Activation of ribulose 1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides: probable role of the small subunit.

The activation properties of the form I and form II ribulose 1,5-bisphosphate carboxylases from Rhodopseudomonas sphaeroides were examined. Both enzymes have a requirement of Mg2+ for optimal activity. Mn2+, Ni2+, and Co2+ can also support activity of the form I enzyme, whereas only Mn2+ can substitute for Mg2+ with the form II enzyme. The effect of different preincubations on the carboxylase reaction was also examined. Both enzymes exhibited a lag when preincubated with other than Mg2+ and CO2 before assay, but the lag was much more pronounced and the rate of the reaction was slower with the form I enzyme under these conditions. Activation of the form I carboxylase By Mg2+ and CO2 occurred more rapidly than that of the form II enzyme. The results obtained with the two distinct forms of carboxylase from R. sphaeroides, as well as studies with the spinach and Rhodospirillum rubrum enzymes, thus indicate that the presence of the small subunit affects the rate of activation by Mg2+ and CO2 as well as the rate of reactivation of ribulose bisphosphate-inactivated enzyme.     

Different molecular forms of D-ribulose-1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides.

Ribulose-1,5-bisphosphate (Rbu-P2) carboxylase isolated from Rhodopseudomonas sphaeroides 2.4.1.Ga was separated into two different forms by DEAE-cellulose column chromatography. Both forms, designated Peak I and Peak II have been purified to homogeneity by the criterion of polyacrylamide disc-gel electrophoresis. The Peak I carboxylase has a molecular weight of 550,000, while the Peak II carboxylase is a smaller protein having a molecular weight of approximately 360,000. Sodium dodecyl sulfate electrophoresis revealed a large subunit for both enzymes which migrates similarly to the large subunit of spinach Rbu-P2 carboxylase. The Peak I enzyme also exhibited a small subunit having a molecular weight of 11,000. No evidence for a smaller polypeptide was found associated with the Peak II enzyme. Antisera prepared against the Peak I enzyme inhibited Peak I enzymatic activity, but had no effect on the activity of the Peak II enzyme. The two enzymes exhibited marked differences in catalytic properties. The Peak I enzyme exhibits optimal activity at pH 8.0 and is inhibited by low concentrations of 6-phosphogluconate, while the Peak II enzyme has a pH optimum of 7.2 and is relatively insensitive to 6-phosphogluconate.     

Transposon mutagenesis and physiological analysis of strains containing inactivated form I and form II ribulose bisphosphate carboxylase/oxygenase genes in Rhodobacter sphaeroides.

Strains of Rhodobacter sphaeroides (Rhodopseudomonas sphaeroides) were constructed such that either the gene encoding form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC-O) or the gene encoding form II RuBPC-O was inactivated. Both strains were capable of photoheterotrophic growth with malate as the electron donor, with only slight differences in growth rate and overall carboxylase specific activity compared with the wild-type strain. Photolithotrophic growth with 1.5% CO2 in hydrogen was also possible for R. sphaeroides strains containing only one of the two RuBPC-O enzyme forms, although the differences in growth rates between wild-type and carboxylase mutant strains were greater under these conditions. These results indicate that the two forms of RuBPC-O are independently regulated. In addition, the regulatory system governing RuBPC-O synthesis may, in some cases, compensate for the lack of the missing enzyme.     

Structural differences in the catalytic subunits of form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodopseudomonas sphaeroides.

There are significant differences in the large subunits of form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase isolated from Rhodopseudomonas sphaeroides. Two-dimensional peptide mapping of carboxymethylated large subunits clearly indicates that there are differences in the primary structure of the two proteins. These results are supported by limited proteolysis with three different proteases and by subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data, in conjunction with immunological studies and investigations on the regulation of the two enzymes, support the conclusion that the large subunits of form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase may be different gene products.     

