scholarly journals In Vivo Photomodification of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Holoenzyme by Ultraviolet-B Radiation (Formation of a 66-Kilodalton Variant of the Large Subunit)

1995 ◽  
Vol 109 (1) ◽  
pp. 221-229 ◽  
Author(s):  
M. I. Wilson ◽  
S. Ghosh ◽  
K. E. Gerhardt ◽  
N. Holland ◽  
T. S. Babu ◽  
...  
Keyword(s):  
Large Subunit ◽  
Ultraviolet B ◽  
Bisphosphate Carboxylase ◽  
Ultraviolet B Radiation

Autologous nitric oxide protects mouse and human keratinocytes from ultraviolet B radiation-induced apoptosis

2003 ◽  
Vol 284 (5) ◽  
pp. C1140-C1148 ◽  
Author(s):  
Richard Weller ◽  
Ann Schwentker ◽  
Timothy R. Billiar ◽  
Yoram Vodovotz
Keyword(s):  
Nitric Oxide ◽  
Soluble Guanylate Cyclase ◽  
Ultraviolet B ◽  
Skin Damage ◽  
Wild Type ◽  
No Donor ◽  
Ultraviolet B Radiation ◽  
Inducible Nos

Nitric oxide (NO) can either prevent or promote apoptosis, depending on cell type. In the present study, we tested the hypothesis that NO suppresses ultraviolet B radiation (UVB)-induced keratinocyte apoptosis both in vitro and in vivo. Irradiation with UVB or addition of the NO synthase (NOS) inhibitor N G-nitro-l-arginine methyl ester (l-NAME) increased apoptosis in the human keratinocyte cell line CCD 1106 KERTr, and apoptosis was greater when the two agents were given in combination. Addition of the chemical NO donor S-nitroso- N-acetyl-penicillamine (SNAP) immediately after UVB completely abrogated the rise in apoptosis induced by l-NAME. An adenoviral vector expressing human inducible NOS (AdiNOS) also reduced keratinocyte death after UVB. Caspase-3 activity, an indicator of apoptosis, doubled in keratinocytes incubated with l-NAME compared with the inactive isomer, d-NAME, and was reduced by SNAP. Apoptosis was also increased on addition of 1,H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase. Mice null for endothelial NOS (eNOS) exhibited significantly higher apoptosis than wild-type mice both in the dermis and epidermis, whereas mice null for inducible NOS (iNOS) exhibited more apoptosis than wild-type mice only in the dermis. These results demonstrate an antiapoptotic role for NO in keratinocytes, mediated by cGMP, and indicate an antiapoptotic role for both eNOS and iNOS in skin damage induced by UVB.

Structural and functional consequences of the replacement of proximal residues Cys172 and Cys192 in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii

2008 ◽  
Vol 411 (2) ◽  
pp. 241-247 ◽  
Author(s):  
María-Jesús García-Murria ◽  
Saeid Karkehabadi ◽  
Julia Marín-Navarro ◽  
Sriram Satagopan ◽  
Inger Andersson ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Higher Plants ◽  
Large Subunit ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Bisphosphate Carboxylase ◽  
Specificity Factor

Proximal Cys172 and Cys192 in the large subunit of the photosynthetic enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) are evolutionarily conserved among cyanobacteria, algae and higher plants. Mutation of Cys172 has been shown to affect the redox properties of Rubisco in vitro and to delay the degradation of the enzyme in vivo under stress conditions. Here, we report the effect of the replacement of Cys172 and Cys192 by serine on the catalytic properties, thermostability and three-dimensional structure of Chlamydomonas reinhardtii Rubisco. The most striking effect of the C172S substitution was an 11% increase in the specificity factor when compared with the wild-type enzyme. The specificity factor of C192S Rubisco was not altered. The Vc (Vmax for carboxylation) was similar to that of wild-type Rubisco in the case of the C172S enzyme, but approx. 30% lower for the C192S Rubisco. In contrast, the Km for CO2 and O2 was similar for C192S and wild-type enzymes, but distinctly higher (approximately double) for the C172S enzyme. C172S Rubisco showed a critical denaturation temperature approx. 2 °C lower than wild-type Rubisco and a distinctly higher denaturation rate at 55 °C, whereas C192S Rubisco was only slightly more sensitive to temperature denaturation than the wild-type enzyme. X-ray crystal structures reveal that the C172S mutation causes a shift of the main-chain backbone atoms of β-strand 1 of the α/β-barrel affecting a number of amino acid side chains. This may cause the exceptional catalytic features of C172S. In contrast, the C192S mutation does not produce similar structural perturbations.

scholarly journals The state of oligomerization of Rubisco controls the rate of synthesis of the Rubisco large subunit in Chlamydomonas reinhardtii

2021 ◽  
Author(s):  
Wojciech Wietrzynski ◽  
Eleonora Traverso ◽  
Francis-André Wollman ◽  
Katia Wostrikoff
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Large Subunit ◽  
Assembly Process ◽  
Bisphosphate Carboxylase ◽  
Photosynthetic Organisms ◽  
The Core ◽  
Key Enzyme ◽  
Assembly Intermediate ◽  
Life On Earth

