Synthesis and assembly of bacterial and higher plant Rubisco subunits in Escherichia coli

The synthesis in Escherichia coli of both the large and small subunits of cereal ribulose bisphosphate carboxylase/oxygenase has been obtained using expression plasmids and bacteriophages. The level and order of synthesis of the large and small subunits were regulated using different promoters, resulting in different subunit pool sizes and ratios that could be controlled in attempts to optimize the conditions for assembly. Neither assembly nor enzyme activity were observed for the higher plant enzyme. In contrast, cyanobacterial large and small subunits can assemble to give an active holoenzyme in Escherichia coli. By the use of deletion plasmids, followed by infection with appropriate phages, it can be demonstrated that the small subunit is essential for catalysis. However, the small subunit is not required for the assembly of a large subunit octomer core in the case of the Synechococcus enzyme; self-assembly of the octomer will occur in an rbcS deletion strain. The cyanobacterial small subunits can be replaced by wheat small subunits to give an active enzyme in Escherichia coli. The hybrid cyanobacterial large/wheat small subunit enzyme has only about 10% of the level of activity of the wild-type enzyme, reflecting the incomplete saturation of the small subunit binding sites on the large subunit octomer, and possibly a mismatch in the subunit interactions of those small subunits that do bind, giving rise to a lower rate of turnover at the active sites.Abbreviations IPTG isopropyl--D-thiogalactopyranoside - L large subunit - Rubisco ribulose bisphosphate carboxylase/oxygenase - S small subunit     

Structural, Functional, and Evolutionary Analysis of Ribulose-1,5-Bisphosphate Carboxylase from the Chromophytic Alga Olisthodiscus luteus

Ribulose-1,5-bisphosphate carboxylase (RuBPCase) was purified from the marine chromophyte Olisthodiscus luteus. This study represents the first extensive analysis of RuBPCase from a chromophytic plant species as well as from an organism where both subunits of the enzyme are encoded on the chloroplast genome. The size of the purified holoenzyme (17.9 Svedberg units, 588 kilodaltons) was determined by sedimentation analysis and the size of the subunits (55 kilodaltons, 15 kilodaltons) ascertained by analytical sodium dodecyl sulfate gel electrophoresis. This data predicts either an 8:9 or 8:8 ratio of the large to small subunits in the holoenzyme. Amino acid analyses demonstrate that the O. luteus RuBPCase large subunit is highly conserved and the small subunit much less so when compared with the chlorophytic plant peptides. The catalytic optima of pH and Mg²⁺ have been determined as well as the response of enzyme catalysis to temperature. The requirements of NaHCO₃ and Mg²⁺ for enzyme activation have also been analyzed. The Michaelis constants for the substrates of the carboxylation reaction (CO₂ and ribulose bisphosphate) were shown to be 45 and 48 micromolar, respectively. Competitive inhibition by oxygen of RuBPCase-catalyzed CO₂ fixation was also demonstrated. These data demonstrate that a high degree of RuBPCase conservation occurs among widely divergent photoautotrophs regardless of small subunit coding site.     

Transcription profiles of hydrogenases related genes in the cyanobacterium Lyngbya majuscula CCAP 1446/4

Background  

Lyngbya majuscula CCAP 1446/4 is a N₂-fixing filamentous nonheterocystous strain that contains two NiFe-hydrogenases: an uptake (encoded by hupSL) and a bidirectional enzyme (encoded by hoxEFUYH). The biosynthesis/maturation of NiFe-hydrogenases is a complex process requiring several accessory proteins for e.g. for the incorporation of metals and ligands in the active center (large subunit), and the insertion of the FeS clusters (small subunit). The last step in the maturation of the large subunit is the cleavage of a C-terminal peptide from its precursor by a specific endopeptidase. Subsequently, the mature large and small subunits can assemble forming a functional enzyme.     

