Reductive Pentose Phosphate Pathway Research Articles

Non-equivalence of Hydrogen Transfer from Glucose to the pro-R and pro-S Methylene Positions of Ethanol during Fermentation by Leuconostoc mesenteroides Quantified by 2H NMR at Natural Abundance

The anaerobic fermentation of glucose by Leuconostoc mesenteroides via the reductive pentose phosphate pathway leads to the accumulation of lactic acid and ethanol. The isotope redistribution coefficients (a(ij)) that characterize the specific derivation of each hydrogen atom in ethanol in relation to the non-exchangeable hydrogen atoms in glucose and the medium water have been determined using quantitative (2)H NMR. First, it is confirmed that the hydrogens of the methylene group are related only to the 1 and 3 positions of glucose via the NAD(P)H pool and not to the 4 position, in contrast to ethanol produced by Saccharomyces cerevisiae. Second, it is found that the conversion factors (C(f)) for the transfer of hydrogen to the pro-S and pro-R positions of the methylene group are not equivalent: the C(f)-1-R:C(f)-1-S ratio is 2.1, whereas the C(f)-3-R:C(f)-3-S ratio is 0.8. It is shown that this non-equivalence is not determined by the stereochemistry of the terminal NADH- and NADPH-dependent alcohol dehydrogenases, but is dependent on the cofactor selectivities of the reductive and oxidative steps of the reduced nucleotide cycle.

Multiple Rubisco forms in proteobacteria: their functional significance in relation to CO2 acquisition by the CBB cycle

Rubisco is the predominant enzymatic mechanism in the biosphere by which autotrophic bacteria, algae, and terrestrial plants fix CO(2) into organic biomass via the Calvin-Benson-Basham reductive pentose phosphate pathway. Rubisco is not a perfect catalyst, suffering from low turnover rates, a low affinity for its CO(2) substrate, and a competitive inhibition by O(2) as an alternative substrate. As a consequence of changing environmental conditions over the past 3.5 billion years, with decreasing CO(2) and increasing O(2) in the atmosphere, Rubisco has evolved into multiple enzymatic forms with a range of kinetic properties, as well as co-evolving with CO(2)-concentrating mechanisms to cope with the different environmental contexts in which it must operate. The most dramatic evidence of this is the occurrence of multiple forms of Rubisco within autotrophic proteobacteria, where Forms II, IC, IBc, IAc, and IAq can be found either singly or in multiple combinations within a particular bacterial genome. Over the past few years there has been increasing availability of genomic sequence data for bacteria and this has allowed us to gain more extensive insights into the functional significance of this diversification. This paper is focused on summarizing what is known about the diversity of Rubisco forms, their kinetic properties, development of bacterial CO(2)-concentrating mechanisms, and correlations with metabolic flexibility and inorganic carbon environments in which proteobacteria perform various types of obligate and facultative chemo- and photoautotrophic CO(2) fixation.

Alkalilimnicola ehrlichii sp. nov., a novel, arsenite-oxidizing haloalkaliphilic gammaproteobacterium capable of chemoautotrophic or heterotrophic growth with nitrate or oxygen as the electron acceptor

A facultative chemoautotrophic bacterium, strain MLHE-1(T), was isolated from Mono Lake, an alkaline hypersaline soda lake in California, USA. Cells of strain MLHE-1(T) were Gram-negative, short motile rods that grew with inorganic electron donors (arsenite, hydrogen, sulfide or thiosulfate) coupled with the reduction of nitrate to nitrite. No aerobic growth was attained with arsenite or sulfide, but hydrogen sustained both aerobic and anaerobic growth. No growth occurred when nitrite or nitrous oxide was substituted for nitrate. Heterotrophic growth was observed under aerobic and anaerobic (nitrate) conditions. Cells of strain MLHE-1(T) could oxidize but not grow on CO, while CH(4) neither supported growth nor was it oxidized. When grown chemoautotrophically, strain MLHE-1(T) assimilated inorganic carbon via the Calvin-Benson-Bassham reductive pentose phosphate pathway, with the activity of ribulose 1,5-bisphosphate carboxylase (RuBisCO) functioning optimally at 0.1 M NaCl and at pH 7.3. Strain MLHE-1(T) grew over broad ranges of pH (7.3-10.0; optimum, 9.3), salinity (15-190 g l(-1); optimum 30 g l(-1)) and temperature (13-40 degrees C; optimum, 30 degrees C). Phylogenetic analysis of 16S rRNA gene sequences placed strain MLHE-1(T) in the class Gammaproteobacteria (family Ectothiorhodospiraceae) and most closely related to Alkalispirillum mobile (98.5 %) and Alkalilimnicola halodurans (98.6 %), although none of these three haloalkaliphilic micro-organisms were capable of photoautotrophic growth and only strain MLHE-1(T) was able to oxidize As(III). On the basis of physiological characteristics and DNA-DNA hybridization data, it is suggested that strain MLHE-1(T) represents a novel species within the genus Alkalilimnicola for which the name Alkalilimnicola ehrlichii is proposed. The type strain is MLHE-1(T) (=DSM 17681(T)=ATCC BAA-1101(T)). Aspects of the annotated full genome of Alkalilimnicola ehrlichii are discussed in the light of its physiology.

