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.

3'end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process

1993 ◽  
Vol 13 (4) ◽  
pp. 2277-2285
Author(s):  
D B Stern ◽  
K L Kindle
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Higher Plants ◽  
Transcription Termination ◽  
Protein Coding ◽  
Coding Regions ◽  
In Vitro Processing ◽  
Mrna Precursor ◽  
Chloroplast Transcripts

Inverted repeat (IR) sequences are found at the 3' ends of most chloroplast protein coding regions, and we have previously shown that the 3'IR is important for accumulation of atpB mRNA in Chlamydomonas reinhardtii (D. B. Stern, E.R. Radwanski, and K. L. Kindle, Plant Cell 3:285-297, 1991). In vitro studies indicate that 3' IRs are inefficient transcription termination signals in higher plants and have furthermore defined processing activities that act on the 3' ends of chloroplast transcripts, suggesting that most chloroplast mRNAs are processed at their 3' ends in vivo. To investigate the mechanism of 3' end processing in Chlamydomonas reinhardtii chloroplasts, the maturation of atpB mRNA was examined in vitro and in vivo. In vitro, a synthetic atpB mRNA precursor is rapidly cleaved at a position 10 nucleotides downstream from the mature 3' terminus. This cleavage is followed by exonucleolytic processing to generate the mature 3' end. In vivo run-on transcription experiments indicate that a maximum of 50% of atpB transcripts are transcriptionally terminated at or near the IR, while the remainder are subject to 3' end processing. Analysis of transcripts derived from chimeric atpB genes introduced into Chlamydomonas chloroplasts by biolistic transformation suggests that in vivo processing and in vitro processing occur by similar or identical mechanisms.

scholarly journals Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance

2006 ◽  
Vol 397 (2) ◽  
pp. 305-312 ◽  
Author(s):  
G. H. Erica Law ◽  
Olga A. Gandelman ◽  
Laurence C. Tisi ◽  
Christopher R. Lowe ◽  
James A. H. Murray
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Green Light ◽  
Firefly Luciferase ◽  
Wild Type ◽  
Photinus Pyralis ◽  
Wild Type Enzyme ◽  
Ph Tolerance

Firefly luciferase catalyses a two-step reaction, using ATP-Mg2+, firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow–green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specific activity of the wild-type enzyme. These were identified by the mutagenesis of solvent-exposed non-conserved hydrophobic amino acids to hydrophilic residues in Photinus pyralis firefly luciferase followed by in vivo activity screening. Mutants F14R, L35Q, V182K, I232K and F465R were found to be the preferred substitutions at the respective positions. The effects of these amino acid replacements are additive, since combination of the five substitutions produced an enzyme with greatly improved pH-tolerance and stability up to 45 °C. All mutants, including the mutant with all five substitutions, showed neither a decrease in specific activity relative to the recombinant wild-type enzyme, nor any substantial differences in kinetic constants. It is envisaged that the combined mutant will be superior to wild-type luciferase for many in vitro and in vivo applications.

scholarly journals The 5' leader of a chloroplast mRNA mediates the translational requirements for two nucleus-encoded functions in Chlamydomonas reinhardtii.

1994 ◽  
Vol 14 (8) ◽  
pp. 5268-5277 ◽  
Author(s):  
W Zerges ◽  
J D Rochaix
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Rna Binding ◽  
Mrna Translation ◽  
Binding Activity ◽  
Leader Sequence ◽  
Sequence Motif ◽  
Wild Type ◽  
Chloroplast Mrna

In the green alga Chlamydomonas reinhardtii, the nuclear mutations F34 and F64 have been previously shown to abolish the synthesis of the photosystem II core polypeptide subunit P6, which is encoded by the chloroplast psbC gene. In this report the functions encoded by F34 and F64 are shown to be required for translation of the psbC mRNA, on the basis of the finding that the expression of a heterologous reporter gene fused to the psbC 5' nontranslated leader sequence requires wild-type F34 and F64 alleles in vivo. Moreover, a point mutation in the psbC 5' nontranslated leader sequence suppresses this requirement for wild-type F34 function. In vitro RNA-protein cross-linking studies reveal that chloroplast protein extracts from strains carrying the F64 mutation contain an approximately 46-kDa RNA-binding protein. The absence of the RNA-binding activity of this protein in chloroplast extracts of wild-type strains suggests that it is related to the role of the F64-encoded function for psbC mRNA translation. The binding specificity of this protein appears to be for an AU-rich RNA sequence motif.

scholarly journals Inhibition of ribulose bisphosphate carboxylase assembly by antibody to a binding protein.