Identification of membrane anchor polypeptides of Escherichia coli fumarate reductase

Fumarate reductase of Escherichia coli has been shown to be a membrane-bound enzyme composed of a 69,000-dalton catalytic-flavin-containing subunit and a 27,000-dalton nonheme-iron-containing subunit. Using gene cloning and amplification techniques, we have observed two additional polypeptides encoded by the frd operon, with apparent molecular weights of 15,000 and 14,000, which are expressed when E. coli is grown anaerobically on glycerol plus fumarate. Expression of these two small polypeptides is necessary for the two large subunits to associate with the membrane. The four subunits remain associated in Triton X-100 extracts of the membrane, and a holoenzyme form of fumarate reductase containing one copy of each of the four polypeptides has been isolated. Unlike the well-characterized two-subunit form, the holoenzyme is not dependent on anions for activity and is not labile at alkaline pH. In these respects, it more closely resembles the membrane-bound activity.     

A point mutation in the gene encoding the Rubisco large subunit interferes with holoenzyme assembly

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), a key enzyme of photosynthetic CO₂ fixation, is composed of 8 large and 8 small subunits. The Rubisco-deficient Nicotiana tabacum mutant Sp25 is able to synthesize the peptides for both subunits but does not contain any active holoenzyme. The phenotype is maternally inherited and thus caused by a mutation in the chloroplast genome, which also encodes the Rubisco large subunit. A comparison of the nucleotide sequences of the large subunit gene of the Sp25 mutant with that of the wild-type tobacco revealed a single nucleotide change in the Sp25 mutant. This resulted in an amino acid substitution at Gly-322, which was replaced by serine.     

Arg-7 mutant x wild-type crosses in Chlamydomonas reinhardi: Study of the enzyme produced in diploid strains

Summary The arg-7 locus is the structural gene for the argininosuccinate lyase (ASL). Interallelic complementation was previously found to occur between several mutants of the locus: this is indicative for the homomultimeric nature of ASL.Two complementing (arg-7-5 and arg-7-7) and two non-complementing (arg-7-1 and arg-7-6) mutants of the arg-7 locus were crossed to the pab-2 strain (which is wild-type for the arg-7 locus). In each cross, heterozygote phenotypically wild-type strains were isolated; their diploid pattern was demonstrated by various criteria: mating type, cell volume, nuclear size.The four heterozygotes were compared to the haploid wild-type and in some experiments, to the diploid strain arg-1xpab-2 homozygous for the arg-7 locus. No difference was found in growth rate and in the Michaelis constant values for ASL. The specific activity of the enzyme produced in the heterozygotes was about 50 percent of the activity found in haploid or diploid wild-type. The heat sensitivity of ASL was also investigated in the different strains: two (containing the complementing mutations arg-7-5 and arg-7-7) of the four heterozygotes produce ASL varieties different from the wild-type enzyme as far as the thermolability is concerned.These results suggest that hybrid ASL can be formed by interaction between the products of wild-type and mutant genes. A clear dominance of the wild-type allele is expected only when the mutant allele has no product of the gene: this could be the case for arg-7-1 and arg-7-6.     

Identification of nonsense mutations in Neurospora: Application to the complex arg-6 locus

Summary The arg-6 locus of Neurospora crassa encodes two enzymes of arginine synthesis, acetylglutamate kinase and acetylglutamyl phosphate reductase. Mutants lacking one or the other enzyme fall into two different complementation groups; a large non-complementing group lacks both enzymes. We wished to survey over 50 alleles for suppressibility by a nonsense suppressor. We compared two methods of assessing suppressibility. One, based on trans-action of a nonsense suppressor, was simple, but not efficient in detecting all suppressible alleles. The other, based on crosses involving a marker linked to the locus surveyed was very efficient in methodology and is suited to all cases, such as the arg-6 locus, in which allelic crosses are sterile. The data indicate that the arg-6 locus encodes a bifunctional protein.     