Molecular Architecture of the Glucose 1-Phosphate Site in ADP-glucose Pyrophosphorylases

ADP-Glc pyrophosphorylase (PPase), a key regulatory enzyme in the biosynthetic pathway of starch and bacterial glycogen, catalyzes the synthesis of ADP-Glc from Glc-1-P and ATP. A homology model of the three-dimensional structure of the Escherichia coli enzyme complexed with ADP-Glc has been generated to study the substrate-binding site in detail. A set of amino acids in the model has been identified to be in close proximity to the glucose moiety of the ADP-Glc ligand. The role of these amino acids (Glu(194), Ser(212), Tyr(216), Asp(239), Phe(240), Trp(274), and Asp(276)) was studied by site-directed mutagenesis through the characterization of the kinetic properties and thermal stability of the designed mutants. All purified alanine mutants had 1 or 2 orders of magnitude lower apparent affinity for Glc-1-P compared with the wild type, indicating that the selected set of amino acids plays an important role in their interaction with the substrate. These amino acids, which are conserved within the ADP-Glc PPase family, were replaced with other residues to investigate the effect of size, hydrophobicity, polarity, aromaticity, or charge on the affinity for Glc-1-P. In this study, the architecture of the Glc-1-P-binding site is characterized. The model overlaps with the Glc-1-P site of other PPases such as Pseudomonas aeruginosa dTDP-Glc PPase and Salmonella typhi CDP-Glc PPase. Therefore, the data reported here may have implications for other members of the nucleotide-diphosphoglucose PPase family.

The Potential Role of Thiamine (Vitamin B1) in Diabetic Complications

Accumulation of triosephosphates arising from high cytosolic glucose concentrations in hyperglycemia is one likely or potential trigger for biochemical dysfunction leading to the development of diabetic complications. This may be prevented by disposal of excess triosephosphates via the reductive pentosephosphate pathway. This pathway is impaired in experimental and clinical diabetes by mild thiamine deficiency. The expression and activity of the thiamine-dependent enzyme, transketolase--the pacemaking enzyme of the reductive pentosephosphate pathway, is consequently decreased. Correction of thiamine deficiency in experimental diabetes by high dose therapy with thiamine and the thiamine monophosphate prodrug, Benfotiamine, restores disposal of triosephosphates by the reductive pentosephosphate pathway in hyperglycemia. This prevented multiple mechanisms of biochemical dysfunction: activation of protein kinase C, activation of the hexosamine pathway, increased glycation and oxidative stress. Consequently, the development of incipient diabetic nephropathy, neuropathy and retinopathy were prevented. Both thiamine and Benfotiamine produced other remarkable effects in experimental diabetes: marked reversals of increased diuresis and glucosuria without change in glycemic status. High dose thiamine also corrected dyslipidemia in experimental diabetes--normalizing cholesterol and triglycerides. Dysfunction of beta-cells and impaired glucose tolerance in thiamine deficiency and suggestion of a link of impaired glucose tolerance with dietary thiamine indicates that thiamine therapy may have a future role in prevention of type 2 diabetes. More immediately, given the emerging multiple benefits of thiamine repletion, even mild thiamine deficiency in diabetes should be avoided and thiamine supplementation to high dose should be considered as adjunct nutritional therapy to prevent dyslipidemia and the development of vascular complications in clinical diabetes.

