Preliminary X-ray crystallographic study of wild-type and mutant ribulose-1,5-bisphosphate carboxylase/oxygenase from<i>Chlamydomonas reinhardtii</i>

Yen, Ann; Haas, Eric J.; Selbo, K.M.; Ross, Charles R.; Spreitzer, Robert J.; Stezowski, John J.

doi:10.1107/s0907444997016211

Cited by 5 publications

(4 citation statements)

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“…It forms ionic and hydrogen bonds with Glu-223 in one large subunit and a hydrogen bond with Lys-161 in a neighboring large subunit (Figure 5A) (8). An atomic structure is not yet available for Chlamydomonas Rubisco (40), but one can assume that the structural interactions between the conserved small-subunit residues and the large subunits are quite similar. The six additional residues in the N-terminal half of the Chlamydomonas βA-βB loop (Figure 2) are likely to reside in the central solvent channel, which is encircled by the βA-βB loops of four small subunits at the top and bottom of the spinach holoenzyme (8).…”

Section: Discussionmentioning

confidence: 99%

Alanine-Scanning Mutagenesis of the Small-Subunit βA−βB Loop of Chloroplast Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase: Substitution at Arg-71 Affects Thermal Stability and CO₂/O₂ Specificity

Spreitzer

Esquível

et al. 2001

Biochemistry

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Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) enzymes from different species differ with respect to carboxylation catalytic efficiency and CO2/O2 specificity, but the structural basis for these differences is not known. Whereas much is known about the chloroplast-encoded large subunit, which contains the alpha/beta-barrel active site, much less is known about the role of the nuclear-encoded small subunit in Rubisco structure and function. In particular, a loop between beta-strands A and B contains 21 or more residues in plants and green algae, but only 10 residues in prokaryotes and nongreen algae. To determine the significance of these additional residues, a mutant of the green alga Chlamydomonas reinhardtii, which lacks both small-subunit genes, was used as a host for transformation with directed-mutant genes. Although previous studies had indicated that the betaA-betaB loop was essential for holoenzyme assembly, Ala substitutions at residues conserved among land plants and algae (Arg-59, Tyr-67, Tyr-68, Asp-69, and Arg-71) failed to block assembly or eliminate function. Only the Arg-71 --> Ala substitution causes a substantial decrease in holoenzyme thermal stability. Tyr-68 --> Ala and Asp-69 --> Ala enzymes have lower K(m)(CO2) values, but these improvements are offset by decreases in carboxylation V(max) values. The Arg-71 --> Ala enzyme has a decreased carboxylation V(max) and increased K(m)(CO2) and K(m)(O2) values, which account for an observed 8% decrease in CO2/O2 specificity. Despite the fact that Arg-71 is more than 20 A from the large-subunit active site, it is apparent that the small-subunit betaA-betaB loop region can influence catalytic efficiency and CO2/O2 specificity.

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Section: Discussionmentioning

confidence: 99%

Alanine-Scanning Mutagenesis of the Small-Subunit βA−βB Loop of Chloroplast Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase: Substitution at Arg-71 Affects Thermal Stability and CO₂/O₂ Specificity

Spreitzer

Esquível

et al. 2001

Biochemistry

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“…1), not specific ionic interactions, that is responsible for the specificity of activase for Rubisco. It may be necessary to compare mutant enzyme crystal structures to address this possibility (33). However, now that it has recently become possible to engineer tobacco Rubisco in vivo (14,15), it would be interest- ing to see whether an R89A substitution would reverse activase specificity, presumably by promoting the formation of an ionic bond between Lys-94 and Asp-95 and mimicking the loop structure of spinach Rubisco.…”

