The synthesis and purification of 2'-carboxy-D-arabinitol 1-phosphate, a natural inhibitor of ribulose 1,5-bisphosphate carboxylase, investigated by 31P n.m.r.
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ABSTRACT:
SUBMITTER: Gutteridge S
PROVIDER: S-EPMC1138735 | biostudies-other | 1989 Jun
REPOSITORIES: biostudies-other
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Project description:The predominant pool of organic matter on earth is derived from the biological reduction and assimilation of carbon dioxide gas, catalyzed primarily by the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). By virtue of its capacity to use molecular oxygen as an alternative and competing gaseous substrate, the catalytic efficiency of RubisCO and the enzyme's ability to assimilate CO2 may be severely limited, with consequent environmental and agricultural effects. Recent genomic sequencing projects, however, have identified putative RubisCO genes from anoxic Archaea. In the present study, these potential RubisCO sequences, from Methanococcus jannaschii and Archaeoglobus fulgidus, were analyzed in order to ascertain whether such sequences might encode functional proteins. We also report the isolation and properties of recombinant RubisCO using sequences obtained from the obligately anaerobic hyperthermophilic methanogen M. jannaschii. This is the first description of an archaeal RubisCO sequence; this study also represents the initial characterization of a RubisCO molecule that has evolved in the absence of molecular oxygen. The enzyme was shown to be a homodimer whose deduced sequence, along with other recently obtained archaeal RubisCO sequences, differs substantially from those of known RubisCO molecules. The recombinant M. jannaschii enzyme has a somewhat low, but reasonable kcat, however, unlike previously isolated RubisCO molecules, this enzyme is very oxygen sensitive yet it is stable to hyperthermal temperatures and catalyzes the formation of the expected carboxylation product. Despite inhibition by oxygen, this unusual RubisCO still catalyzes a weak yet demonstrable oxygenase activity, with perhaps the lowest capacity for CO2/O2 discrimination ever encountered for any RubisCO.
| S-EPMC93548 | biostudies-literature
Project description:Abscisic acid ((+)-ABA) is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC), x-ray crystallography and in silico modelling to identify putative (+)-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+)-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP) substrate. Functionally, (+)-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM), but more potent inhibition of Rubisco activation (Ki of ~ 130 μM). Comparative structural analysis of Rubisco in the presence of (+)-ABA with RuBP in the active site revealed only a putative low occupancy (+)-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+)-ABA binding site in the RuBP binding pocket. Overall we conclude that (+)-ABA interacts with Rubisco. While the low occupancy (+)-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation.
| S-EPMC4510133 | biostudies-literature
Project description:Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the cornerstone of atmospheric CO2 fixation by the biosphere. It catalyzes the addition of CO2 onto enolized ribulose 1,5-bisphosphate (RuBP), producing 3-phosphoglycerate which is then converted to sugars. The major problem of this reaction is competitive O2 addition, which forms a phosphorylated product (2-phosphoglycolate) that must be recycled by a series of biochemical reactions (photorespiratory metabolism). However, the way the enzyme activates O2 is still unknown. Here, we used isotope effects (with 2H, 25Mg, and 18O) to monitor O2 activation and assess the influence of outer sphere atoms, in two Rubisco forms of contrasted O2/CO2 selectivity. Neither the Rubisco form nor the use of solvent D2O and deuterated RuBP changed the 16O/18O isotope effect of O2 addition, in clear contrast with the 12C/13C isotope effect of CO2 addition. Furthermore, substitution of light magnesium (24Mg) by heavy, nuclear magnetic 25Mg had no effect on O2 addition. Therefore, outer sphere protons have no influence on the reaction and direct radical chemistry (intersystem crossing with triplet O2) does not seem to be involved in O2 activation. Computations indicate that the reduction potential of enolized RuBP (near 0.49 V) is compatible with superoxide (O2 •-) production, must be insensitive to deuteration, and yields a predicted 16O/18O isotope effect and energy barrier close to observed values. Overall, O2 undergoes single electron transfer to form short-lived superoxide, which then recombines to form a peroxide intermediate.