Diatom plastids possess a phosphoribulokinase with an altered regulation and no oxidative pentose phosphate pathway.

The chloroplast enzyme phosphoribulokinase (PRK; EC 2.7.1.19) is part of the Calvin cycle (reductive pentose phosphate pathway) responsible for CO(2) fixation in photosynthetic organisms. In green algae and vascular plants, this enzyme is light regulated via reversible reduction by reduced thioredoxin. We have sequenced and characterized the gene of the PRK from the marine diatom Odontella sinensis and found that the enzyme has the conserved cysteine residues necessary for thioredoxin-dependent regulation. Analysis of enzymatic activity of partially purified diatom enzyme and of purified protein obtained by native overexpression in Escherichia coli, however, revealed that under natural redox conditions the diatom enzyme is generally active. Treatment of the enzyme with strong oxidants results in inhibition of the enzyme, which is reversible by subsequent incubation with reducing agents. We determined the redox midpoint potentials of the regulatory cysteine in the PRK from O. sinensis in comparison to the respective spinach (Spinacia oleracea) enzyme and found a more positive redox potential for the diatom PRK, indicating that in vivo this enzyme might not be regulated by thioredoxin. We also demonstrate that in protease-treated diatom plastids, activities of enzymes of the oxidative pentose phosphate pathway are not detectable, thus reducing the need for a tight regulation of the Calvin cycle in diatoms. We discuss our results in the context of rearrangements of the subcellular compartmentation of metabolic pathways due to the peculiar evolution of diatoms by secondary endocytobiosis.

Effector-Mediated Interaction of CbbR I and CbbR II Regulators with Target Sequences in Rhodobacter capsulatus

In Rhodobacter capsulatus, genes encoding enzymes of the Calvin-Benson-Bassham reductive pentose phosphate pathway are located in the cbb(I) and cbb(II) operons. Each operon contains a divergently transcribed LysR-type transcriptional activator (CbbR(I) and CbbR(II)) that regulates the expression of its cognate cbb promoter in response to an as yet unidentified effector molecule(s). Both CbbR(I) and CbbR(II) were purified, and the ability of a variety of potential effector molecules to induce changes in their DNA binding properties at their target promoters was assessed. The responses of CbbR(I) and CbbR(II) to potential effectors were not identical. In gel mobility shift assays, the affinity of both CbbR(I) and CbbR(II) for their target promoters was enhanced in the presence of ribulose-1,5-bisphosphate (RuBP), phosphoenolpyruvate, 3-phosphoglycerate, 2-phosphoglycolate. ATP, 2-phosphoglycerate, and KH(2)PO(4) were found to enhance only CbbR(I) binding, while fructose-1,6-bisphosphate enhanced the binding of only CbbR(II). The DNase I footprint of CbbR(I) was reduced in the presence of RuBP, while reductions in the CbbR(II) DNase I footprint were induced by fructose-1,6-bisphosphate, 3-phosphoglycerate, and KH(2)PO(4). The current in vitro results plus recent in vivo studies suggest that CbbR-mediated regulation of cbb transcription is controlled by multiple metabolic signals in R. capsulatus. This control reflects not only intracellular levels of Calvin-Benson-Bassham cycle metabolic intermediates but also the fixed (organic) carbon status and energy charge of the cell.

Modified pathway to synthesize ribulose 1,5-bisphosphate in methanogenic archaea.