Section: Discussionmentioning

confidence: 99%

Activase Region on Chloroplast Ribulose-1,5-bisphosphate Carboxylase/Oxygenase

Ott¹,

Smith²,

Portis³

et al. 2000

Journal of Biological Chemistry

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In the active form of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), a carbamate at lysine 201 binds Mg 2؉ , which then interacts with the carboxylation transition state. Rubisco activase facilitates this spontaneous carbamylation/metal-binding process by removing phosphorylated inhibitors from the Rubisco active site. Activase from Solanaceae plants (e.g. tobacco) fails to activate Rubisco from non-Solanaceae plants (e.g. spinach and Chlamydomonas reinhardtii), and non-Solanaceae activase fails to activate Solanaceae Rubisco. Directed mutagenesis and chloroplast transformation previously showed that a proline 89 to arginine substitution on the surface of the large subunit of Chlamydomonas Rubisco switched its specificity from non-Solanaceae to Solanaceae activase activation. To define the size and function of this putative activase binding region, substitutions were created at positions flanking residue 89. As in the past, these substitutions changed the identities of Chlamydomonas residues to those of tobacco. Whereas an aspartate 86 to arginine substitution had little effect, aspartate 94 to lysine Rubisco was only partially activated by spinach activase but now fully activated by tobacco activase. In an attempt to eliminate the activase/Rubisco interaction, proline 89 was changed to alanine, which is not present in either non-Solanaceae or Solanaceae Rubisco. This substitution also caused reversal of activase specificity, indicating that amino acid identity alone does not determine the specificity of the interaction.Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) (Rubisco) 1 catalyzes carboxylation of RuBP in the first step of the Calvin cycle of photosynthesis (reviewed in Refs. 1 and 2). To be active, Lys-201 on the Rubisco large subunit must be carbamylated and coordinated with Mg 2ϩ prior to binding RuBP (reviewed in Refs. 2 and 3). Thus, Rubisco is similar to urease, which also uses a carbamate as a ligand for metal binding (5). However, whereas urease requires a set of proteins for the addition of Ni 2ϩ to its active site (6), Rubisco appears to require only one. This protein, Rubisco activase, removes inhibitory sugar phosphates, including RuBP, from the nonactivated active site, thereby facilitating subsequent, spontaneous carbamylation and Mg 2ϩ binding (reviewed in Refs. 3 and 4). In some cases, fully activated (metal-bound) Rubisco may bind inhibitory sugar phosphates that mimic the carboxylation transition state (7,8). These molecules are also removed by Rubisco activase to restore a functional active site (8, 9). Like one of the urease activation proteins, UreG (5), Rubisco activase hydrolyzes nucleoside triphosphate during the activation process (10, 11). However, except for a P-loop motif, there is little homology between these two proteins.Rubisco activase from plants in the family Solanaceae (e.g. tobacco) fails to activate Rubisco from plants outside the family (e.g. spinach and the green alga Chlamydomonas reinhardtii), and activase from non-Solanacea...

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“…3B) , all three residues interact with the same region of the small-subunit ␤A͞␤B loop. Because the x-ray crystal structure of Chlamydomonas Rubisco has not yet been solved (44), and because the N54S and A57V small-subunit substitutions replace residues that are apparently absent from the spinach Rubisco structure (Fig. 3A), it is difficult to deduce the location of the Asn 54 and Ala 57 residues within the Chlamydomonas enzyme.…”

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RbcS suppressor mutations improve the thermal stability and CO ₂ /O ₂ specificity of rbcL - mutant ribulose-1,5-bisphosphate carboxylase/oxygenase

