A kinetic study of ribulose bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum.
(1/1070)
The activation kinetics of purified Rhodospirillum rubrum ribulose bisphosphate carboxylase were analysed. The equilibrium constant for activation by CO(2) was 600 micron and that for activation by Mg2+ was 90 micron, and the second-order activation constant for the reaction of CO(2) with inactive enzyme (k+1) was 0.25 X 10(-3)min-1 . micron-1. The latter value was considerably lower than the k+1 for higher-plant enzyme (7 X 10(-3)-10 X 10(-3)min-1 . micron-1). 6-Phosphogluconate had little effect on the active enzyme, and increased the extent of activation of inactive enzyme. Ribulose bisphosphate also increased the extent of activation and did not inhibit the rate of activation. This effect might have been mediated through a reaction product, 2-phosphoglycolic acid, which also stimulated the extent of activation of the enzyme. The active enzyme had a Km (CO2) of 300 micron-CO2, a Km (ribulose bisphosphate) of 11--18 micron-ribulose bisphosphate and a Vmax. of up to 3 mumol/min per mg of protein. These data are discussed in relation to the proposed model for activation and catalysis of ribulose bisphosphate carboxylase. (+info)
The localisation of 2-carboxy-D-arabinitol 1-phosphate and inhibition of Rubisco in leaves of Phaseolus vulgaris L.
(2/1070)
A recent controversial report suggests that the nocturnal inhibitor of Rubisco, 2-carboxy-D-arabinitol 1-phosphate (CAIP), does not bind to Rubisco in vivo and therefore that CA1P has no physiological relevance to photosynthetic regulation. It is now proved that a direct rapid assay can be used to distinguish between Rubisco-bound and free CA1P, as postulated in the controversial report. Application of this direct assay demonstrates that CA1P is bound to Rubisco in vivo in dark-adapted leaves. Furthermore, CA1P is shown to be in the chloroplasts of mesophyll cells. Thus, CA1P does play a physiological role in the regulation of Rubisco. (+info)
Unusual ribulose 1,5-bisphosphate carboxylase/oxygenase of anoxic Archaea.
(3/1070)
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. (+info)
Subfamily divergence in the multigene family of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS) in Triticeae and its relatives.
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To investigate genetic mechanisms acting on multigene family in plants, we analyzed sequence variation in the rbcS gene of 13 species of Triticeae and one species each of related tribes (Bromeae and Aveneae). A total of 36 rbcS genes were analyzed. Based on dimorphism in the length of intron, the rbcSs of investigated species were classified into two subfamilies A and B. The difference in intron length was caused by an indel of about 200 bp in the middle of the intron. The two subfamilies of rbcS were present in the three tribes, indicating that the divergence of rbcS subfamilies occurred before the split of these tribes. Generally, variation between the two subfamilies of rbcS was larger than that within subfamily, but these two measures were about the same at the tribe level. This result suggested that divergence of the subfamilies of rbcS occurred at about the same time of tribe diversification. The level of nucleotide variation in the exon region between subfamilies was reduced in the Triticeae, but clear change was not detected in the intron sequence. This result suggested that the exon sequences between subfamilies of rbcS were homogenized without affecting the intron sequence in the Triticeae lineage. (+info)
The stromal protein large subunit of ribulose-1,5-bisphosphate carboxylase is translated by membrane-bound ribosomes.
(5/1070)
Translation of the large subunit of ribulose-1,5-bisphosphate carboxylase (LSU) was investigated by labeling of isolated barley plastids with [35S]-methionine. In both chloroplasts and etioplasts, labeling of LSU was severely impaired if plastid membranes were removed from the reaction mixtures. Removal of membrane-bound polysomes with high salt or puromycin greatly decreased translation of LSU. Pulse-labeled chloroplast membranes were shown to release LSU if chased with unlabeled methionine in the presence of stroma. Immunoprecipitation detected higher amounts of labeled LSU translation intermediates associated with the membrane fraction than in the soluble fraction. We therefore conclude that, in plastids, membrane-bound polysomes are required not only for translation of membrane-intrinsic proteins but also for translation of a soluble protein. (+info)
Chaperonin function: folding by forced unfolding.
