Preparative-scale enzymic synthesis of D-[14C]ribulose 1,5-bisphosphate.
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ABSTRACT:
SUBMITTER: Kuehn GD
PROVIDER: S-EPMC1186152 | biostudies-other | 1978 Dec
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:The first structure of a ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from a pulse crop is reported. Rubisco was purified from Pisum sativum (garden pea) and diffraction-quality crystals were obtained by hanging-drop vapour diffusion in the presence of the substrate ribulose 1,5-bisphosphate. X-ray diffraction data were recorded to 2.20?Å resolution from a single crystal at the Canadian Light Source. The overall quaternary structure of non-activated P. sativum Rubisco highlights the conservation of the form I Rubisco hexadecameric complex. The electron density places the substrate in the active site at the interface of the large-subunit dimers. Lys201 in the active site is not carbamylated as expected for this non-activated structure. Some heterogeneity in the small-subunit sequence is noted, as well as possible variations in the conformation and contacts of ribulose 1,5-bisphosphate in the large-subunit active sites. Overall, the active-site conformation most closely correlates with the `closed' conformation observed in other substrate/inhibitor-bound Rubisco structures.
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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 ?M). 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.
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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.
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Project description:During the last years we have witnessed progressive evolution of preparation of acenes with length up to dodecacene by on-surface synthesis in ultra-high vacuum or generation of acenes up to decacene in solid matrices at low temperatures. While these protocols with very specific conditions produce the acenes in amount of few molecules, the strategies leading to the acenes in large quantities dawdle behind. Only recently and after 70 years of synthetic attempts, heptacene has been prepared in bulk phase. However, the preparative scale synthesis of higher homologues still remains a formidable challenge. Here we report the preparation and characterisation of nonacene and show its excellent thermal and in-time stability.
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Project description:The CO2-concentrating mechanism (CCM) has evolved to improve the efficiency of photosynthesis in autotrophic cyanobacteria. CmpR, a LysR-type transcriptional regulator (LTTR) from Synechococcus elongatus PCC 7942, was found to regulate CCM-related genes under low-CO2 conditions. Here, the dimeric structure of the effector-binding domain of CmpR (CmpR-EBD) in complex with the co-activator ribulose 1,5-bisphosphate (RuBP) is reported at 2.15 Å resolution. One RuBP molecule binds to the inter-domain cleft between the two subunits of the CmpR-EBD dimer. Structural comparison combined with sequence analyses demonstrated that CmpR-EBD has an overall structure similar to those of LTTRs of known structure, but possesses a distinctly different effector-binding pattern.
| S-EPMC6096476 | 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.
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