| 1. |
Andralojc PJ,
Harris DA,
( 1992 ) Isolation and characterisation of a functional alpha beta heterodimer from the ATP synthase of Rhodospirillum rubrum. PMID : 1327870 : DOI : 10.1016/0014-5793(92)81326-h Abstract >>
An alpha beta heterodimer of the F1-ATPase of Rhodospirillum rubrum was isolated by extraction of chromatophores with LiCl. Each alpha beta heterodimer contains one tightly bound ADP, which is released upon removal of medium Mg2+. The dimer can be reversibly dissociated by removal of Mg(2+)-ions. The alpha beta heterodimer restores both ATP-synthetic and -hydrolytic activities to LiCl-treated chromatophores, saturation being achieved at approximately 2 mmol alpha beta.mol BChl-1. The heterodimer itself hydrolyses Mg-ATP with an activity distinct from RF1, being unaffected by azide or sulphite ions. The Vmax and Km (ATP) for this Mg(2+)-dependent activity were 110 +/- 10 nmol.min-1.mg protein-1 and 100 +/- 30 microM, respectively. The Km did not differ significantly from that of RF1.
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2. |
Heo J,
Wolfe MT,
Staples CR,
Ludden PW,
( 2002 ) Converting the NiFeS carbon monoxide dehydrogenase to a hydrogenase and a hydroxylamine reductase. PMID : 12374822 : DOI : 10.1128/jb.184.21.5894-5897.2002 PMC : PMC135374 Abstract >>
Substitution of one amino acid for another at the active site of an enzyme usually diminishes or eliminates the activity of the enzyme. In some cases, however, the specificity of the enzyme is changed. In this study, we report that the changing of a metal ligand at the active site of the NiFeS-containing carbon monoxide dehydrogenase (CODH) converts the enzyme to a hydrogenase or a hydroxylamine reductase. CODH with alanine substituted for Cys(531) exhibits substantial uptake hydrogenase activity, and this activity is enhanced by treatment with CO. CODH with valine substituted for His(265) exhibits hydroxylamine reductase activity. Both Cys(531) and His(265) are ligands to the active-site cluster of CODH. Further, CODH with Fe substituted for Ni at the active site acquires hydroxylamine reductase activity.
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3. |
Addlesee HA,
Hunter CN,
( 2002 ) Rhodospirillum rubrum possesses a variant of the bchP gene, encoding geranylgeranyl-bacteriopheophytin reductase. PMID : 11872709 : DOI : 10.1128/jb.184.6.1578-1586.2002 PMC : PMC134874 Abstract >>
The bchP gene product of Rhodobacter sphaeroides is responsible for the reduction of the isoprenoid moiety of bacteriochlorophyll (Bchl) from geranylgeraniol (GG) to phytol; here, we show that this enzyme also catalyzes the reduction of the isoprenoid moiety of bacteriopheophytin (Bphe). In contrast, we demonstrate that a newly identified homolog of this gene in Rhodospirillum rubrum encodes an enzyme, GG-Bphe reductase, capable of reducing the isoprenoid moiety of Bphe only. We propose that Rhodospirillum rubrum is a naturally occurring bchP mutant and that an insertion mutation may have been the initial cause of a partial loss of function. Normal BchP function can be restored to Rhodospirillum rubrum, creating a new transconjugant strain possessing Bchl esterified with phytol. We speculate on the requirement of Rhodospirillum rubrum for phytylated Bphe and on a potential link between the absence of LH2 and of phytylated Bchl from the wild-type bacterium. The identification of a second role for the fully functional BchP in catalyzing the synthesis of phytylated Bphe strongly suggests that homologs of this enzyme may be similarly responsible for the synthesis of phytylated pheophytin in organisms possessing photosystem 2. In addition to bchP, other members of a photosynthesis gene cluster were identified in Rhodospirillum rubrum, including a bchG gene, demonstrated to encode a functional Bchl synthetase by complementation of a Rhodobacter sphaeroides mutant.
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4. |
Drennan CL,
Heo J,
Sintchak MD,
Schreiter E,
Ludden PW,
( 2001 ) Life on carbon monoxide: X-ray structure of Rhodospirillum rubrum Ni-Fe-S carbon monoxide dehydrogenase. PMID : 11593006 : DOI : 10.1073/pnas.211429998 PMC : PMC59822 Abstract >>
A crystal structure of the anaerobic Ni-Fe-S carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum has been determined to 2.8-A resolution. The CODH family, for which the R. rubrum enzyme is the prototype, catalyzes the biological oxidation of CO at an unusual Ni-Fe-S cluster called the C-cluster. The Ni-Fe-S C-cluster contains a mononuclear site and a four-metal cubane. Surprisingly, anomalous dispersion data suggest that the mononuclear site contains Fe and not Ni, and the four-metal cubane has the form [NiFe(3)S(4)] and not [Fe(4)S(4)]. The mononuclear site and the four-metal cluster are bridged by means of Cys(531) and one of the sulfides of the cube. CODH is organized as a dimer with a previously unidentified [Fe(4)S(4)] cluster bridging the two subunits. Each monomer is comprised of three domains: a helical domain at the N terminus, an alpha/beta (Rossmann-like) domain in the middle, and an alpha/beta (Rossmann-like) domain at the C terminus. The helical domain contributes ligands to the bridging [Fe(4)S(4)] cluster and another [Fe(4)S(4)] cluster, the B-cluster, which is involved in electron transfer. The two Rossmann domains contribute ligands to the active site C-cluster. This x-ray structure provides insight into the mechanism of biological CO oxidation and has broader significance for the roles of Ni and Fe in biological systems.
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5. |
Cotton NP,
White SA,
Peake SJ,
McSweeney S,
Jackson JB,
( 2001 ) The crystal structure of an asymmetric complex of the two nucleotide binding components of proton-translocating transhydrogenase. PMID : 11250201 : Abstract >>
Membrane-bound ion translocators have important functions in biology, but their mechanisms of action are often poorly understood. Transhydrogenase, found in animal mitochondria and bacteria, links the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. Linkage is achieved through changes in protein conformation at the nucleotide binding sites. The redox reaction takes place between two protein components located on the membrane surface: dI, which binds NAD(H), and dIII, which binds NADP(H). A third component, dII, provides a proton channel through the membrane. Intact membrane-located transhydrogenase is probably a dimer (two copies each of dI, dII, and dIII). We have solved the high-resolution crystal structure of a dI:dIII complex of transhydrogenase from Rhodospirillum rubrum-the first from a transhydrogenase of any species. It is a heterotrimer, having two polypeptides of dI and one of dIII. The dI polypeptides fold into a dimer. The loop on dIII, which binds the nicotinamide ring of NADP(H), is inserted into the NAD(H) binding cleft of one of the dI polypeptides. The cleft of the other dI is not occupied by a corresponding dIII component. The redox step in the transhydrogenase reaction is readily visualized; the NC4 atoms of the nicotinamide rings of the bound nucleotides are brought together to facilitate direct hydride transfer with A-B stereochemistry. The asymmetry of the dI:dIII complex suggests that in the intact enzyme there is an alternation of conformation at the catalytic sites associated with changes in nucleotide binding during proton translocation.
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6. |
Lanzilotta WN,
Schuller DJ,
Thorsteinsson MV,
Kerby RL,
Roberts GP,
Poulos TL,
( 2000 ) Structure of the CO sensing transcription activator CooA. PMID : 11017196 : DOI : 10.1038/82820 Abstract >>
CooA is a homodimeric transcription factor that belongs to the catabolite activator protein (CAP) family. Binding of CO to the heme groups of CooA leads to the transcription of genes involved in CO oxidation in Rhodospirillum rubrum. The 2.6 A structure of reduced (Fe2+) CooA reveals that His 77 in both subunits provides one heme ligand while the N-terminal nitrogen of Pro 2 from the opposite subunit provides the other ligand. A structural comparison of CooA in the absence of effector and DNA (off state) with that of CAP in the effector and DNA bound state (on state) leads to a plausible model for the mechanism of allosteric control in this class of proteins as well as the CO dependent activation of CooA.
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7. |
Jeeves M,
Smith KJ,
Quirk PG,
Cotton NP,
Jackson JB,
( 2000 ) Solution structure of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase from Rhodospirillum rubrum. PMID : 11004437 : DOI : 10.1016/s0005-2728(00)00159-6 Abstract >>
Transhydrogenase is a proton pump found in the membranes of bacteria and animal mitochondria. The solution structure of the expressed, 21.5 kDa, NADP(H)-binding component (dIII) of transhydrogenase from Rhodospirillum rubrum has been solved by NMR methods. This is the first description of the structure of dIII from a bacterial source. The protein adopts a Rossmann fold: an open, twisted, parallel beta-sheet, flanked by helices. However, the binding of NADP(+) to dIII is profoundly different to that seen in other Rossmann structures, in that its orientation is reversed: the adenosine moiety interacts with the first betaalphabetaalphabeta motif, and the nicotinamide with the second. Features in the structure that might be responsible for changes in nucleotide-binding affinity during catalysis, and for interaction with other components of the enzyme, are identified. The results are compared with the recently determined, high-resolution crystal structures of human and bovine dIII which also show the reversed nucleotide orientation.
