Crystal structure of activated ribulose-1,5-bisphosphate carboxylase/oxygenase from green alga Chlamydomonas reinhardtii complexed with 2-carboxyarabinitol-1,5-bisphosphate

Mizohata, Eiichi; Matsumura, Hideki; Okano, Yasuhiro; Kumei, Maki; Takuma, Hiroki; Onodera, Jun; Kato, Ko; Shibata, Naoki; Inoue, Tsuyoshi; Yokota, Akiho

doi:10.1006/jmbi.2001.5381

Cited by 68 publications

(46 citation statements)

References 64 publications

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“…Despite the increased mobility (higher Bs) in the active site region, we detected two sizable patches of difference ED that were identified and refined as phosphate/sulfate anions (∼60% occupied). Both phosphate positions correlate well with phosphate groups of substrates or analogs that were found in the other RuBisCO structures (28) (Fig. 3A).…”

Section: +supporting

confidence: 74%

Structural mechanism of RuBisCO activation by carbamylation of the active site lysine

Stec

2012

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

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Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a crucial enzyme in carbon fixation and the most abundant protein on earth. It has been studied extensively by biochemical and structural methods; however, the most essential activation step has not yet been described. Here, we describe the mechanistic details of Lys carbamylation that leads to RuBisCO activation by atmospheric CO 2 . We report two crystal structures of nitrosylated RuBisCO from the red algae Galdieria sulphuraria with O 2 and CO 2 bound at the active site. G. sulphuraria RuBisCO is inhibited by cysteine nitrosylation that results in trapping of these gaseous ligands. The structure with CO 2 defines an elusive, preactivation complex that contains a metal cation Mg 2+ surrounded by three H 2 O/OH molecules. Both structures suggest the mechanism for discriminating gaseous ligands by their quadrupole electric moments. We describe conformational changes that allow for intermittent binding of the metal ion required for activation. On the basis of these structures we propose the individual steps of the activation mechanism. Knowledge of all these elements is indispensable for engineering RuBisCO into a more efficient enzyme for crop enhancement or as a remedy to global warming. nitrosylation in algae | activity control | intermediate trapping R ibulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) (EC 4.1.1.39) is a dominant contributor to conversion of gaseous CO 2 into biomass (1). Aerobic life forms, including heterotrophs, primarily derive their carbon through an authotrophic/ photosynthetic route using CO 2 as a carbon source. RuBisCO accomplishes this task by incorporating CO 2 into a phospho-sugar, ribulose 1,5-bisphosphate (RuBP). Incorporation of CO 2 into RuBP generates two molecules of 3-phosphoglycerate. This simple compound is subsequently used to build other organic molecules of life. Synthesized RuBisCO does not have a fully functional active site (2-4). It needs to be activated by a CO 2 molecule that carbamylates its catalytic Lys to bind Mg 2+ that completes the activation process (5, 6). The mechanism of RuBisCO activation by the CO 2 molecule is presented in this work. This knowledge will contribute to acceleration of the progress in remodeling the RuBisCO for practical uses (7-10).RuBisCO is present in many photosynthetic organisms including bacteria, algae, plants (2-4), and archaea (11). It accounts for roughly a half of the soluble protein mass in C3 plant leaves (1). However, RuBisCO is an inefficient enzyme with a low, 1-10/s, turnover rate (12). A side reaction with O 2 reduces its functional activity even further. The side reaction happens when a promiscuous enodiolate intermediate of RuBP is attacked by O 2 (versus CO 2 ) and produces 2-phosphoglycolate (13). This reaction drains a pool of RuBP and decreases the efficiency of carbon fixation by up to 50%. The efficiency of RuBisCO is thus dependent on the ability to discriminate between CO 2 and O 2 or on the specificity ratio. The specificity ratio S is defin...

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Section: +supporting

confidence: 74%

Structural mechanism of RuBisCO activation by carbamylation of the active site lysine

Stec

2012

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

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“…The crystal structure of Rubisco has been previously solved (26,27), yet no RNA binding motifs were identified. The large subunit of the protein consists of two domains, a TIM barrel carboxyl-terminal domain (aa 151-475) and a smaller amino terminus domain (aa 1-150) that was classified as a ferredoxinlike domain by SCOP (25).…”

Section: Discussionmentioning

confidence: 99%

“…The Putative RNA Binding Domain of Rubisco LSU-To understand the structural basis for the RNA binding activity observed in Chlamydomonas Rubisco LSU, we tried to gain some insight from its crystal structure (26,27). The crystal structure of Rubisco consists of two domains, a TIM barrel carboxyl-terminal domain (aa 151-475) and a smaller amino terminus domain (aa 1-150).…”

Section: Rna Binding Activity Of Protein Extracts To the Rbcl 5ј-utr;mentioning

confidence: 99%

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RNA Binding Activity of the Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit from Chlamydomonas reinhardtii