1986 ◽  
Vol 103 (4) ◽  
pp. 1327-1335 ◽  
Author(s):  
S Cannon ◽  
P Wang ◽  
H Roy
Keyword(s):  
Higher Plants ◽  
Binding Protein ◽  
Large Subunit ◽  
Ribulose Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate ◽  
Dual Effect ◽  
Bisphosphate Carboxylase ◽  
Low Concentrations ◽  
The One

We have developed an assay to monitor in vitro the posttranslational assembly of the chloroplast protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Most of the newly synthesized 55-kD catalytic ("large") subunits of this enzyme occur in a 29S complex together with 60- and 61-kD "binding" proteins. When the 29S complex is incubated with ATP and MgCl2 it dissociates into subunits, and the formerly bound large subunits now sediment at 7S (still faster than expected for a monomer). Upon incubation at 24 degrees C, these large subunits assemble into RuBisCO. The minority of newly made large subunits which are not bound to the 29S complex also sediment at 7S. When endogenous ATP was removed by addition of hexokinase and glucose, the dissociation of the 29S complex was inhibited. Nevertheless, the 7S large subunits assembled into RuBisCO, and did so to a greater extent than in controls retaining endogenous ATP. Thus the 7S large subunits are also assembly competent, at least when ATP is removed. Apparently, in chloroplast extracts, ATP can have a dual effect on the assembly of RuBisCO: on the one hand, even at low concentrations it can inhibit incorporation of 7S large subunits RuBisCO; on the other hand, at higher concentrations it can lead to substantial buildup of the 7S large subunit pool by causing dissociation of the 29S complex, and stimulate overall assembly. At both high and zero concentrations of ATP, however, antibody to the binding protein inhibited the assembly of endogenous large subunits into RuBisCO. Thus it appears that all assembly-competent large subunits are associated with the binding protein, either in the 7S complex or in the 29S complex. The involvement of the binding protein in RuBisCO assembly may represent the first example of non-autonomous protein assembly in higher plants and may pose problems for the genetic engineering of RuBisCO from these organisms.

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 3'end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process.

1993 ◽  
Vol 13 (4) ◽  
pp. 2277-2285 ◽  
Author(s):  
D B Stern ◽  
K L Kindle
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Higher Plants ◽  
Transcription Termination ◽  
Protein Coding ◽  
Coding Regions ◽  
In Vitro Processing ◽  
Mrna Precursor ◽  
Chloroplast Transcripts

Inverted repeat (IR) sequences are found at the 3' ends of most chloroplast protein coding regions, and we have previously shown that the 3'IR is important for accumulation of atpB mRNA in Chlamydomonas reinhardtii (D. B. Stern, E.R. Radwanski, and K. L. Kindle, Plant Cell 3:285-297, 1991). In vitro studies indicate that 3' IRs are inefficient transcription termination signals in higher plants and have furthermore defined processing activities that act on the 3' ends of chloroplast transcripts, suggesting that most chloroplast mRNAs are processed at their 3' ends in vivo. To investigate the mechanism of 3' end processing in Chlamydomonas reinhardtii chloroplasts, the maturation of atpB mRNA was examined in vitro and in vivo. In vitro, a synthetic atpB mRNA precursor is rapidly cleaved at a position 10 nucleotides downstream from the mature 3' terminus. This cleavage is followed by exonucleolytic processing to generate the mature 3' end. In vivo run-on transcription experiments indicate that a maximum of 50% of atpB transcripts are transcriptionally terminated at or near the IR, while the remainder are subject to 3' end processing. Analysis of transcripts derived from chimeric atpB genes introduced into Chlamydomonas chloroplasts by biolistic transformation suggests that in vivo processing and in vitro processing occur by similar or identical mechanisms.

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.

scholarly journals Transcriptional and post-transcriptional regulation of ribulose 1,5-bisphosphate carboxylase gene expression in light- and dark-grown amaranth cotyledons.

1985 ◽  
Vol 5 (9) ◽  
pp. 2238-2246 ◽  
Author(s):  
J O Berry ◽  
B J Nikolau ◽  
J P Carr ◽  
D F Klessig
Keyword(s):  
Specific Activity ◽  
Large Subunit ◽  
Growth Conditions ◽  
Small Subunit ◽  
Northern Analysis ◽  
Mrna Levels ◽  
Regulation Of Expression ◽  
Bisphosphate Carboxylase