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8. |
Dennis D,
Brückener K,
Padgette S,
Stark D,
Tran M,
Shah D,
Clemente T,
( 2000 ) Sequence of PHA synthase gene from two strains of Rhodospirillum rubrum and in vivo substrate specificity of four PHA synthases across two heterologous expression systems. PMID : 10803898 : Abstract >>
A 3.0-kb genomic fragment has been isolated from Rhodospirillum rubrum (ATCC 25903) that contains an open reading frame (ORF) with strong homology to other known polyhydroxyalkanoate (PHA) synthase genes. This ORF has lower homology to the R. rubrum strain Ha PHA synthase than would be expected within the same species. We have conducted a series of heterologous expression studies evaluating the in vivo substrate specificity of PHA synthase genes from Rhodobacter sphaeroides, Ralstonia eutropha (formerly Alcaligenes eutrophus), Thiocystis violacea, and Nocardia corrallina, within the PHA-synthase-negative hosts, Ralstonia eutropha DSM541 and Pseudomonas putida GpP104. The N. corrallina PHA synthase incorporated the highest percentage of C5 monomers in the polymer when fermented in medium supplemented with 0.1% heptanoate as the sole carbon source. When the T. violacea and R. sphaeroides were expressed in the PHA-negative host DSM541, a greater percentage of C5 monomer was observed in the polymer as compared to the expression of the PHA synthase of R. eutropha, when the transconjugants were fermented in medium supplemented with 0.4% propionate. Evaluation for preference of medium-chain-length monomers demonstrated the flexibility of the N. corrallina, T. violacea, and R. eutropha synthase genes to polymerize a copolyester composed of short- and medium-chain-length monomers when the respective transconjugants were fermented in medium supplemented with 0.5% octanoate. These studies demonstrate that the PHA synthase from N. corrallina, T. violacea, and R. eutropha are able to polymerize a copolyester composed of short- and medium-chain-length monomers, while the PHA synthase from R. sphaeroides lacks this ability and only produces a short-chain-length polymer. These observations suggest that the composition of the PHA from the PHA-producing organisms does not necessarily reflect the inherent specificity of the PHA synthase.
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9. |
Igarashi RY,
( 2000 ) Cloning and sequencing of glycogen metabolism genes from Rhodobacter sphaeroides 2.4.1. Expression and characterization of recombinant ADP-glucose pyrophosphorylase. PMID : 10729189 : DOI : 10.1006/abbi.1999.1689 Abstract >>
A 6-kb DNA fragment of the Rhodobacter sphaeroides 2.4.1 glg operon was cloned from a genomic library using a polymerase chain reaction probe coding for part of the ADP-glucose pyrophosphorylase (glgC) gene. The DNA fragment was sequenced and found to harbor complete open reading frames for the glgC and glgA (glycogen synthase) genes and partial sequences corresponding to glgP (glycogen phosphorylase) and glgX (glucan hydrolase/transferase) genes. The genomic fragment also contained an apparent truncated sequence corresponding to the C-terminus of the glgB gene (branching enzyme). The presence of active branching enzyme activity in crude sonicates of Rb. sphaeroides cells indicates that the genome contains a full-length glgB at another location. The structure of this operon in relation to other glg operons is further discussed. The deduced sequence of the ADP-glucose pyrophosphorylase enzyme is compared to other known ADP-glucose pyrophosphorylase sequences and discussed in relation to the allosteric regulation of this enzyme family. The glgC gene was subcloned in the vector pSE420 (Invitrogen) for high-level expression in E. coli. The successful overexpression of the recombinant enzyme allowed for the purification of over 35 mg of protein from 10 g of cells, representing a dramatic improvement over enzyme isolation from the native strain. The recombinant enzyme was purified to near homogeneity and found to be physically, immunologically, and kinetically identical to the native enzyme, verifying the fidelity of the cloning step.
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10. |
Brantner CA,
Cheng YS,
( 2000 ) Role of the H protein in assembly of the photochemical reaction center and intracytoplasmic membrane in Rhodospirillum rubrum. PMID : 10671438 : DOI : 10.1128/jb.182.5.1200-1207.2000 PMC : PMC94403 Abstract >>
Rhodospirillum rubrum is a model for the study of membrane formation. Under conditions of oxygen limitation, this facultatively phototrophic bacterium forms an intracytoplasmic membrane that houses the photochemical apparatus. This apparatus consists of two pigment-protein complexes, the light-harvesting antenna (LH) and photochemical reaction center (RC). The proteins of the photochemical components are encoded by the puf operon (LHalpha, LHbeta, RC-L, and RC-M) and by puhA (RC-H). R. rubrum puf interposon mutants do not form intracytoplasmic membranes and are phototrophically incompetent. The puh region was cloned, and DNA sequence determination identified open reading frames bchL and bchM and part of bchH; bchHLM encode enzymes of bacteriochlorophyll biosynthesis. A puhA/G115 interposon mutant was constructed and found to be incapable of phototrophic growth and impaired in intracytoplasmic membrane formation. Comparison of properties of the wild-type and the mutated and complemented strains suggests a model for membrane protein assembly. This model proposes that RC-H is required as a foundation protein for assembly of the RC and highly developed intracytoplasmic membrane. In complemented strains, expression of puh occurred under semiaerobic conditions, thus providing the basis for the development of an expression vector. The puhA gene alone was sufficient to restore phototrophic growth provided that recombination occurred.
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11. |
Pohlmann EL,
Zhang Y,
( 2000 ) Mutagenesis and functional characterization of the glnB, glnA, and nifA genes from the photosynthetic bacterium Rhodospirillum rubrum. PMID : 10648524 : DOI : 10.1128/jb.182.4.983-992.2000 PMC : PMC94374 Abstract >>
Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two different levels: transcriptional regulation of nif expression and posttranslational regulation of dinitrogenase reductase by reversible ADP-ribosylation catalyzed by the DRAT-DRAG (dinitrogenase reductase ADP-ribosyltransferase-dinitrogenase reductase-activating glycohydrolase) system. We report here the characterization of glnB, glnA, and nifA mutants and studies of their relationship to the regulation of nitrogen fixation. Two mutants which affect glnB (structural gene for P(II)) were constructed. While P(II)-Y51F showed a lower nitrogenase activity than that of wild type, a P(II) deletion mutant showed very little nif expression. This effect of P(II) on nif expression is apparently the result of a requirement of P(II) for NifA activation, whose activity is regulated by NH(4)(+) in R. rubrum. The modification of glutamine synthetase (GS) in these glnB mutants appears to be similar to that seen in wild type, suggesting that a paralog of P(II) might exist in R. rubrum and regulate the modification of GS. P(II) also appears to be involved in the regulation of DRAT activity, since an altered response to NH(4)(+) was found in a mutant expressing P(II)-Y51F. The adenylylation of GS plays no significant role in nif expression or the ADP-ribosylation of dinitrogenase reductase, since a mutant expressing GS-Y398F showed normal nitrogenase activity and normal modification of dinitrogenase reductase in response to NH(4)(+) and darkness treatments.
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12. |
Bishop PE,
( 1999 ) Identification of genes unique to Mo-independent nitrogenase systems in diverse diazotrophs. PMID : 10420583 : Abstract >>
A number of nitrogen-fixing bacteria were screened using PCR for genes (vnfG and anfG) unique to the V-containing nitrogenase (vnf) and the Fe-only nitrogenase (anf) systems. Products with sequences similar to that of vnfG were obtained from Azotobacter paspali and Azotobacter salinestris genomic DNAs, and products with sequences similar to that of anfG were obtained from Azomonas macrocytogenes, Rhodospirillum rubrum, and Azotobacter paspali DNAs. Phylogenetic analysis of the deduced amino acid sequences of anfG and vnfG genes shows that each gene product forms a distinct cluster. Furthermore, amplification of an internal 839-bp region in anfD and vnfD yielded a product similar to anfD from Heliobacterium gestii and a product similar to vnfD from Azotobacter paspali and Azotobacter salinestris. Phylogenetic analysis of NifD, VnfD, and AnfD amino acid sequences indicates that AnfD and VnfD sequences are more closely related to each other than either is to NifD. The results of this study suggest that Azotobacter salinestris possesses the potential to express the vanadium (V)-containing nitrogenase (nitrogenase 2) and that R. rubrum, Azomonas macrocytogenes, and H. gestii possess the potential to express the Fe-only nitrogenase (nitrogenase 3). Like Azotobacter vinelandii, Azotobacter paspali appears to have the potential to express both the V-containing nitrogenase and the Fe-only nitrogenase.
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13. |
Schneider G,
Lindqvist Y,
Brändén CI,
Lorimer G,
( 1986 ) Three-dimensional structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum at 2.9 A resolution. PMID : 16453738 : PMC : PMC1167373 Abstract >>
The three-dimensional structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum has been determined at 2.9 A resolution by X-ray crystallographic methods. The MIR-electron density map was substantially improved by two-fold non-crystallographic symmetry averaging. The polypeptide chains in the dimer were traced using a graphics display system with the help of the BONES option in FRODO. The dimer has approximate dimensions of 50 x 72 x 105 A. The enzyme subunit is a typical two-domain protein. The smaller, N-terminal domain consists of 137 amino acid residues and forms a central, mixed five-stranded beta-sheet with alpha-helices on both sides of the sheet. The larger C-terminal domain consists of 329 amino acid residues. This domain has an eight-stranded parallel alpha/beta barrel structure as found in triosephosphate isomerase and a number of other functionally non-related proteins. The active site in Rubisco determined by difference Fourier techniques and fitting of active site residues to the electron density map, is located at the carboxy-end of the beta-strands in the alpha/beta barrel of the C-terminal domain. There are few domain-domain interactions within the subunit. The interactions at the interface between the two subunits of the dimer are tight and extensive. There are tight contacts between the two C-terminal domains, which build up the core of the molecule. There are also interactions between the N-terminal domain of one subunit and the C-terminal domain of the second subunit, close to the active site.