Several sequencing projects unexpectedly uncovered the presence of genes that encode ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) in anaerobic archaea. RubisCO is the key enzyme of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway, a scheme that does not appear to contribute greatly, if at all, to net CO2 assimilation in these organisms. Recombinant forms of the archaeal enzymes do, however, catalyze a bona fide RuBP-dependent CO2 fixation reaction, and it was recently shown that Methanocaldococcus (Methanococcus) jannaschii and other anaerobic archaea synthesize catalytically active RubisCO in vivo. To complete the CBB pathway, there is a need for an enzyme, i.e., phosphoribulokinase (PRK), to catalyze the formation of RuBP, the substrate for the RubisCO reaction. Homology searches, as well as direct enzymatic assays with M. jannaschii, failed to reveal the presence of PRK. The apparent lack of PRK raised the possibility that either there is an alternative pathway to generate RuBP or RubisCO might use an alternative substrate in vivo. In the present study, direct enzymatic assays performed with alternative substrates and extracts of M. jannsachii provided evidence for a previously uncharacterized pathway for RuBP synthesis from 5-phospho-D-ribose-1-pyrophosphate (PRPP) in M. jannaschii and other methanogenic archaea. Proteins and genes involved in the catalytic conversion of PRPP to RuBP were identified in M. jannaschii (Mj0601) and Methanosarcina acetivorans (Ma2851), and recombinant Ma2851 was active in extracts of Escherichia coli. Thus, in this work we identified a novel means to synthesize the CO2 acceptor and substrate for RubisCO in the absence of a detectable kinase, such as PRK. We suggest that the conversion of PRPP to RuBP might be an evolutional link between purine recycling pathways and the CBB scheme.

Quantitative 2H NMR at Natural Abundance Can Distinguish the Pathway Used for Glucose Fermentation by Lactic Acid Bacteria

For any given metabolic pathway, isotope redistribution coefficients (a(ij)) that characterize the specific derivation of each hydrogen atom can be defined. By using quantitative deuterium NMR, the redistribution of deuterium at natural abundance in lactic acid produced by the bacterial fermentation of glucose has been determined for each non-labile hydrogen atom of glucose or water and the hydrogen atoms of lactic acid. Distinct differences are observed in the lactic acid isolated from Lactococcus lactis and Leuconostoc mesenteroides that can be interpreted in terms of the different fermentative pathways used. Specifically, the affiliations observed between the H1, H3, and H4 positions of glucose with methyl and hydroxymethylene of lactic acid can give quantitative information on whether the glycolytic or the reductive pentose-phosphate pathway was involved in glucose catabolism.

Oxyanion Hole-stabilized Stereospecific Isomerization in Ribose-5-phosphate Isomerase (Rpi)

Ribose-5-phosphate isomerase (Rpi) acts as a key enzyme in the oxidative and reductive pentose-phosphate pathways for the conversion of ribose-5-phosphate (R5P) to ribulose-5-phosphate and vice versa. We have determined the crystal structures of Rpi from Thermus thermophilus HB8 in complex with the open chain form of the substrate R5P and the open chain form of the C2 epimeric inhibitor arabinose-5-phosphate as well as the apo form at high resolution. The crystal structures of both complexes revealed that these ring-opened epimers are bound in the active site in a mirror symmetry binding mode. The O1 atoms are stabilized by an oxyanion hole composed of the backbone amide nitrogens in the conserved motif. In the structure of the Rpi.R5P complex, the conversion moiety O1-C1-C2-O2 in cis-configuration interacts with the carboxyl oxygens of Glu-108 in a water-excluded environment. Furthermore, the C2 hydroxyl group is presumed to be highly polarized by short hydrogen bonding with the side chain of Lys-99. R5P bound as the ring-opened reaction intermediate clarified the high stereoselectivity of the catalysis and is consistent with an aldose-ketose conversion by Rpi that proceeds via a cis-enediolate intermediate.

Prevention of incipient diabetic nephropathy by high-dose thiamine and benfotiamine.

Accumulation of triosephosphates arising from high cytosolic glucose concentrations in hyperglycemia is the trigger for biochemical dysfunction leading to the development of diabetic nephropathy-a common complication of diabetes associated with a high risk of cardiovascular disease and mortality. Here we report that stimulation of the reductive pentosephosphate pathway by high-dose therapy with thiamine and the thiamine monophosphate derivative benfotiamine countered the accumulation of triosephosphates in experimental diabetes and inhibited the development of incipient nephropathy. High-dose thiamine and benfotiamine therapy increased transketolase expression in renal glomeruli, increased the conversion of triosephosphates to ribose-5-phosphate, and strongly inhibited the development of microalbuminuria. This was associated with decreased activation of protein kinase C and decreased protein glycation and oxidative stress-three major pathways of biochemical dysfunction in hyperglycemia. Benfotiamine also inhibited diabetes-induced hyperfiltration. This was achieved without change in elevated plasma glucose concentration and glycated hemoglobin in the diabetic state. High-dose thiamine and benfotiamine therapy is a potential novel strategy for the prevention of clinical diabetic nephropathy.