(6/1070)
The ability of the GroEL chaperonin to unfold a protein trapped in a misfolded condition was detected and studied by hydrogen exchange. The GroEL-induced unfolding of its substrate protein is only partial, requires the complete chaperonin system, and is accomplished within the 13 seconds required for a single system turnover. The binding of nucleoside triphosphate provides the energy for a single unfolding event; multiple turnovers require adenosine triphosphate hydrolysis. The substrate protein is released on each turnover even if it has not yet refolded to the native state. These results suggest that GroEL helps partly folded but blocked proteins to fold by causing them first to partially unfold. The structure of GroEL seems well suited to generate the nonspecific mechanical stretching force required for forceful protein unfolding. (+info)
Thiomicrospira kuenenii sp. nov. and Thiomicrospira frisia sp. nov., two mesophilic obligately chemolithoautotrophic sulfur-oxidizing bacteria isolated from an intertidal mud flat.
(7/1070)
Two new members of the genus Thiomicrospira were isolated from an intertidal mud flat sample with thiosulfate as the electron donor and CO2 as carbon source. On the basis of differences in genotypic and phenotypic characteristics, it is proposed that strain JB-A1T (= DSM 12350T) and strain JB-A2T (= DSM 12351T) are members of two new species, Thiomicrospira kuenenii and Thiomicrospira frisia, respectively. The cells were Gram-negative vibrios or slightly bent rods. Strain JB-A1T was highly motile, whereas strain JB-A2T showed a much lower degree of motility combined with a strong tendency to form aggregates. Both organisms were obligately autotrophic and strictly aerobic. Nitrate was not used as electron acceptor. Chemolithoautotrophic growth was observed with thiosulfate, tetrathionate, sulfur and sulfide. Neither isolate was able to grow heterotrophically. For strain JB-A1T, growth was observed between pH values of 4.0 and 7.5 with an optimum at pH 6.0, whereas for strain JB-A2T, growth was observed between pH 4.2 and 8.5 with an optimum at pH 6.5. The temperature limits for growth were between 3.5 and 42 degrees C and 3.5 and 39 degrees C, respectively. The optimum growth temperature for strain JB-A1T was between 29 and 33.5 degrees C, whereas strain JB-A2T showed optimal growth between 32 and 35 degrees C. The mean maximum growth rate on thiosulfate was 0.35 h-1 for strain JB-A1T and 0.45 h-1 for strain JB-A2T. (+info)
Thiomicrospira chilensis sp. nov., a mesophilic obligately chemolithoautotrophic sulfuroxidizing bacterium isolated from a Thioploca mat.
(8/1070)
A new member of the genus Thiomicrospira, which utilizes thiosulfate as the electron donor and CO2 as the carbon source, was isolated from a sediment sample dominated by the filamentous sulfur bacterium Thioploca. Although the physiological properties investigated are nearly identical to other described species of the genus, it is proposed that strain Ch-1T is a member of a new species, Thiomicrospira chilensis sp. nov., on the basis of differences in genotypic characteristics (16S rRNA sequence, DNA homology, G + C content). Strain Ch-1T was highly motile with a slight tendency to form aggregates in the stationary growth phase. The organism was obligately autotrophic and strictly aerobic. Nitrate was not used as an electron acceptor. Chemolithoautotrophic growth was observed with thiosulfate, tetrathionate, sulfur and sulfide. The isolate was not able to grow heterotrophically. Growth of strain Ch-1T was observed between pH 5.3 and 8.5 with an optimum at pH 7.0. The temperature range for growth was between 3.5 and 42 degrees C; the optimal growth temperature was between 32 and 37 degrees C. The mean maximum growth rate on thiosulfate was 0.4 h-1. This is the second Thiomicrospira species described that has a rod-shaped morphology; therefore discrimination between vibrio-shaped Thiomicrospira and rod-shaped Thiobacilli is no longer valid. (+info)