Hong²,

Spreitzer³

2000

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

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In the green alga Chlamydomonas reinhardtii, a Leu 290 -to-Phe (L290F) substitution in the large subunit of ribulose-1,5-bisphosphate carboxylase͞oxygenase (Rubisco), which is coded by the chloroplast rbcL gene, was previously found to be suppressed by second-site Ala 222 -to-Thr and Val 262 -to-Leu substitutions. These substitutions complement the photosynthesis deficiency of the L290F mutant by restoring the decreased thermal stability, catalytic efficiency, and CO 2͞O2 specificity of the mutant enzyme back to wild-type values. Because residues 222, 262, and 290 interact with the loop between ␤ strands A and B of the Rubisco small subunit, which is coded by RbcS1 and RbcS2 nuclear genes, it seemed possible that substitutions in this loop might also suppress L290F. A mutation in a nuclear gene, Rbc-1, was previously found to suppress the biochemical defects of the L290F enzyme at a posttranslational step, but the nature of this gene and its product remains unknown. In the present study, three nuclear-gene suppressors were found to be linked to each other but not to the Rbc-1 locus. DNA sequencing revealed that the RbcS2 genes of these suppressor strains have mutations that cause either Asn 54 -to-Ser or Ala 57 -to-Val substitutions in the small-subunit ␤A͞␤B loop. When present in otherwise wild-type cells, with or without the resident RbcS1 gene, the mutant small subunits improve the thermal stability of wild-type Rubisco. These results indicate that the ␤A͞␤B loop, which is unique to eukaryotic Rubisco, contributes to holoenzyme thermal stability, catalytic efficiency, and CO 2͞O2 specificity. The small subunit may be a fruitful target for engineering improved Rubisco. T he green alga Chlamydomonas reinhardtii serves as an excellent model for the study of photosynthesis because photosynthesis-deficient mutants can be maintained with acetate as an alternative source of carbon and energy. Mutations can be assigned to the nuclear or chloroplast genetic compartments by virtue of Mendelian or uniparental inheritance, and both compartments can be transformed with homologous or heterologous DNA (1). These attributes have been particularly useful for examining the structure-function relationships of eukaryotic ribulose-1,5-bisphosphate carboxylase͞oxygenase (Rubisco, Enzyme Commission 4.1.1.39) (2). Because land plants cannot be maintained in the complete absence of photosynthesis, and the subunits of eukaryotic Rubisco cannot be assembled in Escherichia coli (3), it has been difficult to examine the effects of random or directed mutations on the function of land-plant Rubisco in vivo or in vitro (4-6). Tobacco is the only land plant in which the chloroplast genome can be transformed (5, 6).By screening for photosynthesis-deficient Chlamydomonas mutants and selecting for photosynthesis-competent revertants, several regions of the Rubisco large subunit have been identified that influence the ratio of ribulose 1,5-bisphosphate (RuBP) carboxylase to oxygenase activities (2, 7). Because CO 2 and O 2 compete at the same la...

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Preliminary X-ray crystallographic study of wild-type and mutant ribulose-1,5-bisphosphate carboxylase/oxygenase fromChlamydomonas reinhardtii

Cited by 5 publications

References 5 publications

Alanine-Scanning Mutagenesis of the Small-Subunit βA−βB Loop of Chloroplast Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase: Substitution at Arg-71 Affects Thermal Stability and CO₂/O₂ Specificity

Alanine-Scanning Mutagenesis of the Small-Subunit βA−βB Loop of Chloroplast Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase: Substitution at Arg-71 Affects Thermal Stability and CO₂/O₂ Specificity

Activase Region on Chloroplast Ribulose-1,5-bisphosphate Carboxylase/Oxygenase

RbcS suppressor mutations improve the thermal stability and CO ₂ /O ₂ specificity of rbcL - mutant ribulose-1,5-bisphosphate carboxylase/oxygenase

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Preliminary X-ray crystallographic study of wild-type and mutant ribulose-1,5-bisphosphate carboxylase/oxygenase fromChlamydomonas reinhardtii

Cited by 5 publications

References 5 publications

Alanine-Scanning Mutagenesis of the Small-Subunit βA−βB Loop of Chloroplast Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase: Substitution at Arg-71 Affects Thermal Stability and CO2/O2 Specificity

Alanine-Scanning Mutagenesis of the Small-Subunit βA−βB Loop of Chloroplast Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase: Substitution at Arg-71 Affects Thermal Stability and CO2/O2 Specificity

Activase Region on Chloroplast Ribulose-1,5-bisphosphate Carboxylase/Oxygenase

RbcS suppressor mutations improve the thermal stability and CO 2 /O 2 specificity of rbcL - mutant ribulose-1,5-bisphosphate carboxylase/oxygenase

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Alanine-Scanning Mutagenesis of the Small-Subunit βA−βB Loop of Chloroplast Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase: Substitution at Arg-71 Affects Thermal Stability and CO₂/O₂ Specificity

Alanine-Scanning Mutagenesis of the Small-Subunit βA−βB Loop of Chloroplast Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase: Substitution at Arg-71 Affects Thermal Stability and CO₂/O₂ Specificity

RbcS suppressor mutations improve the thermal stability and CO ₂ /O ₂ specificity of rbcL - mutant ribulose-1,5-bisphosphate carboxylase/oxygenase