| S-EPMC7533879 | biostudies-literature
Project description:The phylogenetic diversity of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, E.C. 4.1.1.39) large-subunit genes of deep-sea microorganisms was analyzed. Bulk genomic DNA was isolated from seven samples, including samples from the Mid-Atlantic Ridge and various deep-sea habitats around Japan. The kinds of samples were hydrothermal vent water and chimney fragment; reducing sediments from a bathyal seep, a hadal seep, and a presumed seep; and symbiont-bearing tissues of the vent mussel, Bathymodiolus sp., and the seep vestimentiferan tubeworm, Lamellibrachia sp. The RuBisCO genes that encode both form I and form II large subunits (cbbL and cbbM) were amplified by PCR from the seven deep-sea sample DNA populations, cloned, and sequenced. From each sample, 50 cbbL clones and 50 cbbM clones, if amplified, were recovered and sequenced to group them into operational taxonomic units (OTUs). A total of 29 OTUs were recorded from the 300 total cbbL clones, and a total of 24 OTUs were recorded from the 250 total cbbM clones. All the current OTUs have the characteristic RuBisCO amino acid motif sequences that exist in other RuBisCOs. The recorded OTUs were related to different RuBisCO groups of proteobacteria, cyanobacteria, and eukarya. The diversity of the RuBisCO genes may be correlated with certain characteristics of the microbial habitats. The RuBisCO sequences from the symbiont-bearing tissues showed a phylogenetic relationship with those from the ambient bacteria. Also, the RuBisCO sequences of known species of thiobacilli and those from widely distributed marine habitats were closely related to each other. This suggests that the Thiobacillus-related RuBisCO may be distributed globally and contribute to the primary production in the deep sea.
| S-EPMC92794 | biostudies-literature
Project description:Like many enzymes, the biogenesis of the multi-subunit CO(2)-fixing enzyme ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) in different organisms requires molecular chaperones. When expressed in Escherichia coli, the large (L) subunits of the Rubisco from the archaeabacterium Methanococcoides burtonii assemble into functional dimers (L(2)). However, further assembly into pentamers of L(2) (L(10)) occurs when expressed in tobacco chloroplasts or E. coli producing RuBP. In vitro analyses indicate that the sequential assembly of L(2) into L(10) (via detectable L(4) and L(6) intermediates) occurs without chaperone involvement and is stimulated by protein rearrangements associated with either the binding of substrate RuBP, the tight binding transition state analog carboxyarabinitol-1,5-bisphosphate, or inhibitory divalent metal ions within the active site. The catalytic properties of L(2) and L(10) M. burtonii Rubisco (MbR) were indistinguishable. At 25 degrees C they both shared a low specificity for CO(2) over O(2) (1.1 mol x mol(-1)) and RuBP carboxylation rates that were distinctively enhanced at low pH (approximately 4 s(-1) at pH 6, relative to 0.8 s(-1) at pH 8) with a temperature optimum of 55 degrees C. Like other archaeal Rubiscos, MbR also has a high O(2) affinity (K(m)(O(2)) = approximately 2.5 microM). The catalytic and structural similarities of MbR to other archaeal Rubiscos contrast with its closer sequence homology to bacterial L(2) Rubisco, complicating its classification within the Rubisco superfamily.