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14. |
Kerby RL,
Hong SS,
Ensign SA,
Coppoc LJ,
Ludden PW,
Roberts GP,
( 1992 ) Genetic and physiological characterization of the Rhodospirillum rubrum carbon monoxide dehydrogenase system. PMID : 1644755 : DOI : 10.1128/jb.174.16.5284-5294.1992 PMC : PMC206364 Abstract >>
A 3.7-kb DNA region encoding part of the Rhodospirillum rubrum CO oxidation (coo) system was identified by using oligonucleotide probes. Sequence analysis of the cloned region indicated four complete or partial open reading frames (ORFs) with acceptable codon usage. The complete ORFs, the 573-bp cooF and the 1,920-bp cooS, encode an Fe/S protein and the Ni-containing carbon monoxide dehydrogenase (CODH), respectively. The four 4-cysteine motifs encoded by cooF are typical of a class of proteins associated with other oxidoreductases, including formate dehydrogenase, nitrate reductase, dimethyl sulfoxide reductase, and hydrogenase activities. The R. rubrum CODH is 67% similar to the beta subunit of the Clostridium thermoaceticum CODH and 47% similar to the alpha subunit of the Methanothrix soehngenii CODH; an alignment of these three peptides shows relatively limited overall conservation. Kanamycin cassette insertions into cooF and cooS resulted in R. rubrum strains devoid of CO-dependent H2 production with little (cooF::kan) or no (cooS::kan) methyl viologen-linked CODH activity in vitro, but did not dramatically alter their photoheterotrophic growth on malate in the presence of CO. Upstream of cooF is a 567-bp partial ORF, designated cooH, that we ascribe to the CO-induced hydrogenase, based on sequence similarity with other hydrogenases and the elimination of CO-dependent H2 production upon introduction of a cassette into this region. From mutant characterizations, we posit that cooH and cooFS are not cotranscribed. The second partial ORF starts 67 bp downstream of cooS and would be capable of encoding 35 amino acids with an ATP-binding site motif.
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15. |
Wang ZY,
Gokan K,
Kobayashi M,
Nozawa T,
( 2005 ) Solution structures of the core light-harvesting alpha and beta polypeptides from Rhodospirillum rubrum: implications for the pigment-protein and protein-protein interactions. PMID : 15740753 : DOI : 10.1016/j.jmb.2005.01.017 Abstract >>
We have determined the solution structures of the core light-harvesting (LH1) alpha and beta-polypeptides from wild-type purple photosynthetic bacterium Rhodospirillum rubrum using multidimensional NMR spectroscopy. The two polypeptides form stable alpha helices in organic solution. The structure of alpha-polypeptide consists of a long helix of 32 amino acid residues over the central transmembrane domain and a short helical segment at the N terminus that is followed by a three-residue loop. Pigment-coordinating histidine residue (His29) in the alpha-polypeptide is located near the middle of the central helix. The structure of beta-polypeptide shows a single helix of 32 amino acid residues in the membrane-spanning region with the pigment-coordinating histidine residue (His38) at a position close to the C-terminal end of the helix. Strong hydrogen bonds have been identified for the backbone amide protons over the central helical regions, indicating a rigid property of the two polypeptides. The overall structures of the R.rubrum LH1 alpha and beta-polypeptides are different from those previously reported for the LH1 beta-polypeptide of Rhodobacter sphaeroides, but are very similar to the structures of the corresponding LH2 alpha and beta-polypeptides determined by X-ray crystallography. A model constructed for the structural subunit (B820) of LH1 complex using the solution structures reveals several important features on the interactions between the LH1 alpha and beta-polypeptides. The significance of the N-terminal regions of the two polypeptides for stabilizing both B820 and LH1 complexes, as clarified by many experiments, may be attributed to the interactions between the short N-terminal helix (Trp2-Gln6) of alpha-polypeptide and a GxxxG motif in the beta-polypeptide.
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16. |
Söderlind E,
Schneider G,
Gutteridge S,
( 1992 ) Substitution of ASP193 to ASN at the active site of ribulose-1,5-bisphosphate carboxylase results in conformational changes. PMID : 1606957 : DOI : 10.1111/j.1432-1033.1992.tb16979.x Abstract >>
The crystal structure of a mutant of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillium rubrum, where Asp193, one of the ligands of the magnesium ion at the activator site, is replaced by Asn, has been determined to a nominal resolution of 0.26 nm. The mutation of Asp to Asn induces both local and global conformation changes as follows. The side chain of Asn193 moves away from the active site and interacts with main-chain oxygen of residue 165, located in the neighbouring strand beta 1 of the alpha/beta barrel. The side chain of Lys166, which forms a salt bridge with Asp193 in the wild-type enzyme, interacts with Asn54 from the second subunit and creates a new subunit-subunit interaction. Another new subunit-subunit interaction is formed, more than 1.2 nm away from the site of the mutation. In the mutant enzyme, the side chain of Asp263 interacts with the side chain of Thr106 from the second subunit. Asp193 is not part of a subunit-subunit interface area or an allosteric regulatory site. Nevertheless, replacement of this residue by Asn results, unexpectedly, in a difference in the packing of the two subunits, which can be described as a slight rotation of one of the subunits relative to the second. The observed structural changes at the active site of the enzyme provide a molecular explanation for the differing behaviour of the Asp193----Asn mutant with respect to activation.
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17. |
Handrick R,
Reinhardt S,
Kimmig P,
Jendrossek D,
( 2004 ) The "intracellular" poly(3-hydroxybutyrate) (PHB) depolymerase of Rhodospirillum rubrum is a periplasm-located protein with specificity for native PHB and with structural similarity to extracellular PHB depolymerases. PMID : 15489436 : DOI : 10.1128/JB.186.21.7243-7253.2004 PMC : PMC523223 Abstract >>
Rhodospirillum rubrum possesses a putative intracellular poly(3-hydroxybutyrate) (PHB) depolymerase system consisting of a soluble PHB depolymerase, a heat-stable activator, and a 3-hydroxybutyrate dimer hydrolase (J. M. Merrick and M. Doudoroff, J. Bacteriol. 88:60-71, 1964). In this study we reinvestigated the soluble R. rubrum PHB depolymerase (PhaZ1). It turned out that PhaZ1 is a novel type of PHB depolymerase with unique properties. Purified PhaZ1 was specific for amorphous short-chain-length polyhydroxyalkanoates (PHA) such as native PHB, artificial PHB, and oligomer esters of (R)-3-hydroxybutyrate with 3 or more 3-hydroxybutyrate units. Atactic PHB, (S)-3-hydroxybutyrate oligomers, medium-chain-length PHA, and lipase substrates (triolein, tributyrin) were not hydrolyzed. The PHB depolymerase structural gene (phaZ1) was cloned. Its deduced amino acid sequence (37,704 Da) had no significant similarity to those of intracellular PHB depolymerases of Wautersia eutropha or of other PHB-accumulating bacteria. PhaZ1 was found to have strong amino acid homology with type-II catalytic domains of extracellular PHB depolymerases, and Ser(42), Asp(138), and His(178) were identified as catalytic-triad amino acids, with Ser(42) as the putative active site. Surprisingly, the first 23 amino acids of the PHB depolymerase previously assumed to be intracellular revealed features of classical signal peptides, and Edman sequencing of purified PhaZ1 confirmed the functionality of the predicted cleavage site. Extracellular PHB depolymerase activity was absent, and analysis of cell fractions unequivocally showed that PhaZ1 is a periplasm-located enzyme. The previously assumed intracellular activator/depolymerase system is unlikely to have a physiological function in PHB mobilization in vivo. A second gene, encoding the putative true intracellular PHB depolymerase (PhaZ2), was identified in the genome sequence of R. rubrum.
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18. |
Spiridonova EM,
Berg IA,
Kolganova TV,
Ivanovskiĭ RN,
Kuznetsov BB,
Turova TP,
( N/A ) [An oligonucleotide primer system for amplification of the ribulose-1,5-bisphosphate carboxylase/oxygenase genes of bacteria of various taxonomic groups]. PMID : 15315232 : Abstract >>
Based on the analysis of GenBank nucleotide sequences of the cbbL and cbbM genes, coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPC), the key enzyme of the Calvin cycle, a primer system was designed that allows about 800-bp-long fragments of these genes to be PCR-ampliflied in various photo- and chemotrophic bacteria. The efficiency of the designed primer system in detection of RuBPC genes was demonstrated in PCR with DNA of taxonomically diverse bacteria possessing RuBPC genes with a known primary structure. Nucleotide sequences of RuBPC gene fragments of bacteria belonging to the genera Acidithiobacillus. Ectothiorhodospira, Magnetospirillum, Methylocapsa, Thioalkalispira, Rhodobacter, and Rhodospirillum were determined to be deposited with GenBank and to be translated into amino acid sequences and subjected to phylogenetic analysis.
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19. |
Edgren T,
Nordlund S,
( 2004 ) The fixABCX genes in Rhodospirillum rubrum encode a putative membrane complex participating in electron transfer to nitrogenase. PMID : 15028689 : DOI : 10.1128/jb.186.7.2052-2060.2004 PMC : PMC374401 Abstract >>
In our efforts to identify the components participating in electron transport to nitrogenase in Rhodospirillum rubrum, we used mini-Tn5 mutagenesis followed by metronidazole selection. One of the mutants isolated, SNT-1, exhibited a decreased growth rate and about 25% of the in vivo nitrogenase activity compared to the wild-type values. The in vitro nitrogenase activity was essentially wild type, indicating that the mutation affects electron transport to nitrogenase. Sequencing showed that the Tn5 insertion is located in a region with a high level of similarity to fixC, and extended sequencing revealed additional putative fix genes, in the order fixABCX. Complementation of SNT-1 with the whole fix gene cluster in trans restored wild-type nitrogenase activity and growth. Using Western blotting, we demonstrated that expression of fixA and fixB occurs only under conditions under which nitrogenase also is expressed. SNT-1 was further shown to produce larger amounts of both ribulose 1,5-bisphosphate carboxylase/oxygenase and polyhydroxy alkanoates than the wild type, indicating that the redox status is affected in this mutant. Using Western blotting, we found that FixA and FixB are soluble proteins, whereas FixC most likely is a transmembrane protein. We propose that the fixABCX genes encode a membrane protein complex that plays a central role in electron transfer to nitrogenase in R. rubrum. Furthermore, we suggest that FixC is the link between nitrogen fixation and the proton motive force generated in the photosynthetic reactions.