The “Green” Form I Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from the Nonsulfur Purple Bacterium Rhodobacter capsulatus

Form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) of the Calvin-Benson-Bassham cycle may be divided into two broad phylogenetic groups, referred to as red-like and green-like, based on deduced large subunit amino acid sequences. Unlike the form I enzyme from the closely related organism Rhodobacter sphaeroides, the form I RubisCO from R. capsulatus is a member of the green-like group and closely resembles the enzyme from certain chemoautotrophic proteobacteria and cyanobacteria. As the enzymatic properties of this type of RubisCO have not been well studied in a system that offers facile genetic manipulation, we purified the R. capsulatus form I enzyme and determined its basic kinetic properties. The enzyme exhibited an extremely low substrate specificity factor, which is congruent with its previously determined sequence similarity to form I enzymes from chemoautotrophs and cyanobacteria. The enzymological results reported here are thus strongly supportive of the previously suggested horizontal gene transfer that most likely occurred between a green-like RubisCO-containing bacterium and a predecessor to R. capsulatus. Expression results from hybrid and chimeric enzyme plasmid constructs, made with large and small subunit genes from R. capsulatus and R. sphaeroides, also supported the unrelatedness of these two enzymes and were consistent with the recently proposed phylogenetic placement of R. capsulatus form I RubisCO. The R. capsulatus form I enzyme was found to be subject to a time-dependent fallover in activity and possessed a high affinity for CO2, unlike the closely similar cyanobacterial RubisCO, which does not exhibit fallover and possesses an extremely low affinity for CO2. These latter results suggest definite approaches to elucidate the molecular basis for fallover and CO2 affinity.     

Isolation of the Rhodopseudomonas sphaeroides form I ribulose 1,5-bisphosphate carboxylase/oxygenase large and small subunit genes and expression of the active hexadecameric enzyme in Escherichia coli

A library of cloned Rhodopseudomonas sphaeroides DNA was screened by colony hybridization for form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) sequences using heterologous RuBPC/O probes. A recombinant plasmid was identified that hybridized to both the Anacystis nidulans and the R. sphaeroides form II RuBPC/O genes. Subcloning of a hybridizing 4-kb SmaI fragment allowed expression of active enzyme in Escherichia coli that was identical to form I RuBPC/O based on polyacrylamide gel electrophoresis and Western immunoblot analysis.     

Synthesis of two different molecular forms of ribulose-bisphosphate carboxylase/oxygenase in Rhodopseudomonas blastica grown in batch culture

The expression of the two different molecular forms (form I and form II) of ribulose-bisphosphate carboxylase/oxygenase (RuBisCO) in Rhodopseudomonas blastica during growth in batch on pyruvate–malate medium was investigated. During the early exponential phase of growth, only form I enzyme was synthesized. At the mid-exponential phase of growth, both forms were expressed, although form I enzyme was predominant. At the late exponential phase, form I and form II enzymes were synthesized but form II enzyme predominated. It is concluded that form I and form II RuBisCO enzymes in R. blastica are differentially expressed and this may be mediated by the level of CO2 in the growth medium.     

Synthesis of two different molecular forms of ribulose-bisphosphate carboxylase/oxygenase in Rhodopseudomonas blastica grown in batch culture

The expression of the two different molecular forms (form I and form II) of ribulose-bisphosphate carboxylase/oxygenase (RuBisCO) in Rhodopseudomonas blastica during growth in batch on pyruvate–malate medium was investigated. During the early exponential phase of growth, only form I enzyme was synthesized. At the mid-exponential phase of growth, both forms were expressed, although form I enzyme was predominant. At the late exponential phase, form I and form II enzymes were synthesized but form II enzyme predominated. It is concluded that form I and form II RuBisCO enzymes in R. blastica are differentially expressed and this may be mediated by the level of CO2 in the growth medium.     

Cloning and characterization of the gene product of the form II ribulose-1,5-bisphosphate carboxylase gene of Rhodopseudomonas sphaeroides.

We report the cloning and characterization of the gene product of the gene for the form II ribulose bisphosphate carboxylase from Rhodopseudomonas sphaeroides. We present evidence that the form II enzyme is encoded by a single gene in R. sphaeroides; however, this gene does hybridize to a second chromosomal locus.     

Expression of endogenous and foreign ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) genes in a RubisCO deletion mutant of Rhodobacter sphaeroides.