The regulation of expression of the genes encoding the large subunit (LSU) and small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase (RuBPCase) was examined in 1- through 8-day-old, dark-grown (etiolated) and light-grown amaranth cotyledons. RuBPCase specific activity in light-grown cotyledons increased during this 8-day period to a level 15-fold higher than in dark-grown cotyledons. Under both growth conditions, the accumulation of the LSU and SSU polypeptides was not coordinated. Initial detection of the SSU occurred 1 and 2 days after the appearance of the LSU in light- and dark-grown cotyledons, respectively. Furthermore, although the levels of the LSU were similar in both light- and dark-grown seedlings, the amount of the SSU followed clearly the changes in enzyme activity. Synthesis of these two polypeptides was dramatically different in etiolated versus light-grown cotyledons. In light the synthesis of both subunits was first observed on day 2 and continued throughout the growth of the cotyledons. In darkness the rate of synthesis of both subunits was much lower than in light and occurred only as a burst between days 2 and 5 after planting. However, mRNAs for both subunits were present in etiolated cotyledons at similar levels on days 4 through 7 (by Northern analysis) and were functional in vitro, despite their apparent inactivity in vivo after day 5. In addition, since both LSU and SSU mRNA levels were lower in dark- than in light-grown seedlings, our results indicate that both transcriptional and post-transcriptional controls modulate RuBPCase production in developing amaranth cotyledons.

Transcriptional and post-transcriptional regulation of ribulose 1,5-bisphosphate carboxylase gene expression in light- and dark-grown amaranth cotyledons

1985 ◽  
Vol 5 (9) ◽  
pp. 2238-2246
Author(s):  
J O Berry ◽  
B J Nikolau ◽  
J P Carr ◽  
D F Klessig
Keyword(s):  
Specific Activity ◽  
Large Subunit ◽  
Growth Conditions ◽  
Small Subunit ◽  
Northern Analysis ◽  
Mrna Levels ◽  
Regulation Of Expression ◽  
Bisphosphate Carboxylase

The regulation of expression of the genes encoding the large subunit (LSU) and small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase (RuBPCase) was examined in 1- through 8-day-old, dark-grown (etiolated) and light-grown amaranth cotyledons. RuBPCase specific activity in light-grown cotyledons increased during this 8-day period to a level 15-fold higher than in dark-grown cotyledons. Under both growth conditions, the accumulation of the LSU and SSU polypeptides was not coordinated. Initial detection of the SSU occurred 1 and 2 days after the appearance of the LSU in light- and dark-grown cotyledons, respectively. Furthermore, although the levels of the LSU were similar in both light- and dark-grown seedlings, the amount of the SSU followed clearly the changes in enzyme activity. Synthesis of these two polypeptides was dramatically different in etiolated versus light-grown cotyledons. In light the synthesis of both subunits was first observed on day 2 and continued throughout the growth of the cotyledons. In darkness the rate of synthesis of both subunits was much lower than in light and occurred only as a burst between days 2 and 5 after planting. However, mRNAs for both subunits were present in etiolated cotyledons at similar levels on days 4 through 7 (by Northern analysis) and were functional in vitro, despite their apparent inactivity in vivo after day 5. In addition, since both LSU and SSU mRNA levels were lower in dark- than in light-grown seedlings, our results indicate that both transcriptional and post-transcriptional controls modulate RuBPCase production in developing amaranth cotyledons.

scholarly journals The fixABCX Genes in Rhodospirillum rubrum Encode a Putative Membrane Complex Participating in Electron Transfer to Nitrogenase

2004 ◽  
Vol 186 (7) ◽  
pp. 2052-2060 ◽  
Author(s):  
Tomas Edgren ◽  
Stefan Nordlund
Keyword(s):  
Electron Transfer ◽  
Electron Transport ◽  
Western Blotting ◽  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Transmembrane Protein ◽  
Wild Type ◽  
Bisphosphate Carboxylase

ABSTRACT In our efforts to identify the components participating in electron transport to nitrogenase in Rhodospirillum rubrum, we used mini-Tn5 mutagenesis followed by metronidazole selection. One of the mutants isolated, SNT-1, exhibited a decreased growth rate and about 25% of the in vivo nitrogenase activity compared to the wild-type values. The in vitro nitrogenase activity was essentially wild type, indicating that the mutation affects electron transport to nitrogenase. Sequencing showed that the Tn5 insertion is located in a region with a high level of similarity to fixC, and extended sequencing revealed additional putative fix genes, in the order fixABCX. Complementation of SNT-1 with the whole fix gene cluster in trans restored wild-type nitrogenase activity and growth. Using Western blotting, we demonstrated that expression of fixA and fixB occurs only under conditions under which nitrogenase also is expressed. SNT-1 was further shown to produce larger amounts of both ribulose 1,5-bisphosphate carboxylase/oxgenase and polyhydroxy alkanoates than the wild type, indicating that the redox status is affected in this mutant. Using Western blotting, we found that FixA and FixB are soluble proteins, whereas FixC most likely is a transmembrane protein. We propose that the fixABCX genes encode a membrane protein complex that plays a central role in electron transfer to nitrogenase in R. rubrum. Furthermore, we suggest that FixC is the link between nitrogen fixation and the proton motive force generated in the photosynthetic reactions.

Sign in / Sign up

Export Citation Format

Share Document