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20. |
Majewski C,
Trebst A,
( 1990 ) The pet genes of Rhodospirillum rubrum: cloning and sequencing of the genes for the cytochrome bc1-complex. PMID : 2176269 : DOI : 10.1007/bf00262431 Abstract >>
A cytochrome bc1-complex of Rs. rubrum was isolated and the three subunits were purified to homogeneity. The N-terminal amino acid sequence of the purified subunits was determined by automatic Edman degradation. The pet genes of Rhodospirillum rubrum coding for the three subunits of the cytochrome bc1-complex were isolated from a genomic library of Rs. rubrum using oligonucleotides specific for conserved regions of the subunits from other organisms and a heterologous probe derived from the genes for the complex of Rb. capsulatus. The complete nucleotide sequence of a 5500 bp SalI/SphI fragment is described which includes the pet genes and three additional unidentified open reading frames. The N-terminal amino acid sequence of the isolated subunits was used for the identification of the three genes. The genes encoding the subunits are organized as follows: Rieske protein, cytochrome b, cytochrome c1. Comparison of the N-terminal protein sequences with the protein sequences deduced from the nucleotide sequence showed that only cytochrome c1 is processed during transport and assembly of the three subunits of the complex. Only the N-terminal methionine of the Rieske protein is cleaved off. The similarity of the deduced amino acid sequence of the three subunits to the corresponding subunits of other organisms is described and implications for structural features of the subunits are discussed.
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21. |
Schneider G,
Lindqvist Y,
Lundqvist T,
( 1990 ) Crystallographic refinement and structure of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum at 1.7 A resolution. PMID : 2107319 : DOI : 10.1016/0022-2836(90)90088-4 Abstract >>
The amino acid sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum has been fitted to the electron density maps. The resulting protein model has been refined to a nominal resolution of 1.7 A using the constrained-restrained least-squares refinement program of Sussman and the restrained least-squares refinement program of Hendrickson & Konnert. The crystallographic refinement, based on 76,452 reflections with F greater than sigma (F) in the resolution range 5.5 to 1.7 A resulted in a crystallographic R-factor of 18.0%. The asymmetric unit contains one dimeric ribulose-1,5-biphosphate carboxylase molecule, consisting of 869 amino acid residues and 736 water molecules. The geometry of the refined model is close to ideal, with root-mean-square deviations of 0.018 A in bond lengths and 2.7 degrees in bond angles. Two loop regions, comprising residues 54 to 63 and 324 to 335, and the last ten amino acid residues at the C terminus are disordered in our crystals. The expected trimodal distribution is obtained for the side-chain chi 1-angles with a marked preference for staggered conformation. The hydrogen-bonding pattern in the N-terminal beta-sheet and the parallel sheet in the beta/alpha-barrel is described. A number of hydrogen bonds and salt bridges are involved in domain-domain and subunit-subunit interactions. The subunit-subunit interface in the dimer covers an area of 2800 A2. Considerable deviations from the local 2-fold symmetry are found at both the N terminus (residues 2 to 5) and the C terminus (residues 422 to 457). Furthermore, loop 8 in the beta/alpha-barrel domain has a different conformation in the two subunits. A number of amino acid side-chains have different conformations in the two subunits. Most of these residues are located at the surface of the protein. An analysis of the individual temperature factors indicates a high mobility of the C-terminal region and for some of the loops at the active site. The positions and B-factors for 736 solvent sites have been refined (average B: 45.9 A2). Most of the solvent molecules are bound as clusters to the protein. The active site of the enzyme, especially the environment of the activator Lys191 in the non-activated enzyme is described. Crystallographic refinement at 1.7 A resolution clearly revealed the presence of a cis-proline at the active site. This residue is part of the highly conserved region Lys166-Pro167-Lys168.
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22. |
Lehman LJ,
Fitzmaurice WP,
Roberts GP,
( 1990 ) The cloning and functional characterization of the nifH gene of Rhodospirillum rubrum. PMID : 1979299 : DOI : 10.1016/0378-1119(90)90426-r Abstract >>
Dinitrogenase reductase (the nifH product) from Rhodospirillum rubrum is regulated by a post-translational modification system encoded by draTG. As demonstrated in this report, the cloning, sequencing, and functional characterization of the nifH gene provides a basis for further analysis as well as revealing interesting features of gene organization. The coding regions of nifH and draT are separated by only 400 bp, though the genes are divergently transcribed and differentially regulated. The construction of a nifH insertion caused a Nif- phenotype and destroyed the mutant's ability to synthesize both dinitrogenase and dinitrogenase reductase, verifying functionality and transcriptional organization of the nifHDK genes.
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23. |
Bérard J,
Gingras G,
( N/A ) The puh structural gene coding for the H subunit of the Rhodospirillum rubrum photoreaction center. PMID : 1903263 : Abstract >>
The Rhodospirillum rubrum structural gene puh, coding for the photoreaction center H polypeptide, and three other putative genes that surround puh were cloned and sequenced. The deduced 257 amino acid H polypeptide has a molecular weight of 27,909, in close agreement with polyacrylamide gel electrophoresis determination. Hydropathy plots predict a single hydrophobic alpha helix. The H polypeptide of Rhodospirillum rubrum shares only 23% of its residues with all three of the H polypeptides from Rhodopseudomonas viridis, Rhodobacter capsulatus, and Rhodobacter sphaeroides. Despite this apparent low degree of similarity, statistical analysis leaves no doubt about their close relatedness. Interspecies evolutionary distance, assessed by this analysis, confirms the closeness of the two Rhodobacter species, Rhodospirillum rubrum and Rhodopseudomonas viridis being approximately equidistant from them. Three regions of the H polypeptide are highly conserved in all four species. They correspond to known contact points of H with the complex of the other two (L and M) subunits on the cytoplasmic side of the membrane. A glutamic acid residue (H polypeptide residue 177), conserved in the other bacteria and suggested to be involved in the binding of secondary quinone QB, is replaced by serine in Rhodospirillum rubrum. The open reading frames G115, I2372, and I3087 are predicted to, respectively, encode polypeptides of 480, 224, and 155 residues coiled in 10, 2, and 1 transmembrane helices. Open reading frame G115 shares 56% identical residues with F1696, a sequence arranged in the genome of Rhodobacter capsulatus. The gene product of ORF I3087 is predicted to share highly similar sequences with nitrogenase reductase (encoded by nifH) of 11 different bacterial species and is suggested to have a regulatory function.
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24. |
Lundqvist T,
Schneider G,
( 1991 ) Crystal structure of the ternary complex of ribulose-1,5-bisphosphate carboxylase, Mg(II), and activator CO2 at 2.3-A resolution. PMID : 1899197 : DOI : 10.1021/bi00218a004 Abstract >>
The activated ternary complex, enzyme-CO2-Mg(II), of the dimeric ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum can be prepared in the same crystal form that was used for the crystallographic structure determination of the native nonactivated enzyme (Schneider, G., Br?nden, C.-I., & Lorimer, G. (1986) J. Mol. Biol. 187, 141-143). The three-dimensional structure of the activated enzyme has been determined to a nominal resolution of 2.3 A by protein crystallographic methods. The activator CO2 forms a carbamate with Lys191, located at the bottom of the funnel-shaped active site. In both subunits, this labile adduct is stabilized by a Mg(II) ion, bound to the carbamate and the side chains of Asp193 and Glu194. One solvent molecule was found within the first coordination sphere of the metal ion. The metal-binding site in ribulose-1,5-bisphosphate carboxylase consists thus of at least three protein ligands, all located on loop 2 of the beta/alpha barrel. One additional metal ligand, the side chain of the conserved Asn111, was observed close to the Mg(II) ion in the B-subunit. Other structural differences at the active site between the activated and nonactivated enzyme are limited to side-chain positions. Nevertheless, it is obvious that the hydrogen-bonding pattern in the vicinity of the activator site is completely altered.
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25. |
Timm J,
Brochier-Armanet C,
Perard J,
Zambelli B,
Ollagnier-de-Choudens S,
Ciurli S,
Cavazza C,
( 2017 ) The CO dehydrogenase accessory protein CooT is a novel nickel-binding protein. PMID : 28447092 : DOI : 10.1039/c7mt00063d Abstract >>
In Rhodospirillum rubrum, maturation of Carbon Monoxide Dehydrogenase (CODH) requires three accessory proteins, CooC, CooT and CooJ, dedicated to nickel insertion into the active site, which is constituted by a distorted [NiFe3S4] cubane coordinated with a mononuclear Fe site. CooC is an ATPase proposed to provide the energy required for the maturation process, while CooJ is described as a metallochaperone with 16 histidines and 2 cysteines at the C-terminus, likely involved in metal binding and/or storage. Prior to the present study, no information was available on CooT at the molecular level. Here, the X-ray structure of RrCooT was obtained, which revealed that this protein is a homodimer featuring a fold that resembles an Sm-like domain, suggesting a role in RNA metabolism that was however not supported by experimental observations. Biochemical and biophysical evidence based on circular dichroism spectroscopy, light scattering, isothermal titration calorimetry and site-directed mutagenesis showed that RrCooT specifically binds a single Ni(ii) per dimer, with a dissociation constant of 9 nM, through the pair of Cys2, highly conserved residues, located at the dimer interface. Despite its role in the activation of RrCODH in vivo, CooT was thought to be a unique protein, found only in R. rubrum, with an unclear function. In this study, we extended the biological impact of CooT, establishing that this protein is a member of a novel Ni(ii)-binding protein family with 111 homologues, linked to anaerobic metabolism in bacteria and archaea, and in most cases to the presence of CODH.