Assembly of NADH: ubiquinone reductase (complex I) in Neurospora mitochondria. Independent pathways of nuclear-encoded and mitochondrially encoded subunits

NADH:ubiquinone reductase, the respiratory chain complex I of mitochondria, consists of some 25 nuclear-encoded and seven mitochondrially encoded subunits, and contains as redox groups one FMN, probably one internal ubiquinone and at least four iron-sulphur clusters. We are studying the assembly of the enzyme in Neurospora crassa. The flux of radioactivity in cells that were pulse-labelled with [35S]methionine was followed through immunoprecipitable assembly intermediates into the holoenzyme. Labelled polypeptides were observed to accumulate transiently in a Mr 350,000 intermediate complex. This complex contains all mitochondrially encoded subunits of the enzyme as well as subunits encoded in the nucleus that have no homologous counterparts in a small, merely nuclear-encoded form of the NADH:ubiquinone reductase made by Neurospora crassa cells poisoned with chloramphenicol. With regard to their subunit compositions, the assembly intermediate and small NADH:ubiquinone reductase complement each other almost perfectly to give the subunit composition of the large complex I. These results suggest that two pathways exist in the assembly of complex I that independently lead to the preassembly of two major parts, which subsequently join to form the complex. One preassembled part is related to the small form of NADH:ubiquinone reductase and contributes most of the nuclear-encoded subunits, FMN, three iron-sulphur clusters and the site for the internal ubiquinone. The other part is the assembly intermediate and contributes all mitochondrially encoded subunits, one iron-sulphur cluster and the catalytic site for the substrate ubiquinone. We discuss the results with regard to the evolution of the electron pathway through complex I.     

Phosphorylation of small subunit plays a crucial role in the regulation of RuBPCase in moss and spinach

RuBPCase has been purified to electrophoretic homogeneity from moss and spinach. On denaturing SDS-polyacrylamide gels the purified enzyme revealed two discrete bands, thereby indicating the presence of large and small subunits. The phosphoprotein nature of RuBPCase was proved by in vivo labelling of enzyme with [³²P]orthophosphate. Autoradiographic analysis of ³²P-labelled RuBPCase on SDS-PAG demonstrated that phosphorylation was restricted to the small subunit. Dephosphorylation of purified RuBPCase with alkaline phosphatase resulted in a dramatic decline (70% decrease) in the biological activity of the enzyme. Fractionation of the dephosphorylated enzyme on denaturing gels revealed only the presence of large subunits of RuBPCase. Thus it became evident that dephosphorylation of RuBPCase brings about the dissociation of small subunits from the catalytic large subunits (octamer). The dephosphorylated small subunits were isolated as dimers. These results clearly indicate that phosphorylation of small subunits is mandatory for the reconstitution of holoenzyme and hence crucial for the activation of RuBPCase.     

Catalysis by cyanobacterial ribulose-bisphosphate carboxylase large subunits in the complete absence of small subunits

An expression plasmid incorporating the structural gene for the large subunit of a cyanobacterial ribulose-bisphosphate carboxylase, but not the gene for its complementary small subunit, directs the synthesis of large subunits in Escherichia coli. This provides a means for obtaining a preparation of large subunits completely devoid of small subunits, which is not otherwise achievable. In extracts, these large subunits were found predominantly in the form of octamers, but intersubunit interactions were weaker than in the holoenzyme, which contains eight small subunits as well as eight large subunits, and tended to be broken by procedures which separated octamers from lower oligomers and monomers. However, partial purification by anion-exchange chromatography was possible. The large subunits recognized the reaction-intermediate analog, 2'-carboxy-D-arabinitol 1,5-bisphosphate, thus enabling measurement of catalytic site concentrations, but the binding was much weaker than to the holoenzyme. E. coli-produced large subunits catalyzed carboxylation with a kcat of 1% of that of the holoenzyme and the substrate affinities were 3- to 5-fold weaker. They also assembled with heterologous small subunits isolated from spinach ribulose-P2 carboxylase with a 100-fold increase in catalytic activity under standard assay conditions. Since catalysis can proceed in their absence, the small subunits cannot be directly involved in the catalytic chemistry. Their stimulative influence upon catalysis must be exerted by conformational means.     