A Rhodobacter sphaeroides ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion strain was constructed that was complemented by plasmids containing either the form I or form II CO2 fixation gene cluster. This strain was also complemented by genes encoding foreign RubisCO enzymes expressed from a Rhodospirillum rubrum RubisCO promoter. In R. sphaeroides, the R. rubrum promoter was regulated, resulting in variable levels of disparate RubisCO molecules under different growth conditions. Photosynthetic growth of the R. sphaeroides deletion strain complemented with cyanobacterial RubisCO revealed physiological properties reflective of the unique cellular environment of the cyanobacterial enzyme. The R. sphaeroides RubisCO deletion strain and R. rubrum promoter system may be used to assess the properties of mutagenized proteins in vivo, as well as provide a potential means to select for altered RubisCO molecules after random mutagenesis of entire genes or gene regions encoding RubisCO enzymes.     

Biochemical characterization of biotin-responsive multiple carboxylase deficiency: heterogeneity within the bio genetic complementation group

Three biotin-dependent enzymes, pyruvate carboxylase (PC), propionyl CoA carboxylase (PCC), and beta-methylcrotonyl CoA carboxylase (beta MCC), were biochemically characterized in fibroblasts from two patients with neonatal multiple carboxylase deficiency. Genetic complementation analyses indicated that both cell lines, designated lines 1 and 2, were deficient in the various carboxylase activities and belonged to the bio complementation group. The activities of the three carboxylases became normal when line 2 cells were incubated in medium supplemented with biotin (1 mg/l) for 24 hrs, whereas 4-6 days were required to achieve maximum activities of PC, PCC, and beta MCC (57%, 46%, and 29% of mean normal enzyme activity, respectively) in line 1 cells incubated in medium containing up to 10 mg/1 biotin. Furthermore, PC activity in line 2 continued to increase under apparent gluconeogenic conditions in culture, but not in line 1. Thermostability studies suggested that biotin stabilizes PC and beta MCC in both cell lines. PC in line 1 cells incubated with or without biotin was less stable than that in normal or line 2 cells, and the less than normal increase of enzyme activities in line 1, especially that of PC, may represent incomplete biotination. These results indicate that there is biochemical heterogeneity within the bio complementation group. Immunotitration with antibodies prepared against purified pig heart PCC demonstrated normal quantities of cross-reacting material in both lines and no differences in the amount of this material after incubation with supplemental biotin, despite the seven- to 20-fold increase in PCC activity. Thus, the increase in carboxylase activity in both bio lines appears to represent activation of rpe-existing apocarboxylase rather than de novo enzyme synthesis. The primary defect in this form of multiple carboxylase deficiency may be in a common holocarboxylase synthetase or in biotin transport. If the defect is in the synthetase, the differences noted between the two bio lines could be explained by a difference in the enzyme's Km for biotin.     

Succinate dehydrogenase in Rhodopseudomonas sphaeroides: subunit composition and immunocross-reactivity with other related bacteria.

Antibodies were raised against the succinate dehydrogenase (SDH) present in the chromatophores of phototrophically grown Rhodopseudomonas sphaeroides. Crossed immunoelectrophoresis experiments indicated that the SDH present in the cytoplasmic membranes of heterotrophically grown R. sphaeroides is probably the same enzyme observed in the chromatophores. The enzyme was extracted by Triton X-100 in a form which consisted of only two subunits (molecular weight, 68,000 and 30,000) and was not associated with a cytochrome b. The antibodies directed against SDH from R. sphaeroides showed no immunocross-reactivity with SDH from phylogenetically related bacterial species, including Rhodopseudomonas capsulata, Paracoccus denitrificans, Rhodopseudomonas palustris, Rhodospirillum rubrum, and Rhodospirillum fulvum.     

Nucleotide and deduced amino acid sequence of the Rhodobacter sphaeroides gene encoding form II ribulose-1,5-bisphosphate carboxylase/oxygenase and comparison with other deduced form I and II sequences

The nucleotide sequence for the Rhodobacter sphaeroides form II ribulose 1,5-bisphosphate carboxylase/oxygenase was determined. The deduced product is highly homologous with the form II-like enzyme of Rhodospirillum rubrum , but appears to be more distantly related to the large subunit of the L₈S₈ enzyme found in autotrophic bacteria, cyanobacteria and higher plants. Several regions highly conserved among L₈S₈ and L_X enzymes correspond with regions previously implicated in catalytic activity and subunit interactions. An imperfect palindrome and a stem loop structure were identified in the 5' and 3' flanking sequences, respectively, of R. sphaeroides rbpL .     