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26. |
Bélanger G,
Bérard J,
Corriveau P,
Gingras G,
( 1988 ) The structural genes coding for the L and M subunits of Rhodospirillum rubrum photoreaction center. PMID : 2836391 : Abstract >>
In Rhodospirillum rubrum, pufL, and pufM, the structural genes coding for the photoreaction center L and M polypeptides, are comprised respectively of 831 and 921 nucleotides. They are separated by a stretch of 12 nucleotides between the TAA stop codon of pufL and the first base of the ATG initiation codon of pufM. The predicted amino acid sequence of the L and M polypeptides, respectively, contain 275 and 305 residues with corresponding molecular weights of 30,473 and 33,978. Their sequences are highly homologous to those of Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas viridis. As can be deduced from the crystallographic structure of other photoreaction centers, the regions of greatest similarity are the binding sites of the cofactors involved in the photochemical reaction rather than the protein secondary structure. L and M contain, at conserved positions of their sequences, three main clusters of positively charged residues on the cytoplasmic side of the membrane. This arrangement may be involved in protein orientation during membrane assembly. Evolutionary distance of pufL and pufM, as assessed by substitution frequency analysis, confirms the closeness of the two Rhodobacter species, the other two species being equidistant from one another. Interspecies evolutionary distance is greater for pufL than for pufM.
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27. |
Fitzmaurice WP,
Saari LL,
Lowery RG,
Ludden PW,
Roberts GP,
( 1989 ) Genes coding for the reversible ADP-ribosylation system of dinitrogenase reductase from Rhodospirillum rubrum. PMID : 2506427 : DOI : 10.1007/bf00331287 Abstract >>
Nitrogen fixation activity in the photosynthetic bacterium Rhodospirillum rubrum is controlled by the reversible ADP-ribosylation of the dinitrogenase reductase component of the nitrogenase enzyme complex. This report describes the cloning and characterization of the genes encoding the ADP-ribosyltransferase (draT) and the ADP-ribosylglycohydrolase (draG) involved in this regulation. These genes are shown to be contiguous on the R. rubrum chromosome and highly linked to the nifHDK genes. Sequence analysis revealed the use of TTG as the initiation codon of the draT gene as well as a potential open reading frame immediately downstream of draG. The mono-ADP-ribosylation system in R. rubrum is the first in which both the target protein and modifying enzymes as well as their structural genes have been isolated, making it the model system of choice for analysis of this post-translational regulatory mechanism.
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28. |
Moure VR,
Costa FF,
Cruz LM,
Pedrosa FO,
Souza EM,
Li XD,
Winkler F,
Huergo LF,
( 2015 ) Regulation of nitrogenase by reversible mono-ADP-ribosylation. PMID : 24934999 : DOI : 10.1007/82_2014_380 Abstract >>
Posttranslational modification of proteins plays a key role in the regulation of a plethora of metabolic functions. Protein modification by mono-ADP-ribosylation was first described as a mechanism of action of bacterial toxins. Since these pioneering studies, the number of pathways regulated by ADP-ribosylation in organisms from all domains of life expanded significantly. However, in only a few cases the full regulatory ADP-ribosylation circuit is known. Here, we review the system where mono-ADP-ribosylation regulates the activity of an enzyme: the regulation of nitrogenase in bacteria. When the nitrogenase product, ammonium, becomes available, the ADP-ribosyltransferase (DraT) covalently links an ADP-ribose moiety to a specific arginine residue on nitrogenase switching-off nitrogenase activity. After ammonium exhaustion, the ADP-ribosylhydrolase (DraG) removes the modifying group, restoring nitrogenase activity. DraT and DraG activities are reversibly regulated through interaction with PII signaling proteins . Bioinformatics analysis showed that DraT homologs are restricted to a few nitrogen-fixing bacteria while DraG homologs are widespread in Nature. Structural comparisons indicated that bacterial DraG is closely related to Archaea and mammalian ADP-ribosylhydrolases (ARH). In all available structures, the ARH active site consists of a hydrophilic cleft carrying a binuclear Mg(2+) or Mn(2+) cluster, which is critical for catalysis.
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29. |
( 1997 ) In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: molecular and physiological characterization of cooCTJ. PMID : 9079911 : DOI : 10.1128/jb.179.7.2259-2266.1997 PMC : PMC178962 Abstract >>
The products of cooCTJ are involved in normal in vivo Ni insertion into the carbon monoxide dehydrogenase (CODH) of Rhodospirillum rubrum. Located on a 1.5-kb DNA segment immediately downstream of the CODH structural gene (cooS), two of the genes encode proteins that bear motifs reminiscent of other (urease and hydrogenase) Ni-insertion systems: a nucleoside triphosphate-binding motif near the N terminus of CooC and a run of 15 histidine residues regularly spaced over the last 30 amino acids of the C terminus of CooJ. A Gm(r)omega-linker cassette was developed to create both polar and nonpolar (60 bp) insertions in the cooCTJ region, and these, along with several deletions, were introduced into R. rubrum by homologous recombination. Analysis of the exogenous Ni levels required to sustain CO-dependent growth of the R. rubrum mutants demonstrated different phenotypes: whereas the wild-type strain and a mutant bearing a partial cooJ deletion (of the region encoding the histidine-rich segment) grew at 0.5 microM Ni supplementation, strains bearing Gm(r)omega-linker cassettes in cooT and cooJ required approximately 50-fold-higher Ni levels and all cooC insertion strains, bearing polar or nonpolar insertions, grew optimally at 550 microM Ni.
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30. |
( 1997 ) A 3-hydroxy-3-methylglutaryl-CoA lyase gene in the photosynthetic bacterium Rhodospirillum rubrum. PMID : 9003443 : DOI : 10.1016/s0167-4838(96)00158-6 Abstract >>
A 1.2 kb long DNA segment from Rhodospirillum rubrum has been sequenced (EMBL/GenBank accession number: U41280). This DNA segment includes the first sequenced gene for a putative 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, termed hmgL, from a photosynthetic organism. The sequenced segment also contains a ribosome-binding site and two clusters of possible-35 and -10 promotor sequences preceding the hmgL gene. Translation of the gene would yield a 303 amino-acid-long protein with a calculated molecular weight of 31.1 kDa. This protein shows 55-60% identity and approx. 75% similarity, including conservative substitutions, with the three eukaryotic and the single prokaryotic HMG-CoA lyases which previously have been sequenced. The R. rubrum enzyme showed stronger homology to the chicken HMG-CoA lyase than to the other bacterial protein. Significant sequence similarity was also found with homocitrate synthases from nitrogen-fixing prokaryotes. In contrast to the other sequenced prokaryotic HMG-CoA lyase (from Pseudomonas mevalonii), the R. rubrum hmgL does not seem to appear in an operon together with a HMG-CoA reductase. The hmgL gene was transcribed in photosynthetically grown cells as judged by amplification of cDNAs synthesised from DNA-free total RNA. In addition, HMG-CoA lyase activity was found in R. rubrum cells grown anaerobically in the light with leucine as the carbon source.
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31. |
( 1996 ) Characterization of the region encoding the CO-induced hydrogenase of Rhodospirillum rubrum. PMID : 8892819 : DOI : 10.1128/jb.178.21.6200-6208.1996 PMC : PMC178490 Abstract >>
In the photosynthetic bacterium Rhodospirillum rubrum, the presence of carbon monoxide (CO) induces expression of several proteins. These include carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. Together these enzymes catalyze the following conversion: CO + H2O --> CO2 + H2. This system enables R. rubrum to grow in the dark on CO as the sole energy source. Expression of this system has been shown previously to be regulated at the transcriptional level by CO. We have now identified the remainder of the CO-regulated genes encoded in a contiguous region of the R. rubrum genome. These genes, cooMKLXU, apparently encode proteins related to the function of the CO-induced hydrogenase. As seen before with the gene for the large subunit of the CO-induced hydrogenase (cooH), most of the proteins predicted by these additional genes show significant sequence similarity to subunits of Escherichia coli hydrogenase 3. In addition, all of the newly identified coo gene products show similarity to subunits of NADH-quinone oxidoreductase (energy-conserving NADH dehydrogenase I) from various eukaryotic and prokaryotic organisms. We have found that dicyclohexylcarbodiimide, an inhibitor of mitochondrial NADH dehydrogenase I (also called complex I), inhibits the CO-induced hydrogenase as well. We also show that expression of the cooMKLXUH operon is regulated by CO and the transcriptional activator CooA in a manner similar to that of the cooFSCTJ operon that encodes the subunits of CODH and related proteins.
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32. |
( 1993 ) Molecular cloning and sequencing of the ferredoxin I fdxN gene of the photosynthetic bacterium Rhodospirillum rubrum. PMID : 8399287 : DOI : 10.1016/0005-2728(93)90131-x Abstract >>
Using an oligonucleotide probe derived from the amino acid sequence of Rhodospirillum rubrum ferredoxin I, the gene (fdxN) was identified, cloned and sequenced. The FdxN coding region is 183 nucleotides which codes for a 61 amino acid (7267 Da) protein. Phylogenetic comparisons between the R. rubrum FdI and other 8Fe-8S nif-coupled ferredoxins showed only moderate degrees of similarity between the amino acid sequences. R. rubrum FdI synthesis was stimulated by nif derepressing conditions, but was not completely repressed by nif repression. Previous reports of an extracellular clostridial-type ferredoxin in R. rubrum could not be confirmed.