| S-EPMC2797158 | biostudies-literature
Project description:Transcriptional activity is often used as a surrogate for gene expression in environmental microbial communities. We developed a real-time PCR assay in which the ABI-Prism (PE Applied Biosystems) detection system is used for quantification of large-subunit ribulose-1,5-bisphosphate caboxylase/oxygenase (rbcL) mRNA in diatoms and pelagophytes both in cultures and from natural phytoplankton communities. Plasmid DNA containing rbcL inserts, as well as in vitro transcribed mRNA of the plasmids, was used to generate standard curves with a dynamic range of more than 6 orders of magnitude with high accuracy and precision (R(2) = 0.998). Expression levels in a cultured diatom (Phaeodactylum tricornutum) were quantified through one light-dark cycle by using traditional 35S-labeled oligonucleotide hybridization and real-time PCR. The mRNA levels detected by the two techniques were similar and correlated well (R(2) = 0.95; slope = 1.2). The quantities obtained by hybridization were slightly, yet significantly, larger (t = 5.29; P = 0.0011) than the quantities obtained by real-time PCR. This was most likely because partially degraded transcripts were not detected by real-time PCR. rbcL mRNA detection by real-time PCR was 3 orders of magnitude more sensitive than rbcL mRNA detection by hybridization. Diatom and pelagophyte rbcL mRNAs were also quantified in a profile from an oligotrophic site in the Gulf of Mexico. We detected the smallest amount of diatom rbcL expression in the surface water and maximum expression at a depth that coincided with the depth of the subsurface chlorophyll maximum. These results indicate that real-time PCR may be utilized for quantification of microbial gene expression in the environment.
| S-EPMC123995 | biostudies-literature
Project description:The ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) enzyme found in plants, algae, and an array of autotrophic bacteria is also encoded by a subset of methanotrophs, but its role in these microbes has largely remained elusive. In this study, we showed that CO2 was requisite for RubisCO-encoding Methylococcus capsulatus strain Bath growth in a bioreactor with continuous influent and effluent gas flow. RNA sequencing identified active transcription of several carboxylating enzymes, including key enzymes of the Calvin and serine cycles, that could mediate CO2 assimilation during cultivation with both CH4 and CO2 as carbon sources. Marker exchange mutagenesis of M. capsulatus Bath genes encoding key enzymes of potential CO2-assimilating metabolic pathways indicated that a complete serine cycle is not required, whereas RubisCO is essential for growth of this bacterium. 13CO2 tracer analysis showed that CH4 and CO2 enter overlapping anaplerotic pathways and implicated RubisCO as the primary enzyme mediating CO2 assimilation in M. capsulatus Bath. Notably, we quantified the relative abundance of 3-phosphoglycerate and ribulose-1,5-bisphosphate 13C isotopes, which supported that RubisCO-produced 3-phosphoglycerate is primarily converted to ribulose-1-5-bisphosphate via the oxidative pentose phosphate pathway in M. capsulatus Bath. Collectively, our data establish that RubisCO and CO2 play essential roles in M. capsulatus Bath metabolism. This study expands the known capacity of methanotrophs to fix CO2 via RubisCO, which may play a more pivotal role in the Earth's biogeochemical carbon cycling and greenhouse gas regulation than previously recognized. Further, M. capsulatus Bath and other CO2-assimilating methanotrophs represent excellent candidates for use in the bioconversion of biogas waste streams that consist of both CH4 and CO2. IMPORTANCE The importance of RubisCO and CO2 in M. capsulatus Bath metabolism is unclear. In this study, we demonstrated that both CO2 and RubisCO are essential for M. capsulatus Bath growth. 13CO2 tracing experiments supported that RubisCO mediates CO2 fixation and that a noncanonical Calvin cycle is active in this organism. Our study provides insights into the expanding knowledge of methanotroph metabolism and implicates dually CH4/CO2-utilizing bacteria as more important players in the biogeochemical carbon cycle than previously appreciated. In addition, M. capsulatus and other methanotrophs with CO2 assimilation capacity represent candidate organisms for the development of biotechnologies to mitigate the two most abundant greenhouse gases, CH4 and CO2.