Characterization of antiserum directed against form II ribulose 1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides.

Antiserum directed against form II ribulose 1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides showed no cross-reactivity towards the form I enzyme as evidenced by a lack of immunopreciptation. In addition, this antiserum failed to inhibit form I enzymatic activity.     

Influence of pH, O2, and temperature on the absorption properties of the secondary light-harvesting antenna in members of the family Rhodospirillaceae

In some Rhodospirillaceae, the primary light-harvesting (LH I) antenna absorbs near-infrared light around 870 nm, whereas LH II (holochrome B800-860) has a major absorption band between 850 and 860 nm (B860) and a minor absorbancy around 800 nm (B800). Results show that, unlike LH I, holochrome B800-860 (LH II) exhibits unstable light absorption properties in whole cells. This was observed in Rhodopseudomonas capsulata grown anaerobically in light in weakly buffered carbohydrate medium; cultures lost both carotenoid-dependent brown-yellow pigmentation and LH II absorbancy. The whole cell spectrophotometric changes were attributed to mild acid conditions generated during sugar metabolism. LH II absorbancy was also destroyed in both R. capsulata and Rhodopseudomonas gelatinosa when cultures growing at neutral pH were acidified to a pH value around 5.0 with HCl. In contrast, during the same time period of exposure to pH 5.0, only a 50% decrease in Rhodopseudomonas sphaeroides LH II B800 absorbancy was measured. At neutral pH, LH II absorbancy in suspensions of nongrowing Rhodopseudomonas spp. was also sensitive to O2 exposure and to incubation at 30 to 40 degrees C. During treatment with O2, the rate of LH II B800 absorption decrease in R. gelatinosa and R. sphaeroides was 60 and 40% per h, respectively, compared with their absorbancy maximum around 860 nm. Both 860-nm absorbancy and the total bacteriochlorophyll content of the cells remained unchanged. On the other hand, no significant decrease in B800 if LH II in R. capsulata occurred during O2 exposure, but a 20% absorption decay rate per h of B800 was observed in cells incubated anaerobically at 40 degrees C. These B800 LH II spectral changes Rhodopseudomonas spp. were prevented by maintaining cells at neutral pH and at 10 degrees C. The near-infrared absorption spectrum of Rhodospirillum rubrum, which does not form LH II, was not significantly influenced by these different pH, aerobic, or temperature conditions.     

Purification and properties of acyl coenzyme A thioesterase II from Rhodopseudomonas sphaeroides

A high molecular weight acyl coenzyme A (acyl-CoA) thioesterase, designated thioesterase II, has been purified 5300-fold from photoheterotrophically grown cells of Rhodopseudomonas sphaeroides. In contrast to R. sphaeroides acyl-CoA thioesterase I [Boyce, S.G., & Lueking, D.R. (1984) Biochemistry 23, 141-147], thioesterase II has a native molecular mass (Mr) of 120,000, is capable of hydrolyzing saturated and unsaturated acyl-CoA substrates with acyl chain lengths ranging from C4 to C18, and is completely insensitive to the serine esterase inhibitor diisopropyl fluorophosphate. Palmitoyl-CoA and stearoyl-CoA are the preferred (lowest Km) saturated acyl-CoA substrates and vaccenoyl-CoA is the preferred unsaturated substrate. However, comparable Vmax values were obtained with a variety of acyl-CoA substrates. Unlike a similar thioesterase present in cells of Escherichia coli [Bonner, W.M., & Bloch, K. (1972) J. Biol. Chem. 247, 3123-3133], R. sphaeroides thioesterase II displays a high ratio of decanoyl-CoA to palmitoyl-CoA activities and exhibits little ability to hydrolyze 3-hydroxyacyl-CoA substrates. Only 3-hydroxydodecanoyl-CoA supported a measurable rate of enzyme activity. With the purification of thioesterase II, the enzymes responsible for greater than 90% of the acyl-CoA thioesterase activity present in cell-free extracts of R. sphaeroides have now been identified.     