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33. |
( 1993 ) Complementation analysis and regulation of CO2 fixation gene expression in a ribulose 1,5-bisphosphate carboxylase-oxygenase deletion strain of Rhodospirillum rubrum. PMID : 8349547 : DOI : 10.1128/jb.175.16.5066-5077.1993 PMC : PMC204973 Abstract >>
A ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) deletion strain of Rhodospirillum rubrum that was incapable of photolithoautotrophic growth was constructed. Photoheterotrophic growth, however, was possible for the R. rubrum RubisCO deletion strain when oxidized carbon compounds such as malate were supplied. The R. rubrum RubisCO-deficient strain was not complemented to photolithoautotrophic growth by various R. rubrum DNA fragments that contain the gene encoding RubisCO, cbbM. When the R. rubrum cbbM deletion strain harbored plasmids containing R. rubrum DNA inserts with at least 2.0 kb preceding the translational start site of the cbbM gene, RubisCO activity and RubisCO antigen were detected. Lack of RubisCO expression was therefore not the cause for the failure to complement the cbbM mutant strain. Interestingly, DNA fragments encoding either of two complete Calvin-Benson-Bassham CO2- fixation (cbb) gene operons from Rhodobacter sphaeroides were able to complement the R. rubrum RubisCO deletion strain to photolithoautotrophic growth. The same R. rubrum DNA fragments that failed to complement the R. rubrum cbbM deletion strain successfully complemented the RubisCO deletion strain of R. sphaeroides, pointing to distinct differences in the regulation of metabolism and the genetics of photolithoautotrophic growth in these two organisms. A number of cbb genes were identified by nucleotide sequence analysis of the region upstream of cbbM. Included among these was an open reading frame encoding a cbbR gene showing a high degree of sequence similarity to known lysR-type CO2 fixation transcriptional activator genes. The placement and orientation of the cbbR transcriptional regulator gene in R. rubrum are unique.
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34. |
( 1995 ) Cloning of the amphibolic Calvin cycle/OPPP enzyme D-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) from spinach chloroplasts: functional and evolutionary aspects. PMID : 8616224 : DOI : 10.1007/bf00020468 Abstract >>
Exploiting the differential expression of genes for Calvin cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin cycle enzyme D-ribulose-5-phosphate 3-epimerase (R5P3E)(EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach. Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E. coli overexpressing the spinach subunit under the control of the bacteriophage T7 promoter, demonstrating that we have cloned the first functional ribulose-5-phosphate 3-epimerase from any eukaryotic source. The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum. A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E. coli genome. A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene.
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35. |
( 1996 ) Characterization of the CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum and the gene encoding the large subunit of the enzyme. PMID : 8626276 : DOI : 10.1128/jb.178.6.1515-1524.1996 PMC : PMC177833 Abstract >>
In the presence of carbon monoxide, the photosynthetic bacterium Rhodospirillum rubrum induces expression of proteins which allow the organism to metabolize carbon monoxide in the net reaction CO + H2O --> CO2 + H2. These proteins include the enzymes carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. In this paper, we present the complete amino acid sequence for the large subunit of this hydrogenase and describe the properties of the crude enzyme in relation to other known hydrogenases. The amino acid sequence deduced from the CO-induced hydrogenase large-subunit gene (cooH) shows significant similarity to large subunits of other Ni-Fe hydrogenases. The closest similarity is with HycE (58% similarity and 37% identity) from Escherichia coli, which is the large subunit of an Ni-Fe hydrogenase (isoenzyme 3). The properties of the CO-induced hydrogenase are unique. It is exceptionally resistant to inhibition by carbon monoxide. It also exhibits a very high ratio of H2 evolution to H2 uptake activity compared with other known hydrogenases. The CO-induced hydrogenase is tightly membrane bound, and its inhibition by nonionic detergents is described. Finally, the presence of nickel in the hydrogenase is addressed. Analysis of wild-type R. rubrum grown on nickel-depleted medium indicates a requirement for nickel for hydrogenase activity. However, analysis of strain UR294 (cooC insertion mutant defective in nickel insertion into CODH) shows that independent nickel insertion mechanisms are utilized by hydrogenase and CODH. CooH lacks the C-terminal peptide that is found in other Ni-Fe hydrogenases; in other systems, this peptide is cleaved during Ni processing.
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36. |
( 1993 ) An open reading frame in the Rhodospirillum rubrum plasmid, pKY1, similar to algA, encoding the bifunctional enzyme phosphomannose isomerase-guanosine diphospho-D-mannose pyrophosphorylase (PMI-GMP). PMID : 8383539 : DOI : 10.1016/0167-4781(93)90224-2 Abstract >>
The nucleotide sequence of a BglII fragment (3188 bp) from the plasmid pKY1 of Rhodospirillum rubrum was determined. A significant similarity was found between the amino acid sequences deduced from the nucleotide sequence of BglII fragment with that of algA, encoding the bifunctional enzyme with both the activities of phosphomannose isomerase and guanosine diphospho-D-mannose pyrophosphorylase of Pseudomonas aeruginosa.
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37. |
( 1996 ) A peptidoglycan binding domain in the porin-associated protein (PAP) of Rhodospirillum rubrum FR1. PMID : 8674970 : DOI : 10.1111/j.1574-6968.1996.tb08134.x Abstract >>
The porin-associated protein of Rhodospirillum rubrum FR1 was found to contain a peptidoglycan binding motif. A partial fragment of 179 amino acids, obtained by cleavage of PAP with trypsin, Asp-N protease, and CNBr, was sequenced. Substantial sequence homology was found of the C-terminal part (residues 126-179) of porin-associated protein with OmpA, the peptidoglycan-associated lipoprotein of several bacteria, protein F of Pseudomonas aeruginosa, and PIII of Neisseria gonorrhoeae, the latter being also a porin-associated protein. The 179 amino acid fragment comprised about 67% of the mass spectrometrically determined total mass of PAP of 27850 Da.
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38. |
( 1996 ) Transcription of the glnB and glnA genes in the photosynthetic bacterium Rhodospirillum rubrum. PMID : 8704966 : DOI : 10.1099/13500872-142-5-1265 Abstract >>
The PII protein, encoded by glnB, has a central role in the control of nitrogen metabolism in nitrogen-fixing prokaryotes. The glnB gene of Rhodospirillum rubrum was isolated and sequenced. The deduced amino acid sequence had very high sequence identity to other PII proteins. The glnA gene, encoding glutamine synthetase, was located 135 bp downstream of glnB and was partially sequenced. glnB is cotranscribed with glnA from a promoter with high similarity to the sigma 54-dependent promoter consensus sequence. A putative sigma 70 promoter was also identified further upstream of glnB. Northern blotting analyses showed that in addition glnA is either transcribed from an unidentified promoter or, more likely, that the glnBA transcript is processed to give the glnA mRNA. The total level of the two transcripts was much higher in nitrogen-fixing cells than in ammonia-grown cells.
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39. |
( 1995 ) CO dehydrogenase. PMID : 8561463 : DOI : 10.1146/annurev.mi.49.100195.001513 Abstract >>
Structurally and functionally diverse CO dehydrogenases are key components of various energy-yielding pathways in aerobic and anaerobic microbes from the Bacteria and Archaea domains. Aerobic microbes utilize Mo-Fe-flavin CO dehydrogenases to oxidize CO in respiratory pathways. Phototrophic anaerobes grow by converting CO to H2, a process initiating with a CO dehydrogenase that contains nickel and iron-sulfur centers. Acetate-producing anaerobes employ a nickel/iron-sulfur CO dehydrogenase to synthesize acetyl-CoA from a methyl group, CO, and CoA. A similar enzyme is responsible for the cleavage of acetyl-CoA by anaerobic Archaea that obtain energy by fermenting acetate to CH4 and CO2. Acetotrophic sulfate reducers from the Bacteria and Archaea also utilize CO dehydrogenase to cleave acetyl-CoA yielding methyl and carbonyl groups. These microbes obtain energy for growth via a respiratory pathway in which the methyl and carbonyl groups are oxidized to CO2, and sulfate is reduced to sulfide.
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40. |
Yamaguchi M,
Hatefi Y,
( 1994 ) Energy-transducing nicotinamide nucleotide transhydrogenase: nucleotide sequences of the genes and predicted amino acid sequences of the subunits of the enzyme from Rhodospirillum rubrum. PMID : 7844118 : Abstract >>
Based on the amino acid sequence of the N-terminus of the soluble subunit of the Rhodospirillum rubrum nicotinamide nucleotide transhydrogenase, two oligonucleotide primers were synthesized and used to amplify the corresponding DNA segment (110 base pairs) by the polymerase chain reaction. Using this PCR product as a probe, one clone with the insert of 6.4 kbp was isolated from a genomic library of R. rubrum and sequenced. This sequence contained three open reading frames, constituting the genes nntA1, nntA2, and nntB of the R. rubrum transhydrogenase operon. The polypeptides encoded by these genes were designated alpha 1, alpha 2, and beta, respectively, and are considered to be the subunits of the R. rubrum transhydrogenase. The predicted amino acid sequence of the alpha 1 subunit (384 residues; molecular weight 40276) has considerable sequence similarity to the alpha subunit of the Escherichia coli and the N-terminal 43-kDa segment of the bovine transhydrogenases. Like the latter, it has a beta alpha beta fold in the corresponding region, and the purified, soluble alpha 1 subunit cross-reacts with antibody to the bovine N-terminal 43-kDa fragment. The predicted amino acid sequence of the beta subunit of the R. rubrum transhydrogenase (464 residues; molecular weight 47808) has extensive sequence identity with the beta subunit of the E. coli and the corresponding C-terminal sequence of the bovine transhydrogenases. The chromatophores of R. rubrum contain a 48-kDa polypeptide, which cross-reacts with antibody to the C-terminal 20-kDa fragment of the bovine transhydrogenase. The predicted amino acid sequence of the alpha 2 subunit of the R. rubrum enzyme (139 residues; molecular weight 14888) has considerable sequence identity in its C-terminal half to the corresponding segments of the bovine and the alpha subunit of the E. coli transhydrogenases.