Metabolic signals that lead to control of CBB gene expression in Rhodobacter capsulatus.

Various mutant strains were used to examine the regulation and metabolic control of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway in Rhodobacter capsulatus. Previously, a ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deficient strain (strain SBI/II) was found to show enhanced levels of cbb(I) and cbb(II) promoter activities during photoheterotrophic growth in the presence of dimethyl sulfoxide. With this strain as the starting point, additional mutations were made in genes encoding phosphoribulokinase and transketolase and in the gene encoding the LysR-type transcriptional activator, CbbR(II). These strains revealed that a product generated by phosphoribulokinase was involved in control of CbbR-mediated cbb gene expression in SBI/II. Additionally, heterologous expression experiments indicated that Rhodobacter sphaeroides CbbR responded to the same metabolic signal in R. capsulatus SBI/II and mutant strain backgrounds.

Maintenance and control of redox poise in Rhodobacter capsulatus strains deficient in the Calvin-Benson-Bassham pathway.

Carbon dioxide serves as the preferred electron acceptor during photoheterotrophic growth of nonsulfur purple photosynthetic bacteria such as Rhodobacter capsulatus and Rhodobacter sphaeroides. This CO2, produced as a result of the oxidation of preferred organic carbon sources, is reduced through reactions of the Calvin-Benson-Bassham reductive pentose phosphate pathway. This pathway is thus crucial to maintain a balanced intracellular oxidation-reduction potential (or redox poise) under photoheterotrophic growth conditions. In the absence of a functional Calvin-Benson-Bassham pathway, either an exogenous electron acceptor, such as dimethylsulfoxide, must be supplied or the organism must somehow develop alternative electron acceptor pathways to preserve the intracellular redox state of the cell. Spontaneous variants of Rba. capsulatus strains deficient in the Calvin-Benson-Bassham pathway that have become photoheterotrophically competent (in the absence of an exogenous electron acceptor) were isolated. These strains (SBP-PHC and RCNd1, RCNd3, and RCNd4) were shown to obviate normal ammonia control and derepress synthesis of the dinitrogenase enzyme complex for the dissipation of excess reducing equivalents and generation of H2 gas via proton reduction. In contrast to previous studies with other organisms, the dinitrogenase reductase polypeptides were maintained in an active and unmodified form in strain SBP-PHC and the respective RCNd strains. Unlike the situation in Rba. sphaeroides, the Rba. capsulatus strains did not regain full ammonia control when complemented with plasmids that reconstituted a functional Calvin-Benson-Bassham pathway. Moreover, dinitrogenase derepression in Rba. capsulatas was responsive to the addition of the auxiliary electron acceptor dimethylsulfoxide. These results indicated a hierarchical control over the removal of reducing equivalents during photoheterotrophic growth that differs from strains of Rba. sphaeroides and Rhodospirillum rubrum deficient in the Calvin-Benson-Bassham pathway.

Multiple regulators and their interactions in vivo and in vitro with the cbb regulons of Rhodobacter capsulatus

The cbbI and cbbII operons encode structural genes which are important for carbon dioxide fixation via the Calvin-Benson-Bassham reductive pentose phosphate pathway in Rhodobacter capsulatus. Each operon is regulated by cognate LysR-type transcriptional activators, CbbRI and CbbRII, with the product of the cbbRI gene, CbbRI, able to control its own transcription under some growth conditions. Furthermore, CbbRI may at least partially regulate the cbbII operon, with significant, yet regulated transcription of the cbbII operon occurring in the absence of any CbbR. These results suggested the importance of additional regulators. Thus, in addition to the rather specific control exerted by CbbR, a more globally significant regulatory system, the RegA-RegB (PrrA-PrrB) two-component system, was found to contribute to transcriptional regulation of each cbb operon. The regA and regB mutant strains were found to contain constitutive levels of form I and form II RubisCO, the major proteins encoded by the cbbI and cbbII operons, respectively. In addition, DNaseI footprint analyses indicated that RegA∗, a constitutively active mutant form of RegA, binds specifically to cbbI and cbbII promoter-operator regions. CbbRI, CbbRII, and RegA binding loci were localized relative to transcription start sites, leading to a coherent picture of how each of these regulators interacts with specific promoter-operator sequences of the cbb operons.