Questions about the complexity of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) has played a central role in our understanding of chloroplast biogenesis and photosynthesis. In particular, its catalysis of the rate-limiting step of CO₂ fixation, and the mutual competition of CO₂ and O₂ at the active site, makes Rubisco a prime focus for genetically engineering an increase in photosynthetic productivity. Although it remains difficult to manipulate the chloroplast-encoded large subunit and nuclear-encoded small subunit of crop plants, much has been learned about the structure/function relationships of Rubisco by expressing prokaryotic genes in Escherichia coli or by exploiting classical genetics and chloroplast transformation of the green alga Chlamydomonas reinhardtii. However, the complexity of chloroplast Rubisco in land plants cannot be completely addressed with the existing model organisms. Two subunits encoded in different genetic compartments have coevolved in the formation of the Rubisco holoenzyme, but the function of the small subunit remains largely unknown. The subunits are posttranslationally modified, assembled via a complex process, and degraded in regulated ways. There is also a second chloroplast protein, Rubisco activase, that is responsible for removing inhibitory molecules from the large-subunit active site. Many of these complex interactions and processes display species specificity. This means that attempts to engineer or discover a better Rubisco may be futile if one cannot transfer the better enzyme to a compatible host. We must frame the questions that address this problem of chloroplast-Rubisco complexity. We must work harder to find the answers.     

Subunit association in acetohydroxy acid synthase isozyme III.

Acetohydroxy acid synthase isozyme III (AHAS III) from Escherichia coli is composed of large and small subunits (encoded by the genes ilvI and ilvH) in an alpha 2 beta 2 structure. The large (61-kDa) subunit apparently contains the catalytic machinery of the enzyme, while the small (17-kDa) subunit is required for specific stabilization of the active conformation of the large subunit as well as for valine sensitivity. The interaction between subunits has been studied by using purified enzyme and extracts containing subcloned subunits. The association between large and small subunits is reversible, with a dissociation constant sufficiently high to have important experimental consequences: the activity of the enzyme shows a concentration dependence curve which is concave upward, and this dependence becomes linear upon the addition of excess large or small subunits. We estimate that at a concentration of 10(-7) M for each subunit (7 micrograms of enzyme ml-1), the large subunits are only half associated as the I2H2 active holoenzyme. This dissociation constant is high enough to cause underestimation of the activity of AHAS III in bacterial extracts. The true activity of this isozyme in extracts is observed in the presence of excess small subunits, which maintain the enzyme in its associated form. Reexamination of an E. coli K-12 ilvBN+ ilvIH+ strain grown in glucose indicates that AHAS III is the major isozyme expressed. As an excess of small subunits does not influence the apparent Ki for valine inhibition of the purified enzyme, it is likely that valine binds to and inhibits I2H2 rather than inducing dissociation. AHAS I and II seem to show a much lower tendency to dissociate than does AHAS III.     

The induction and characterization of recessive suppressors of arg-2 in Schizophyllum commune

Summary Genetic analyses have been made to detect recessive suppressor mutations in eight prototrophic strains derived by treating an arginine dependent strain with hydroxylamine. The results indicate that one strain possesses a recessive suppressor, su-1, which maps outside the arg-2 locus and is capable of suppressing auxotrophy conferred by the arg-2 mutation. This suppressor is incapable of suppressing auxotrophy conferred by eight other loci. Prototrophy in the remaining seven strains resulted from either intragenic suppression, reversion, or from a suppressor mutation that is closely linked to the arg-2 locus. The results of heterokaryotic allelic tests with the seven strains indicate that the mutation to prototrophy is recessive.     

Amino-terminal truncations of the ribulose-bisphosphate carboxylase small subunit influence catalysis and subunit interactions.