Abstract Ribulose 1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) is present in all photosynthetic organisms and is a key enzyme for photosynthesis-driven life on Earth. Its most prominent form is a hetero-oligomer in which small subunits (SSU) stabilize the core of the enzyme built from large subunits (LSU), yielding, after a chaperone-assisted multistep assembly process, an LSU8SSU8 hexadecameric holoenzyme. Here we use Chlamydomonas reinhardtii and a combination of site-directed mutants to dissect the multistep biogenesis pathway of Rubisco in vivo. We identify assembly intermediates, in two of which LSU are associated with the RAF1 chaperone. Using genetic and biochemical approaches we further unravel a major regulation process during Rubisco biogenesis, in which LSU translation is controlled by its ability to assemble with the SSU, via the mechanism of Control by Epistasy of Synthesis (CES). Altogether this leads us to propose a model whereby the last assembly intermediate, an LSU8-RAF1 complex, provides the platform for SSU binding to form the Rubisco enzyme, and when SSU is not available, converts to a key regulatory form that exerts negative feed-back on the initiation of LSU translation.

scholarly journals Discovery of a readily heterologously expressed Rubisco from the deep sea with potential for CO2 capture

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Junli Zhang ◽  
Guoxia Liu ◽  
Alonso I. Carvajal ◽  
Robert H. Wilson ◽  
Zhen Cai ◽  
...  
Keyword(s):  
Deep Sea ◽  
Carbon Capture ◽  
Simple Structure ◽  
Large Subunit ◽  
Carboxylation Efficiency ◽  
Riftia Pachyptila ◽  
Bisphosphate Carboxylase ◽  
High Functioning ◽  
Highly Active

AbstractRibulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key CO2-fixing enzyme in photosynthesis, is notorious for its low carboxylation. We report a highly active and assembly-competent Form II Rubisco from the endosymbiont of a deep-sea tubeworm Riftia pachyptila (RPE Rubisco), which shows a 50.5% higher carboxylation efficiency than that of a high functioning Rubisco from Synechococcus sp. PCC7002 (7002 Rubisco). It is a simpler hexamer with three pairs of large subunit homodimers around a central threefold symmetry axis. Compared with 7002 Rubisco, it showed a 3.6-fold higher carbon capture efficiency in vivo using a designed CO2 capture model. The simple structure, high carboxylation efficiency, easy heterologous soluble expression/assembly make RPE Rubisco a ready-to-deploy enzyme for CO2 capture that does not require complex co-expression of chaperones. The chemosynthetic CO2 fixation machinery of chemolithoautotrophs, CO2-fixing endosymbionts, may be more efficient than previously realized with great potential for next-generation microbial CO2 sequestration platforms.

Protein synthesis in flax following inoculation with flax rust

1986 ◽  
Vol 64 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Ben C. S. Sutton ◽  
Michael Shaw
Keyword(s):  
Protein Synthesis ◽  
Large Subunit ◽  
Two Dimensional ◽  
Bisphosphate Carboxylase ◽  
Two Dimensional Electrophoresis ◽  
Host Pathogen Interaction ◽  
Melampsora Lini ◽  
In Vivo Labelling ◽  
Dimensional Electrophoresis

Resistance to flax rust Melampsora lini (Ehrenb.) Lév. in flax carrying the N resistance gene is determined by 24 h postinoculation, at which time hypersensitivity is observed. We have examined protein synthesis in cotyledons inoculated with both virulent and avirulent races of rust by in vivo labelling with [35S]methionine. The pattern of protein synthesis was assessed by one- and two-dimensional electrophoresis 8, 13, and 18 h after inoculation. No changes in protein synthesis were observed in the first 14 h following inoculation; however, by 18 h after inoculation the susceptible combination showed a marked decrease in protein synthesis (22%; P = 0.01). This could be largely accounted for by the reduced synthesis of the ribulose 1,5-bisphosphate carboxylase large subunit, which was readily quantified on electrophoresis gels. In addition, a 30-kDa polypeptide also declined in the susceptible combination. Two-dimensional electrophoresis enabled changes to be detected in the synthesis of other minor polypeptides. None of these changes were observed in the resistant combination in which a small increase in the synthesis of the ribulose 1,5-bisphosphate carboxylase large subunit and the 30-kDa polypeptide was found. These results indicate that the outcome of the host–pathogen interaction has already been determined by 18 h after inoculation.

scholarly journals The state of oligomerization of Rubisco controls the rate of LSU translation in Chlamydomonas reinhardtii

2020 ◽  
Author(s):  
Wojciech Wietrzynski ◽  
Eleonora Traverso ◽  
Francis-André Wollman ◽  
Katia Wostrikoff
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Large Subunit ◽  
Small Subunit ◽  
Bisphosphate Carboxylase ◽  
Photosynthetic Organisms ◽  
Initiation Of Translation ◽  
Key Enzyme ◽  
Assembly Intermediate ◽  
Life On Earth

AbstractRibulose 1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) is a key enzyme for photosynthesis-driven life on Earth. While present in all photosynthetic organisms, its most prominent form is a hetero-oligomer in which a Small Subunit (SSU) stabilizes the core of the enzyme built from Large Subunits (LSU), yielding, after a chaperone-assisted multistep assembly, a LSU8SSU8 hexadecameric holoenzyme. Here we use Chlamydomonas reinhardtii, and a combination of site-directed mutants, to dissect the multistep biogenesis pathway of Rubisco in vivo. We identify assembly intermediates, in two of which LSU is associated with the RAF1 chaperone. Using genetic and biochemical approaches we further unravel a major regulation process during Rubisco biogenesis which places translation of its large subunit under the control of its ability to assemble with the small subunit, by a mechanism of Control by Epistasy of Synthesis (CES). Altogether this leads us to propose a model where the last assembly intermediate, an octameric LSU8-RAF1 complex which delivers LSU to SSU to form the Rubisco enzyme, converts to a key regulator form able to exert a negative feed-back on the initiation of translation of LSU, when SSU is not available.

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