Maximum activity of recombinant ribulose 1,5-bisphosphate carboxylase/oxygenase of Anabaena sp. strain CA requires the product of the rbcX gene

Abstract

Filamentous cyanobacteria of the genus Anabaena contain a unique open reading frame, rbcX, which is juxtaposed and cotranscribed with the genes (rbcL and rbcS) encoding form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Plasmid constructions containing the genes from Anabaena sp. strain CA were prepared, and expression studies in Escherichia coli indicated that the product of the rbcX gene mimicked the ability of chaperonin proteins to facilitate the proper folding of recombinant RubisCO proteins. The purified recombinant Anabaena sp. strain CA RubisCO, much like the RubisCO enzymes from other cyanobacteria, was shown not to undergo inhibition of activity during a time course experiment, and the properties of this chaperoned recombinant protein appear to be consistent with those of the enzyme isolated from the native organism.Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) is an important catalyst which serves as the cornerstone enzyme of the Calvin-Benson-Bassham reductive pentose phosphate pathway of carbon dioxide assimilation. Two major structural forms of this enzyme, form I and form II, have been described, the catalytic (large) subunits of which are only slightly related (25). Unlike the form II enzyme, the form I RubisCO is also comprised of a second polypeptide, the small subunit. In cyanobacteria, the large and small subunits are encoded by the rbcL and rbcS genes, respectively, while in proteobacteria, these proteins are encoded by the cbbL and cbbS genes, which often are found associated with many additional structural and regulatory genes of the Calvin-BensonBassham pathway (8). Recently it was found that the genes in and around the rbcL and rbcS genes of three species of Anabaena are organized differently from the corresponding genes in other cyanobacteria (15,16). Specifically, a gene encoding RubisCO activase (rca) is located downstream from rbcS (16), with another open reading frame, rbcX, juxtaposed and cotranscribed with the rbcL and rbcS genes of Anabaena sp. strain PCC 7120 (12) as well as Anabaena variabilis and Anabaena sp. strain CA (16). Transcription of the rbcLXS operon is differentially regulated relative to rca expression in Anabaena sp. strain CA (16), with rca transcription preceding the appearance of rbc message after a dark-to-light shift. These studies suggest that RubisCO activase might play an important physiological role. Indeed, since various phosphorylated intermediates often accumulate in cyanobacteria in the dark, by analogy to eukaryotic systems (20), the early transcription of rca might signify the need of the organism to use RubisCO activase to catalyze the removal of such metabolic inhibitors (23) from RubisCO. Inasmuch as it is difficult to obtain from recombinant and native sources, large amounts of RubisCO required to examine the role of RubisCO activase in mediating the catalytic properties of Anabaena RubisCO (2, 12, 23), elucidation of the factors which affect the recovery of active recombinant enzyme is crucial. Previous difficul...

Global Gene Expression Profiles of the Cyanobacterium Synechocystis sp. Strain PCC 6803 in Response to Irradiation with UV-B and White Light

“…The transcription of ribulose bisphosphate carboxylase-oxygenase (Rubisco) genes (rbcL and rbcS) was synchronized with the level of photosynthetic activity: rbcL, rbcS, and rbcX were four-to fivefold down-regulated after 2 h of intense UV-B exposure. RbcX acts analogously to the GroEL/GroES chaperone proteins for Rubisco folding (21). An inorganic carbon-concentrating mechanism in cyanobacteria allows the cells to adapt to a wide range of CO 2 concentrations in the environment via a light-dependent process (17).…”

Structure-Function Studies with the Unique Hexameric Form II Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) from Rhodopseudomonas palustris

“…[1-3 H]RuBP used in these assays was synthesized and purified as described elsewhere (30). Rates of Rubisco activation were measured with unactivated Rubisco enzymes using a modified procedure adapted from elsewhere (31). Purified enzymes (500 -2500 g) were unactivated by four to five buffer exchanges against a 100 mM HEPES-NaOH buffer, pH 8.0 using 0.5-ml Amicon Ultracel filters (50-kDa molecular mass cutoff; Millipore).…”

“…The rapid reactivation of the wild-type enzyme indicates weak inactivation by RuBP binding. Unlike eukaryotic and cyanobacterial Rubisco enzymes, which are known to interact with accessory proteins to relieve sugar phosphate inhibition in vivo (31,51), no such regulatory proteins have been identified in R. palustris or R. capsulatus. It might be tentatively concluded that inactivation of the wild-type enzyme by RuBP (and potentially other sugar phosphates) is probably too weak to have a potential regulatory role in vivo.…”

Assessing host specialization in symbiotic cyanobacteria associated with four closely related species of the lichen fungusPeltigera

“…The 16S rDNA, trnL (a group I intron found in a conserved position of tRNA Leu (UAA) gene across the cyanobacteria, including plant chloroplasts; Kuhsel et al, 1990) and rbcLX region (which includes the last 82 amino acids of the RUBISCO large subunit (rbcL), a putative chaperone gene (rbcX) and two intergenic spacers; Li & Tabita, 1997) were amplified using published primers (Wilmotte et al, 1993;Nu¨bel et al, 1997;Rudi et al, 1998;Turner et al, 1999). Each 25-ml PCR reaction consisted of: 25 mg BSA, 0.625 U Taq DNA polymerase (Abgene, Rochester, NY, USA), 1.5 mM MgCl 2 , dNTPs (0.2 mM each), primers (0.5 mM each) and PCR buffer.…”