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41. |
Shelver D,
Kerby RL,
He Y,
Roberts GP,
( 1995 ) Carbon monoxide-induced activation of gene expression in Rhodospirillum rubrum requires the product of cooA, a member of the cyclic AMP receptor protein family of transcriptional regulators. PMID : 7721706 : DOI : 10.1128/jb.177.8.2157-2163.1995 PMC : PMC176861 Abstract >>
Induction of the CO-oxidizing system of the photosynthetic bacterium Rhodospirillum rubrum is regulated at the level of gene expression by the presence of CO. In this paper, we describe the identification of a gene that is required for CO-induced gene expression. An 11-kb deletion of the region adjacent to the previously characterized cooFSCTJ region resulted in a mutant unable to synthesize CO dehydrogenase in response to CO and unable to grow utilizing CO as an energy source. A 2.5-kb region that corresponded to a portion of the deleted region complemented this mutant for its CO regulation defect, restoring its ability to grow utilizing CO as an energy source. When the 2.5-kb region was sequenced, one open reading frame, designated cooA, predicted a product showing similarity to members of the cyclic AMP receptor protein (CRP) family of transcriptional regulators. The product, CooA, is 28% identical (51% similar) to CRP and 18% identical (45% similar) to FNR from Escherichia coli. The insertion of a drug resistance cassette into cooA resulted in a mutant that could not grow utilizing CO as an energy source. CooA contains a number of cysteine residues substituted at, or adjacent to, positions that correspond to residues that contact cyclic AMP in the crystal structure of CRP. A model based on this observation is proposed for the recognition of CO by Cooa. Adjacent to cooA are two genes, nadB and nadC, with predicted products similar to proteins in other bacteria that catalyze reactions in the de novo synthesis of NAD.(ABSTRACT TRUNCATED AT 250 WORDS)
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42. |
Lee IY,
Collins ML,
( 1993 ) Identification and partial sequence of the BchA gene of Rhodospirillum rubrum. PMID : 7763790 : Abstract >>
The DNA sequence was determined for a region upstream of the puf operon of Rhodospirillum rubrum. A partial ORF was identified. The DNA sequence and the inferred amino acid sequences were aligned with those of bchA of Rhodobacter capsulatus and other phototrophic bacteria. Based on this alignment and genetic evidence, this ORF was identified as R. rubrum bchA, which encodes an enzyme involved in bacteriochlorophyll biosynthesis. An additional ORF was identified in the intergenic region between bchA and pufB. Sequences possibly having a role in puf expression were identified.
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43. |
Dus K,
Sletten K,
Kamen MD,
( 1968 ) Cytochrome c2 of Rhodospirillum rubrum. II. Complete amino acid sequence and phylogenetic relationships. PMID : 4883098 : Abstract >>
N/A
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44. |
Fraij B,
Hartman FC,
( 1983 ) Isolation and sequencing of an active-site peptide from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase after affinity labeling with 2-[(bromoacetyl)amino]pentitol 1,5-bisphosphate. PMID : 6404301 : DOI : 10.1021/bi00275a028 Abstract >>
2-[(Bromoacetyl)amino]pentitol 1,5-bisphosphate was reported to be a highly selective affinity label for ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum [Fraij, B., & Hartman, F. C. (1982) J. Biol. Chem. 257, 3501-3505]. The enzyme has now been inactivated with a 14C-labeled reagent in order to identify the target residue at the sequence level. Subsequent to inactivation, the enzyme was carboxymethylated with iodoacetate and then digested with trypsin. The only radioactive peptide in the digest was obtained at a high degree of purity by successive chromatography on DEAE-cellulose, SP-Sephadex, and Sephadex G-25. On the basis of amino acid analysis of the purified peptide, the derivatized residue was a methionyl sulfonium salt. Automated Edman degradation confirmed the purity of the labeled peptide and established its sequence as Leu-Gln- Gly-Ala-Ser-Gly-Ile-His-Thr-Gly-Thr-Met-Gly-Phe-Gly-Lys-Met-Glu-Gly-Glu-Ser-Ser - Asp-Arg. Cleavage of this peptide with cyanogen bromide showed that the reagent moiety was covalently attached to the second methionyl residue. Sequence homology with the carboxylase/oxygenase from spinach indicates that the lysyl residue immediately preceding the alkylated methionine corresponds to Lys-334, a residue previously implicated at the active site.
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45. |
Salemme FR,
Freer ST,
Xuong NH,
Alden RA,
Kraut J,
( 1973 ) The structure of oxidized cytochrome c 2 of Rhodospirillum rubrum. PMID : 4350650 : DOI : 10.2210/pdb1c2c/pdb Abstract >>
N/A
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46. |
Brunisholz RA,
Suter F,
Zuber H,
( 1984 ) The light-harvesting polypeptides of Rhodospirillum rubrum. I. The amino-acid sequence of the second light-harvestng polypeptide B 880-beta (B 870-beta) of Rhodospirillum rubrum S 1 and the carotenoidless mutant G-9+. carotenoidless mutant G-9+. PMID : 6434396 : Abstract >>
The light-harvesting complex B 880 from Rhodospirillum rubrum S 1 (wild type) and B 870 from the carotenoidless mutant G-9+ was shown to consist mainly of an organic solvent-(chloroform/methanol-) soluble and an organic solvent-insoluble polypeptide. The isolation and separation of these two low-molecular-mass polypeptides (Mr 6101 and Mr 6079) were achieved by a two-step extraction procedure of chromatophores using in the first step chloroform/methanol containing 0.1M ammonium acetate. Following Sephadex LH-60 chromatography of this first extract a light-harvesting polypeptide (B 870-alpha) was isolated and its complete amino acid sequence was determined (R. Brunisholz et al. (1981) FEBS Lett. 129/1, 150-154, B 880-alpha: G. Gogel et al. (1983) Biochim. Biophys. Acta 746, 32-39). Upon reextraction of the chromatophore pellet with chloroform/methanol/ammonium acetate containing in addition acetic acid a second low-molecular-mass polypeptide (B 880-beta of B 870-beta) was generated. The complete amino acid sequences of the chloroform/methanol-insoluble light-harvesting polypeptide of Rs. rubrum S 1 (B 880-beta) and of Rs. rubrum G-9+ (B 870-beta) were determined. They are identical and consist of 54 amino acid residues. The conserved histidine residue within the hydrophobic stretch raises more evidence for ligand complexation of bacteriochlorophyll to this specific histidine residue which therefore possibly plays the key role in pigment-protein interactions. Both polypeptides (B 880-alpha and B 880-beta) are part of the light-harvesting complex B 880 in an apparent ratio of 1:1. Based on the primary structure data a possible arrangement of both light-harvesting polypeptides within the membrane will be discussed.
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47. |
Matsubara H,
Inoue K,
Hase T,
Hiura H,
Kakuno T,
Yamashita J,
Horio T,
( 1983 ) Structure of the extracellular ferredoxin from Rhodospirillum rubrum: close similarity to clostridial ferredoxins. PMID : 6411697 : DOI : 10.1093/oxfordjournals.jbchem.a134273 Abstract >>
The amino acid sequence of an [8Fe-8S] ferredoxin isolated from the culture medium of Rhodospirillum rubrum, a photosynthetic purple non-sulfur bacterium, was determined by a combination of various conventional procedures. The sequence was A-Y-K-I-E-E-T-C-I-S-C-G-A-C-A-A-E-C-P-V-N-A-I-E-Q-G-D-T-I-F-V-V-N-A-D-T-C-I-D-C - G-N-C-A-N-V-C-P-V-G-A-P-V-A-E (55 amino acid residues). It lacked methionine, leucine, histidine, arginine, and tryptophan. The molecular weight was calculated to be 5,568 excluding iron and sulfur atoms. The distribution of 8 cysteine residues was exactly the same as that of clostridial-type ferredoxin, suggesting retention of the duplication of the bacterial ancestral ferredoxin gene. The extracellular ferredoxin of R. rubrum was compared with other ferredoxins observed in closely related photosynthetic bacteria and the evolutionary significance of this ferredoxin is discussed.
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48. |
Salemme FR,
Freer ST,
Alden RA,
Kraut J,
( 1973 ) Atomic coordinates for ferricytochrome c2 of Rhodospirillum rubrum. PMID : 4200400 : DOI : 10.1016/0006-291x(73)90886-3 Abstract >>
N/A
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49. |
Zhang Y,
Cummings AD,
Burris RH,
Ludden PW,
Roberts GP,
( 1995 ) Effect of an ntrBC mutation on the posttranslational regulation of nitrogenase activity in Rhodospirillum rubrum. PMID : 7665521 : DOI : 10.1128/jb.177.18.5322-5326.1995 PMC : PMC177326 Abstract >>
Homologs of ntrB and ntrC genes from Rhodospirillum rubrum were cloned and sequenced. A mutant lacking ntrBC was constructed, and this mutant has normal nitrogenase activity under nif-derepressing conditions, indicating that ntrBC are not necessary for the expression of the nif genes in R. rubrum. However, the post-translational regulation of nitrogenase activity by ADP-ribosylation in response to NH4+ was partially abolished in this mutant. More surprisingly, the regulation of nitrogenase activity in response to darkness was also affected, suggesting a physiological link between the ntr system and energy signal transduction in R. rubrum. The expression of glutamine synthetase, as well as its posttranslational regulation, was also altered in this ntrBC mutant.