| S-EPMC8388818 | biostudies-literature
Project description:Diel periodicity in the expression of key genes involved in carbon and nitrogen assimilation in marine Synechococcus spp. was investigated in a natural population growing in the surface waters of a cyclonic eddy in the northeast Atlantic Ocean. Synechococcus sp. cell concentrations within the upper mixed layer showed a net increase of three- to fourfold during the course of the experiment (13 to 22 July 1991), the population undergoing approximately one synchronous division per day. Consistent with the observed temporal pattern of phycoerythrin (CpeBA) biosynthesis, comparatively little variation was found in cpeBA mRNA abundance during either of the diel cycles investigated. In marked contrast, the relative abundance of transcripts originating from the genes encoding the large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL) and glutamine synthetase (glnA) showed considerable systematic temporal variation and oscillated during the course of each diel cycle in a reciprocal rhythm. Whereas activation of rbcL transcription was clearly not light dependent, expression of glnA appeared sensitive to endogenous changes in the physiological demands for nitrogen that arise as a natural consequence of temporal periodicity in photosynthetic carbon assimilation. The data presented support the hypothesis that a degree of temporal separation may exist between the most active periods of carbon and nitrogen assimilation in natural populations of marine Synecoccoccus spp.
| S-EPMC91547 | biostudies-literature
Project description:A pilot field experiment to assess the relationship between traditional biogeochemical rate measurements and transcriptional activity of microbial populations was carried out at the LEO 15 site off Tuckerton, N.J. Here, we report the relationship between photosynthetic capacity of autotrophic plankton and transcriptional activity of the large subunit gene (rbcL) for ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the enzyme responsible for primary carbon fixation during photosynthesis. Similar diel patterns of carbon fixation and rbcL gene expression were observed in three of four time series, with maxima for photosynthetic capacity (P(max)) and rbcL mRNA occurring between 10 a.m. and 1 p.m. The lowest P(max) and rbcL levels were detected between 6 p.m. and 10:30 p.m. A significant correlation was found between P(max) and form ID rbcL mRNA (R(2) = 0.56) and forms IA and IB (R(2) = 0.41 and 0.47, respectively). The correlation between the abundance of "diatom" rbcL and P(max) mRNA was modest (R(2) = 0.49; n = 12) but improved dramatically (R(2) = 0.97; n = 10) upon removal of two outliers which represented afternoon samples with high P(max) but lower mRNA levels. Clone libraries from reverse transcription-PCR-amplified rbcL mRNA indicated the presence of several chromophytic algae (diatoms, prymnesiophytes, and chrysophytes) and some eukaryotic green flagellates. Analogous results were obtained from amplified small rRNA sequences and secondary pigment analysis. These results suggest that diatoms were a major contributor to carbon fixation at LEO 15 at the time of sampling and that photosynthetic carbon fixation was partially controlled by transcriptional regulation of the RubisCO gene.
| S-EPMC520902 | biostudies-literature
Project description:The first x-ray crystal structure has been solved for an activated transition-state analog-bound form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This enzyme, from Rhodopseudomonas palustris, assembles as a unique hexamer with three pairs of catalytic large subunit homodimers around a central 3-fold symmetry axis. This oligomer arrangement is unique among all known Rubisco structures, including the form II homolog from Rhodospirillum rubrum. The presence of a transition-state analog in the active site locked the activated enzyme in a "closed" conformation and revealed the positions of critical active site residues during catalysis. Functional roles of two form II-specific residues (Ile(165) and Met(331)) near the active site were examined via site-directed mutagenesis. Substitutions at these residues affect function but not the ability of the enzyme to assemble. Random mutagenesis and suppressor selection in a Rubisco deletion strain of Rhodobacter capsulatus identified a residue in the amino terminus of one subunit (Ala(47)) that compensated for a negative change near the active site of a neighboring subunit. In addition, substitution of the native carboxyl-terminal sequence with the last few dissimilar residues from the related R. rubrum homolog increased the enzyme's kcat for carboxylation. However, replacement of a longer carboxyl-terminal sequence with termini from either a form III or a form I enzyme, which varied both in length and sequence, resulted in complete loss of function. From these studies, it is evident that a number of subtle interactions near the active site and the carboxyl terminus account for functional differences between the different forms of Rubiscos found in nature.
| S-EPMC4118107 | biostudies-literature