The first 20 residues at the amino terminus of the small subunit of spinach ribulose-1,5-bisphosphate carboxylase form an irregular arm that makes extensive contacts with the large subunit and also with another small subunit (S. Knight, I. Andersson, and C.-I. Brändén [1990] J Mol Biol 215: 113-160). The influence of these contacts on subunit binding and, indirectly, on catalysis was investigated by constructing truncations from the amino terminus of the small subunit of the highly homologous enzyme from Synechococcus PCC 6301 expressed in Escherichia coli. Removal of the first six residues (and thus the region of contact with a neighboring small subunit) affected neither the affinity with which the small subunits bound to the large subunits nor the catalytic properties of the assembled holoenzyme. Extending the truncation to include the first 12 residues (which encroaches into a highly conserved region that interacts with the large subunit) also did not weaken intersubunit binding appreciably, but it reduced the catalytic activity of the holoenzyme nearly 5-fold. Removal of an additional single residue (i.e. removal of a total of 13 residues) weakened intersubunit binding approximately 80-fold. Paradoxically, this partially restored catalytic activity to approximately 40% of that of the wild-type holoenzyme. None of these truncations materially affected the Km values for ribulose-1,5-bisphosphate or CO2. Removal of all 20 residues of the irregular arm (thereby deleting the conserved region of contact with large subunits) totally abolished the small subunit's ability to bind to large subunits to form a stable holoenzyme. However, this truncated small subunit was still synthesized by the E. coli cells. These data are interpreted in terms of the role of the amino-terminal arm of the small subunit in maintaining the structure of the holoenzyme.     

The two genes for the small subunit of RuBP Carboxylase/oxygenase are closely linked in Chlamydomonas reinhardtii

Summary Ribulose bisphosphate carboxylase-oxygenase (Rubisco) is a key enzyme in the photosynthetic fixation of CO₂ by the chloroplast. The synthesis of the enzyme is an example of the cooperation between the chloroplast and the nucleocytoplasmic compartments, as it is assembled from subunits encoded in the two respective genomes. I have used a synthetic oligonucleotide probe to isolate the nuclear Rubisco small subunit genes (rbcS) directly from a genomic library of Chlamydomonas reinhardtii DNA. They constitute only a small family: there are two rbcS genes, and an additional related sequence, in the C. reinhardtii genome. All three are clustered within 11kb at a single locus, and should thus be particularly well suited for genetic manipulation. The pattern of expression of rbcS RNA is dependent on the growth conditions.     

Studies on the functions of the RNA polymerase components by means of mutations

Summary It had been shown earlier, that RNA polymerase 13 S particles contain the large components with a molecular weight of about 3–10⁵ and small subunits with a molecular weight of 4·10⁴-1·10⁵. These polymerase components easily dissociate and reassociate with restoration of the enzyme activity.Both temperature-sensitive (tsX) and rifamycin-resistant (rif-r-I) mutations proved to affect the large polymerase component without changing the small subunits. These mutations were mapped at different, though closely linked, loci of metB-thi region of E. coli K12 chromosome. These results as well as certain literature data allow to conclude that the large RNA polymerase component consists of at least two polypeptides, one being altered by ts mutation, and the other—by rif-r mutation.The large polymerase component when separated from the small subunits retain the ability to bind to T2 phage DNA while the separate small subunits lack this property. Rifamycin does not affect RNA polymerase-T2 DNA binding while ts mutation leads to inability of the enzyme to form stable complexes with DNA. Therefore, it is likely that the polypeptide affected by ts mutation is responsible for the attachment of RNA polymerase to specific sites of DNA template. On the other hand, the small subunits as well as polypeptide of the large component, which determines RNA polymerase sensitivity to rifamycin, seem not to participate in the enzyme binding to DNA template. It is suggested, that the catalytic site of RNA polymerase is located in the large component and formed by rifamycin-binding polypeptide. The small subunits are supposed to have regulatory function and activate the large components.     