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50. |
Theiler R,
Suter F,
Zuber H,
( 1983 ) N-terminal sequences of subunits L and M of the photosynthetic reaction centre from Rhodospirillum rubrum G-9+. Separation of the subunits by gel filtration on hydroxypropylated Sephadex G 100 in organic solvents. PMID : 6199280 : Abstract >>
A new method has been developed by which subunits L and M of the photosynthetic reaction centre from Rhodospirillum rubrum G-9+ can be obtained in pure form, starting form freeze-dried chromatophore membranes. The method employs extraction into a mixture of chloroform/methanol and gel permeation chromatography on a column of hydroxypropylated Sephadex G 100. Cross-contamination of the purified subunits was less than 5% (mol/mol), as estimated by manual Edman degradation. Automated Edman degradation has been carried out with both subunits in a liquid-phase sequencer. 36 amino-acid residues of subunit L and 50 residues of subunit M could be unequivocally identified. In both cases, the sequence analyses came to a premature end as the signal sudden by dropped to the level of the accidental fluctuations of the phenylthiohydantoin-derivatives background. This effect is explained by the unusual susceptibility to peptide bond cleavage of certain threonine residues which probably underwent N leads to O acyl shift during the cleavage reactions. The N-terminal sequences have been compared to those of subunits L and M of the photosynthetic reactions centre from Rhodopseudomonas sphaeroides R-26 (sutton, M.R., Rosen, D., Feher, G. & Steiner, L.A. (1982) Biochemistry 21, 3842-3849). The homology among subunits L is close to 90% and thus markedly higher than that among subunits M (32%). This finding indicates a pre-eminent role of subunit L in the primary events of photosynthetic energy conversion.
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51. |
Pope MR,
Murrell SA,
Ludden PW,
( 1985 ) Covalent modification of the iron protein of nitrogenase from Rhodospirillum rubrum by adenosine diphosphoribosylation of a specific arginine residue. PMID : 3923473 : DOI : 10.1073/pnas.82.10.3173 PMC : PMC397737 Abstract >>
Nitrogenase in Rhodospirillum rubrum is inactivated in vivo by the covalent modification of the Fe protein with a nucleotide. The preparation of two modified peptides derived from proteolytic digestion of the inactive Fe protein is described. The modifying group is shown to be adenosine diphosphoribose, linked through the terminal ribose to a guanidino nitrogen of arginine. The structural features were established by using proton and phosphorus NMR, positive- and negative-ion fast atom bombardment mass spectrometry, and fast atom bombardment/collisionally activated decomposition mass spectrometry. Spectral methods along with chromatographic analysis and sequential degradation established the sequence of the modification site of Fe protein as Gly-Arg(ADR-ribose)-Gly-Val-Ile-Thr. This corresponds to the sequence in the Fe protein from Azotobacter vinelandii for amino acid residues 99 to 104.
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52. |
Johnson TC,
Yee BC,
Carlson DE,
Buchanan BB,
Johnson RS,
Mathews WR,
Biemann K,
( 1988 ) Thioredoxin from Rhodospirillum rubrum: primary structure and relation to thioredoxins from other photosynthetic bacteria. PMID : 3129411 : DOI : 10.1128/jb.170.5.2406-2408.1988 PMC : PMC211140 Abstract >>
Thioredoxin was isolated from a photosynthetic purple nonsulfur bacterium, Rhodospirillum rubrum, and its primary structure was determined by high-performance tandem mass spectrometry. The sequence identity of R. rubrum thioredoxin to Escherichia coli thioredoxin was intermediate to those of the Chlorobium thiosulfatophilum and Chromatium vinosum proteins. The results indicate that R. rubrum has an NADP-thioredoxin system similar to that of other photosynthetic purple bacteria.
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53. |
Lowery RG,
Ludden PW,
( 1988 ) Purification and properties of dinitrogenase reductase ADP-ribosyltransferase from the photosynthetic bacterium Rhodospirillum rubrum. PMID : 3141411 : Abstract >>
The enzyme that catalyzes the ADP-ribosylation and concomitant inactivation of dinitrogenase reductase in Rhodospirillum rubrum has been purified greater than 19,000-fold to near homogeneity. We propose dinitrogenase reductase ADP-ribosyltransferase (DRAT) as the working name for the enzyme. DRAT activity is stabilized by NaCl and ADP. The enzyme is a monomer with a molecular mass of 30 kDa and is a different polypeptide than dinitrogenase reductase activating glycohydrolase. NAD (Km = 2 mM), etheno-NAD, nicotinamide hypoxanthine dinucleotide, and nicotinamide guanine dinucleotide will serve as donor molecules in DRAT-catalyzed ADP-ribosylation reaction, and dinitrogenase reductases from R. rubrum, Azotobacter vinelandii, Klebsiella pneumoniae, and Clostridium pasteurianium will serve as acceptors. No other proteins or small molecules, including water, have been found to be effective as acceptors. Nicotinamide is released stoichiometrically with formation of the ADP-ribosylated product. DRAT is inhibited by NaCl and has maximal activity at a pH of 7.0.
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54. |
Bérard J,
Bélanger G,
Corriveau P,
Gingras G,
( 1986 ) Molecular cloning and sequence of the B880 holochrome gene from Rhodospirillum rubrum. PMID : 3001063 : Abstract >>
Restriction fragments of genomic Rhodospirillum rubrum DNA were selected according to size by electrophoresis followed by hybridization with [32P]mRNA encoding the two B880 holochrome polypeptides. The fragments were cloned into Escherchia coli C600 with plasmid pBR327 as a vector. The clones were selected by colony hybridization with 32P-holochrome-mRNA and counterselected by hybridization with Rs. rubrum ribosomal RNA, a minor contaminant of the mRNA preparation. Chimeric plasmid pRR22 was shown to contain the B880 genes by hybrid selection of B880 holochrome-mRNA. We report a restriction map of its 2.2-kilobase insert and the sequence of a 430 base pair fragment thereof. Genes alpha and beta are nearly contiguous, indicating that they are transcribed as a single operon. The predicted amino acid sequences coincide with the sequences of the alpha and beta polypeptides established in other laboratories, except for additional C-terminal tails of 10 and 13 amino acid residues, respectively. We suggest that these tail sequences may serve, during membrane assembly, to give these intrinsic membrane proteins their peculiar orientation with their C-terminus facing the periplasm and their N terminus facing the cytoplasm. Intraspecific sequence homology between the alpha and beta genes of R. rubrum is low, showing no evolutionary relatedness. This is in contrast to the high interspecific homology between the corresponding sequences of Rs. rubrum and Rhodopseudomonas capsulata B880 genes.
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55. |
Falk G,
Walker JE,
( 1988 ) DNA sequence of a gene cluster coding for subunits of the F0 membrane sector of ATP synthase in Rhodospirillum rubrum. Support for modular evolution of the F1 and F0 sectors. PMID : 2902844 : DOI : 10.1042/bj2540109 PMC : PMC1135046 Abstract >>
A region was cloned from the genome of the purple non-sulphur photobacterium Rhodospirillum rubrum that contains genes coding for the membrane protein subunits of the F0 sector of ATP synthase. The clone was identified by hybridization with a synthetic oligonucleotide designed on the basis of the known protein sequence of the dicyclohexylcarbodi-imide-reactive proteolipid, or subunit c. The complete nucleotide sequence of 4240 bp of this region was determined. It is separate from an operon described previously that encodes the five subunits of the extrinsic membrane sector of the enzyme, F1-ATPase. It contains a cluster of structural genes encoding homologues of all three membrane subunits a, b and c of the Escherichia coli ATP synthase. The order of the genes in Rsp. rubrum is a-c-b'-b where b and b' are homologues. A similar gene arrangement for F0 subunits has been found in two cyanobacteria, Synechococcus 6301 and Synechococcus 6716. This suggests that the ATP synthase complexes of all these photosynthetic bacteria contain nine different polypeptides rather than eight found in the E. coli enzyme; the chloroplast ATP synthase complex is probably similar to the photosynthetic bacterial enzymes in this respect. The Rsp. rubrum b subunit is modified after translation. As shown by N-terminal sequencing of the protein, the first seven amino acid residues are removed before or during assembly of the ATP synthase complex. The subunit-a gene is preceded by a gene coding for a small hydrophobic protein, as has been observed previously in the atp operons in E. coli, bacterium PS3 and cyanobacteria. A number of features suggest that the Rsp. rubrum cluster of F0 genes is an operon. On its 5' side are found sequences resembling the -10 (Pribnow) and -35 boxes of E. coli promoters, and the gene cluster is followed by a sequence potentially able to form a stable stem-loop structure, suggesting that it acts as a rho-independent transcription terminator. These features and the small intergenic non-coding sequences suggest that the genes are cotranscribed, and so the name atp2 is proposed for this second operon coding for ATP synthase subunits in Rsp. rubrum. The finding that genes for the F0 and F1 sectors of the enzyme are in separate clusters supports the view that these represent evolutionary modules.
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56. |
( 1998 ) Substitution of valine for histidine 265 in carbon monoxide dehydrogenase from Rhodospirillum rubrum affects activity and spectroscopic states. PMID : 9461598 : DOI : 10.1074/jbc.273.7.4059 Abstract >>
In carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum, histidine 265 was replaced with valine by site-directed mutagenesis of the cooS gene. The altered form of CODH (H265V) had a low nickel content and a dramatically reduced level of catalytic activity. Although treatment with NiCl2 and CoCl2 increased the activity of H265V CODH by severalfold, activity levels remained more than 1000-fold lower than that of wild-type CODH. Histidine 265 was not essential for the formation and stability of the Fe4S4 clusters. The Km and KD for CO as well as the KD for cyanide were relatively unchanged as a result of the amino acid substitution in CODH. The time-dependent reduction of the [Fe4S4]2+ clusters by CO occurred on a time scale of hours, suggesting that, as a consequence of the mutation, a rate-limiting step had been introduced prior to the transfer of electrons from CO to the cubanes in centers B and C. EPR spectra of H265V CODH lacked the gav = 1.86 and gav = 1.87 signals characteristic of reduced forms of the active site (center C) of wild-type CODH. This indicates that the electronic properties of center C have been modified possibly by the disruption or alteration of the ligand-mediated interaction between the nickel site and Fe4S4 chromophore.
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