Cloning and expression of the d-ribulose-1,5-bisphosphate carboxylase/oxygenase form II gene from Thiobacillus intermedius in Escherichia coli

Abstract Both form I and II ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes were detected in Thiobacillus intermedius by heterologous hybridization using specific probes from Anacystis nidulans and Rhodobacter sphaeroides , respectively. However, only the previously reported from I enzyme could be demonstrated in cells grown under a number of different conditions. The reason(s) why the form II gene is not expressed in T. intermedius is/are not clear at this time. The form II gene was isolated from a lambda library by screening with the Rb. sphaeroides probe. A Sal I fragment from this clone was ligated into pUC8 and transformed into Escherichia coli DH5α. Subclones pTi20IIA and pTi20IIB representing both orientations relative to the lac promoter were isolated. Low levels of RuBisCO activity were detected in both induced and non-induced pTi20IIA indicating the probable expression from a T. intermedius promoter. Induced pTi20IIB produced much higher levels of enzyme activity. Analysis of cell-free extracts using sucrose density gradients confirmed the expression of a form II RuBisCO similar in size to that found in Rhodobacter capsulatus . Other Calvin cycle genes were not clustered with either the form I or form II genes.     

Interaction of inactivated and active ribulose 1,5-bisphosphate carboxylase/oxygenase of Rhodobacter sphaeroides with nucleotides and the chaperonin 60 (GroEL) protein.

Purified inactivated form I ribulose 1,5-bisphosphate carboxylase/oxygenase (form I RubisCO) of Rhodobacter sphaeroides was activated by ATP and, to some extent, by other adenylates and nucleotides. Reactivation in the presence of ATP occurred by a time-dependent and concentration-dependent process which appeared to be irreversible. The carbamylated form of inactivated form I RubisCO was less susceptible to ATP-mediated reactivation than the uncarbamylated inactivated enzyme. In some cases, ATP analogs could mimic the reactivation process; one analog, adenylyl(beta, gamma-methylene)-diphosphonate, was found to partially block ATP-mediated reactivation but could not block reactivation induced by Mg(II). Concomitant with the recovery of enzymatic activity, the migration of the inactivated form I RubisCO on nondenaturing and sodium dodecyl sulfate gels changed from a pattern that was characteristic of inactivated enzyme to a pattern that was identical to that of the active protein. It was further found that discrete proportions of active enzyme and the chaperonin 60 protein of R. sphaeroides aggregated in the presence of ATP. The form I RubisCO is thus proposed to contain a specific ATP-binding site that may contribute to both the regulation of activity and the assembly of active enzyme.     

Multiple acyl-coenzyme A carboxylases in Pseudomonas citronellolis.

Pseudomonas citronellolis was shown to contain four different acyl-coenzyme A carboxylases, including acetyl-, propionyl-, 3-methylcrotonyl-, and geranyl-CoA carboxylases, when grown on the appropriate carbon sources. Acetyl-CoA carboxylase activity in crude extracts was stimulated approximately 40-fold by inclusion of 0.4-0.5 M ammonium sulfate in the assay. Unexpectedly high levels of propionyl-CoA carboxylase activity, also stimulated by ammonium sulfate, were found in acetate-grown cells. That these acetyl- and propionyl-CoA carboxylase activities were due to different enzymes was shown by their resolution during purification by a procedure that stabilized acetyl-CoA carboxylase as a complex and separated propionyl-CoA carboxylase into two required protein fractions. Propionate- or valine-grown cells contained a propionyl-CoA carboxylase activity that was strongly inhibited by ammonium sulfate in the assay, and which may represent an inducible form of the enzyme. Geranyl- and 3-methylcrotonyl-CoA carboxylases that catalyze the carboxylation of the 3-methyl groups of homologous acyl-CoA acceptors, were induced by growth on the monoterpenes, citronellic or geranoic acid; only 3-methylcrotonyl-CoA carboxylase was induced by growth on leucine or isovaleric acid. Induction of either carboxylase was associated with the appearance of similar high-molecular-weight, biotin-containing proteins as measured by gel filtration. These two carboxylases are probably distinct enzymes since 3-methyl-crotonyl-CoA carboxylase from isovalerate-grown cells does not carboxylate geranyl-CoA, while geranyl-CoA carboxylase will carboxylate both acyl-CoA homologues. P. citronellolis appears to be a useful system for studying the structural aspects of pairs of homologous acyl-CoA carboxylases.