Polymeric structure of the cyclic AMP-dependent protein kinase from the dimorphic fungus Mucor rouxii and purification of its catalytic subunit

Summary The polymeric structure of the cyclic AMP-dependent protein kinase (E.C.2.7.1.37) from the dimorphic fungus Mucor rouxii was analyzed through studies of gel filtration and sucrose gradient centrifugation of the holoenzyme and its subunits and by photoaffinity labeling of the regulatory subunit. It was demonstrated that it is a tetramer composed by two regulatory subunits (R) of mol. wt. 75 000 and two catalytic subunits (C) of mol. wt. 41 000 forming a holoenzyme R₂C₂ of mol. wt. 242 000. Frictional coefficients of 1.55 and 1.62 for the holoenzyme and for the regulatory dimer, respectively, indicate a significant degree of dimensional asymmetry in both molecules. A procedure for the purification of the catalytic subunit of the kinase is presented. The holoenzyme could be bound to a cyclic AMP-agarose column and the catalytic subunit could be eluted by 0.5 M NaCl, well resolved from the bulk of protein. This particular behaviour of the holoenzyme in cyclic AMP-agarose chromatography allowed the inclusion of this step in the purification of the catalytic subunit and corroborated that the holoenzyme was not dissociated by cyclic AMP alone. The isolated catalytic subunit displays Michaelis-Menten behaviour towards kemptide, protamine and histone and is inhibited by sulfhydryl reagents, indicating that the molecule has at least one cysteine residue essential for enzyme activity. The catalytic activity of the isolated C subunit is inactivated by the mammalian protein kinase inhibitor, and is inhibited by the regulatory subunit from homologous and heterologous sources. In general, the properties of the catalytic subunit suggest a structural similarity between Mucor and mammalian C subunits.Abbreviations C catalytic subunit monomer of protein kinase - R regulatory subunit monomer of protein kinase - 8-N₃-cyclic AMP 8-azido-cylic AMP - SDS sodium dodecyl sulfate - Pipes piperazine-N,N-bis(2-ethanesulfonic acid) See AcknowledgementsCareer Investigators from the CONICET     

Subunits polypeptide composition of Rubisco in Avena species

Abstract

Rubisco was extracted and purified from leaves of nineteen Avena species. The holoenzyme shows the already known “slow” and “fast” forms after native electrophoresis. The electrophoresis in denaturing conditions revealed a unique polypeptide for the large subunit and two types of polypeptides for the small subunit. The addition of leupeptin, a thiol proteinase inhibitor, during purification of the holoenzyme, reduced microheterogeneity within both subunit types. Subunit composition of the enzyme appears to be a taxonomic tool for distinguishing three species: A. clauda Dur., A. eriantha Dur. and A. canariensis Baum. Rajh. et Baum.     

Imported large subunits of ribulose bisphosphate carboxylase/oxygenase, but not imported beta-ATP synthase subunits, are assembled into holoenzyme in isolated chloroplasts

The large subunit of ribulose bisphosphate carboxylase from Anacystis nidulans 6301, and the β subunit of chloroplast ATP synthase from maize, were fused to the transit peptide of the small subunit of ribulose bisphosphate carboxylase from soybean. These proteins were assayed for post-translational import into isolated pea chloroplasts. Both proteins were imported into chloroplasts. Imported large subunits were associated with two distinct macromolecular structures. The smaller of these structures was a hybrid ribulose bisphosphate carboxylase holoenzyme, and the larger was the binding protein oligomer. Time-course experiments following import of the large subunit revealed that the amount of large subunit associated with the binding protein oligomer decreased over time, and that the amount of large subunit present in the assembled holoenzyme increased. We also observed that imported small subunits of ribulose bisphosphate carboxylase, although predominantly present in the holoenzyme, were also found associated with the binding protein oligomer. In contrast, the imported β subunit of chloroplast ATP synthase did not assemble into a thylakoid-bound coupling factor complex.