Induction of carbon monoxide dehydrogenase to facilitate redox balancing in a ribulose bisphosphate carboxylase/oxygenase-deficient mutant strain of Rhodospirillum rubrum.

A ribulose-1,5-bisphosphate carboxylase/oxygenase-deficient mutant strain (strain I-19) of Rhodospirillum rubrum was capable of growth under photoheterotrophic conditions in the absence of exogenous electron acceptors. These results suggested that alternative means of removing reducing equivalents have been acquired that allow this strain to remove reducing equivalents in the absence of a functional Calvin-Benson-Bassham reductive pentose phosphate pathway. Previously, the proton-reducing activity of the dinitrogenase complex was implicated in helping to maintain redox balance. However, since considerable amounts of CO2 were still fixed in this strain, the complete profile of enzymes involved in alternative CO2 fixation schemes was assessed. A specific and substantial induction of carbon monoxide dehydrogenase (CO dehydrogenase) synthesis was found in the mutant strain; although none of the other CO2 fixation pathways or enzyme activities were altered. These results suggested that CO dehydrogenase contributes to the photoheterotrophic success of strain I-19. Furthermore, the data implicate interacting and complex regulatory processes required to maintain the proper redox balance of this organism and other nonsulfur purple bacteria.

Microbial ribulose 1,5-bisphosphate carboxylase/oxygenase: A different perspective

Marine and terrestrial photosynthetic and chemoautotrophic microorganisms assimilate considerable amounts of carbon dioxide. Like green plastids, the predominant means by which this process occurs is via the Calvin-Benson-Bassham reductive pentose phosphate pathway, where ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a paramount role. Recent findings indicate that this enzyme is subject to diverse means of control, including specific and elaborate means to guarantee its high rate and extent of synthesis. In addition, powerful and specific means to regulate Rubisco activity is a characteristic feature of many microbial systems. In many respects, the diverse properties of microbial Rubisco enzymes suggest interesting strategies to elucidate the molecular basis of CO2/O2 specificity, the ‘holy grail’ of Rubisco biochemistry. These systems thus provide, as the title suggests, ‘different perspectives’ to this fundamental problem. These include vast possibilities for imaginative biological selection using metabolically versatile organisms with well-defined genetic transfer capabilities to solve important issues of Rubisco specificity and molecular control. This review considers the major issues of Rubisco biochemistry and regulation in photosynthetic microoganisms including proteobacteria, cyanobacteria, marine nongreen algae, as well as other interesting prokaryotic and eukaryotic microbial systems recently shown to possess this enzyme.

Identification of a Catalytic Aspartyl Residue ofd-Ribulose 5-Phosphate 3-Epimerase by Site-directed Mutagenesis

Guided by comparative sequence considerations, we have examined the possibility of a catalytic role of Asp186 of D-ribulose 5-phosphate epimerase by site-directed mutagenesis of the recombinant spinach enzyme. Accordingly, D186A, D186N, and D186E mutants of the epimerase were constructed, purified, and characterized; as judged by their electrophoretic mobilities the mutants are properly assembled into octamers like the wild-type enzyme. Based on the extent of internal quenching of Trp fluorescence, the conformational integrity of the wild-type enzyme is preserved in the mutants. The wild-type kcat of 7.1 x 10(3) s-1 is lowered to 3.3 x 10(-4) s-1 in D186A, 0.13 s-1 in D186N, and 1.1 s-1 in D186E; as gauged by D186A, altogether lacking a functional side chain at position 186, the beta-carboxyl of Asp186 facilitates catalysis by >10(7)-fold. Relative to the wild-type enzyme, the Km for D-ribulose 5-phosphate is essentially unaltered with D186N and D186E but increased 10-fold with D186A. Apart from their impairments in epimerase activity, the mutants are unable to catalyze exchange between solvent protons and the C3 proton of substrates. This deficiency and the differential alterations of kinetic parameters among the mutants are consistent with Asp186 serving as an electrophile to facilitate alpha-proton abstraction. This study is the first to identify a catalytic group of the epimerase.

