of the enzyme in heterologous hosts. This precluded application of the power of directed mutagenesis to the higher plant enzyme until Whitney et al. (8) constructed the first such mutant by chloroplast transformation in the natural host, tobacco. Because it was desirable to maintain photosynthetic viability, a mutation (a Leu to Val substitution at residue 335 of the plastid-encoded large subunit) was chosen with the aim of changing the kinetic parameters (particularly the CO 2 /O 2 specificity (9)) as much as possible without seriously disabling catalytic performance at elevated CO 2 concentration. The substitution successfully reduced the CO 2 /O 2 specificity ...

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“…2). Although never observed with wildtype Rubisco, DiMP is generated by mutants with replacements for residues that constitute the binding pocket for the C1 phosphate group of RuBP (11,12). Given the absence of forward processing of the enediol and its compromised binding due to the truncated loop 6, this intermediate is relegated to dissociation from the active site and subsequent decomposition.…”

Section: Resultsmentioning

confidence: 99%

Catalytic Roles of Flexible Regions at the Active Site of Ribulose-Bisphosphate Carboxylase/Oxygenase (Rubisco)

Hartman¹,

Harpel²,

Chen³

et al. 1995

Photosynthesis: From Light to Biosphere

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Rubisco: Maladapted or Misunderstood

Cited by 42 publications

References 24 publications

Linked Rubisco Subunits Can Assemble into Functional Oligomers without Impeding Catalytic Performance

Linked Rubisco Subunits Can Assemble into Functional Oligomers without Impeding Catalytic Performance

The Relationship between Side Reactions and Slow Inhibition of Ribulose-bisphosphate Carboxylase Revealed by a Loop 6 Mutant of the Tobacco Enzyme

Catalytic Roles of Flexible Regions at the Active Site of Ribulose-Bisphosphate Carboxylase/Oxygenase (Rubisco)

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