Yosef

Irihimovitch

Knopf

et al. 2004

Journal of Biological Chemistry

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Transfer of the green algae Chlamydomonas reinhardtii from low light to high light generated an oxidative stress that led to a dramatic arrest in the synthesis of the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The translational arrest correlated with transient changes in the intracellular levels of reactive oxygen species and with shifting the glutathione pool toward its oxidized form (Irihimovitch, V., and Shapira, M. (2000) J. Biol. Chem. 275, 16289 -16295). Here we examined how the redox potential of glutathione affected the RNA-protein interactions with the 5-untranslated region of rbcL. This RNA region specifically binds a group of proteins with molecular masses of 81, 62, 51, and 47 kDa in UV-cross-linking experiments under reducing conditions. Binding of these proteins was interrupted by exposure to oxidizing conditions (GSSG), and a new protein of 55 kDa was shown to interact with the RNA. The 55-kDa protein comigrated with Rubisco LSU in one-and two-dimensional gels, and its RNA binding activity was further verified by using the purified protein in UV-cross-linking experiments under oxidizing conditions. However, the LSU of purified and oxidized Rubisco bound to RNA in a sequence-independent manner. A remarkable structural similarity was found between the amino-terminal domain of Rubisco LSU in C. reinhardtii and the RNA binding domain, a highly prevailing motif among RNAbinding proteins. It appears from the crystal structure of Rubisco that the amino terminus of LSU is buried within the holoenzyme. We propose that under oxidizing conditions it is exposed to the surface and can, therefore, bind RNA. Accordingly, a recombinant form of the polypeptide domain that corresponds to the amino terminus of LSU was found to bind RNA in vitro with or without GSSG.When plants and algae absorb light energy that exceeds the level of electron carrier saturation they generate reactive oxygen species (ROS), 1 that cause a variety of cellular and molecular damage. This phenomenon is referred to as photoinhibition and is common to all photosynthetic organisms (1-3). Recovery from photoinhibition can be achieved by decreasing the chlorophyll content and by activating a variety of antioxidant pathways that involve ascorbate and glutathione (4, 5).Ribulose-1,5-bisphosphate carboxylase (Rubisco) is the key enzyme in photosynthetic carbon assimilation. In Chlamydomonas reinhardtii and in land plants the enzyme is composed of eight large subunits (LSU) encoded by the chloroplast rbcL gene and eight small subunits encoded by the nuclear rbcS gene family. Assembly of the holoenzyme is mediated by the chloroplast chaperonins cpn60 and cpn10 (6, 7). We previously showed that transfer of the green algae C. reinhardtii from low light (70 mol m Ϫ2 s Ϫ1 ) to high light (700 mol m Ϫ2 s Ϫ1 ) generates an oxidative stress that leads to photoinhibition and a dramatic arrest in the synthesis of the LSU of Rubisco (8). These light-induced effects were found to be transient, with cell recovery taking pla...

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“…We focus on RuBisCO found in the leaves of spinach, for which several X-ray structures are available, e.g., [6][7][8]. Our investigation considers the carboxylation step only, i.e., the electrophilic attack of CO 2 to enolized RuBP.…”

Section: Introductionmentioning

confidence: 99%

Quantum chemical modeling of the kinetic isotope effect of the carboxylation step in RuBisCO

Götze

Saalfrank

2011

J Mol Model

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Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most important enzyme for the assimilation of carbon into biomass, features a well-known isotope effect with regards to the CO(2) carbon atom. This kinetic isotope effect α = k(12)/k(13) for the carboxylation step of the RuBisCO reaction sequence, and its microscopic origin, was investigated with the help of cluster models and quantum chemical methods [B3LYP/6-31G(d,p)]. We use a recently proposed model for the RuBisCO active site, in which a water molecule remains close to the reaction center during carboxylation of ribulose-1,5-bisphosphate [B. Kannappan, J.E. Gready, J. Am. Chem. Soc. 130 (2008), 15063]. Alternative active-site models and/or computational approaches were also tested. An isotope effect alpha for carboxylation is found, which is reasonably close to the one measured for the overall reaction, and which originates from a simple frequency shift of the bending vibration of (12)CO(2) compared to (13)CO(2). The latter is the dominant mode for the product formation at the transition state.

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Crystal structure of activated ribulose-1,5-bisphosphate carboxylase/oxygenase from green alga Chlamydomonas reinhardtii complexed with 2-carboxyarabinitol-1,5-bisphosphate

Cited by 68 publications

References 64 publications

Structural mechanism of RuBisCO activation by carbamylation of the active site lysine

Structural mechanism of RuBisCO activation by carbamylation of the active site lysine

RNA Binding Activity of the Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit from Chlamydomonas reinhardtii

Quantum chemical modeling of the kinetic isotope effect of the carboxylation step in RuBisCO

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