Physiological control and regulation of the Rhodobacter capsulatus cbb operons.

The genes encoding enzymes of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway in Rhodobacter capsulatus are organized in at least two operons, each preceded by a separate cbbR gene, encoding potential LysR-type transcriptional activators. As a prelude to studies of cbb gene regulation in R. capsulatus, the nucleotide sequence of a 4,537-bp region, which included cbbRII, was determined. This region contained the following open reading frames: a partial pgm gene (encoding phosphoglucomutase) and a complete qor gene (encoding NADPH:quinone oxidoreductase), followed by cbbRII, cbbF (encoding fructose 1,6-bisphosphatase), cbbP (encoding phosphoribulokinase), and part of cbbT (encoding transketolase). Physiological control of the CBB pathway and regulation of the R. capsulatus cbb genes were studied by using a combination of mutant strains and promoter fusion constructs. Characterization of mutant strains revealed that either form I or form II ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO), encoded by the cbbLS and cbbM genes, respectively, could support photoheterotrophic and autotrophic growth. A strain with disruptions in both cbbL and cbbM could not grow autotrophically and grew photoheterotrophically only when dimethyl sulfoxide was added to the culture medium. Disruption of cbbP resulted in a strain that did not synthesize form II RubisCO and had a phenotype similar to that observed in the RubisCO-minus strain, suggesting that there is only one cbbP gene in R. capsulatus and that this gene is cotranscribed with cbbM. Analysis of RubisCO activity and synthesis in strains with disruptions in either cbbRI or cbbRII, and beta-galactosidase determinations from wild-type and mutant strains containing cbbIp- and cbbIIp-lacZ fusion constructs, indicated that the cbbI and cbbII operons of R. capsulatus are within separate CbbR regulons.

13] Chaperonins of the purple nonsulfur bacterium Rhodobacter sphaeroides

Publisher Summary The chapter presents a study related to the chaperonins of the purple nonsulfur bacterium Rhodobacter sphaeroides. The purple nonsulfur bacterium Rhodobacter sphaeroides is a metabolically versatile organism capable of being cultured under widely different growth conditions. Used as a model to study the regulation and biochemistry of carbon fixation in prokaryotes, R. sphaeroides , possesses two ribulose-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) enzymes. Rubisco, the key enzyme of the Calvin reductive pentose phosphate pathway, is a multimeric enzyme that has been demonstrated to require chaperonins for folding and subsequent assembly both in vivo and in vitro . The Escherichia coli and R. sphaeroides cpn60s have 68% identity, and several of the key amino acid residues identified in the E. coli protein are conserved in the R. sphaeroides cpn60. Of the 14 E. coli cpn60 amino acid residues identified by site-directed mutagenesis that are involved in ATP hydrolysis are conserved in R. sphaeroides cpn60. Similarly, most of the residues involved in E. coli GroEL binding, polypeptide binding, and folding of heterologous proteins are conserved in the R. sphaeroides cpn60. The relationships between cpn60s of diverse eubacteria show that the R. sphaeroides cpn60 is most closely related to proteins from α subdivision of the purple bacteria.

The molecular regulation of the reductive pentose phosphate pathway in Proteobacteria and Cyanobacteria.

In phototrophic and chemoautotrophic proteobacteria, genes encoding enzymes of the Calvin-Benson-Bassham pathway of CO2 fixation are often found in clusters that are transcribed from a single promoter under control of the LysR-type transcriptional activator, CbbR. Mutations affecting CbbR prevent induction of cbb genes. Gel-retardation assays have demonstrated CbbR binding to putative regulatory regions of cbb operons, and in two cases, footprinting experiments have delimited the nucleotide sequence protected by CbbR. Fusion of cbb control sequences to reporter genes has allowed the regions required for promoter activity to be defined, and recent experiments indicate that the cbb regulon in Rhodobacter is controlled by a global two-component signal transduction system that also regulates other metabolic processes in this organism. Different ways of regulating CBB cycle enzymes that also have roles in heterotrophic metabolism have recently been discovered. In cyanobacteria, the genes of the CBB pathway are organized and regulated differently, and these oxygen-evolving phototrophic bacteria have evolved different strategies to control the assimilation of CO2.