The single superoxide dismutase of Rhodobacter capsulatus is a cambialistic,manganese-containing enzyme

The phototrophic bacterium Rhodobacter capsulatus contains a single, oxygen-responsive superoxide dismutase (SOD(Rc)) homologous to iron-containing superoxide dismutase enzymes. Recombinant SOD(Rc), however, displayed higher activity after refolding with Mn(2+), especially when the pH of the assay mixture was raised. SOD(Rc) isolated from Rhodobacter cells also preferentially contains manganese, but metal discrimination depends on the culture conditions, with iron fractions increasing from 7% in aerobic cultures up to 40% in photosynthetic cultures. Therefore, SOD(Rc) behaves as a Mn-containing dismutase with cambialistic properties.     

FeMo cofactor biosynthesis in a nifE- mutant of Rhodobacter capsulatus.

In all diazotrophic micro-organisms investigated so far, mutations in nifE, one of the genes involved in the biosynthesis of the FeMo cofactor (FeMoco), resulted in the accumulation of cofactorless inactive dinitrogenase. In this study, we have found that strains of the phototrophic non-sulfur purple bacterium Rhodobacter capsulatus with mutations in nifE, as well as in the operon harbouring the nifE gene, were capable of reducing acetylene and growing diazotrophically, although at distinctly lower rates than the wild-type strain. The diminished rates of substrate reduction were found to correlate with the decreased amounts of the dinitrogenase component (MoFe protein) expressed in R. capsulatus. The in vivo activity, as measured by the routine acetylene-reduction assay, was strictly Mo-dependent. Maximal activity was achieved under diazotrophic growth conditions and by supplementing the growth medium with molybdate (final concentration 20-50 microM). Moreover, in these strains a high proportion of ethane was produced from acetylene ( approximately 10% of ethylene) in vivo. However, in in vitro measurements with cell-free extracts as well as purified dinitrogenase, ethane production was always found to be less than 1%. The isolation and partial purification of the MoFe protein from the nifE mutant strain by Q-Sepharose chromatography and subsequent analysis by EPR spectroscopy and inductively coupled plasma MS revealed that FeMoco is actually incorporated into the protein (1.7 molecules of FeMoco per tetramer). On the basis of the results presented here, the role of NifNE in the biosynthetic pathway of the FeMoco demands reconsideration. It is shown for the first time that NifNE is not essential for biosynthesis of the cofactor, although its presence guarantees formation of a higher content of intact FeMoco-containing MoFe protein molecules. The implications of our findings for the biosynthesis of the FeMoco will be discussed.     

Ornithine lipid is required for optimal steady-state amounts of c-type cytochromes in Rhodobacter capsulatus

The c-type cytochromes are haemoproteins that are subunits or physiological partners of electron transport chain components, like the cytochrome bc(1) complex or the cbb(3)-type cytochrome c oxidase. Their haem moieties are covalently attached to the corresponding apocytochromes via a complex post-translational maturation process. During our studies of cytochrome biogenesis, we uncovered a novel class of mutants that are unable to produce ornithine lipid and that lack several c-type cytochromes. Molecular analyses of these mutants led us to the ornithine lipid biosynthesis genes of Rhodobacter capsulatus. Herein, we have characterized these mutants, and established the chemical structure of this non-phosphorus membrane lipid from R. capsulatus. Ornithine lipids are known to induce potent host immune responses, including B-lymphocyte mitogenicity, adjuvanticity and macrophage activation. Yet, despite their widespread occurrence in Eubacteria, and the diverse biological effects they elicit in mammals, their physiological role in bacterial cells remained hitherto poorly defined. Our findings now indicate that under certain bacterial growth conditions ornithine lipids are crucial for optimal steady-state amounts of some extracytoplasmic proteins, including several c-type cytochromes, and attribute them a novel and important biological function.     

Cobalamin (vitamin B(12)) biosynthesis in Rhodobacter capsulatus

In Rhodobacter capsulatus, cobalamin biosynthesis has been shown to occur when the bacteria are grown either aerobically or anaerobically. However, a comparison of the main cobalamin biosynthetic operon found within R. capsulatus would suggest that the encoded proteins belong to the oxygen-dependent pathway for cobalamin biosynthesis, although, significantly, no homologue of the essential mono-oxygenase CobG has yet been detected. Nonetheless, within this main cob operon is found a large open reading frame termed orf663 that is not found in any other cobalamin biosynthetic operon. When overproduced in Escherichia coli, orf663 was found to encode a 90 kDa integral membrane protein. Some of this protein is cleaved within E. coli to give a soluble N-terminal region that can easily be purified and yields a 50 kDa flavoprotein. When expressed in harness with the genes for precorrin-3a synthesis, ORF663 appears to mediate the transformation of precorrin-3a into a new chromophoric compound. Another open reading frame in close proximity to orf663 is termed orf647, and was found to encode a 2Fe-2S ferredoxin-like protein. We suggest that these two proteins may provide an alternative oxygen-independent mechanism for ring contraction within R. capsulatus.     

An overlap between operons involved in carotenoid and bacteriochlorophyll biosynthesis in Rhodobacter capsulatus

A new example of superoperonal gene arrangement has been documented in the Rhodobacter capsulatus photosynthetic gene cluster. The promoter for the operon initiated by the bchI gene is embedded within an upstream operon for carotenoid synthesis. The stop codon for the crtA gene, the only gene in the first operon, overlaps the start codon of the downstream bchI gene. As a consequence of this overlap, the promoter(s) for the bch operon must be located within the crtA structural gene. The bchI gene is shown here for the first time to be required for the conversion of protoporphyrin IX to subsequent intermediates in bacteriochlorophyll biosynthesis.     

Dynamin is membrane-active: lipid insertion is induced by phosphoinositides and phosphatidic acid

Dynamin is a large GTPase involved in the regulation of membrane constriction and fission during receptor-mediated endocytosis. Dynamin contains a pleckstrin-homology domain which is essential for endocytosis and which binds to anionic phospholipids. Here, we show for the first time that dynamin is a membrane-active molecule capable of penetrating into the acyl chain region of membrane lipids. Lipid penetration is strongly stimulated by phosphatidic acid (PA), phosphatidylinositol 4-phosphate, and phosphatidylinositol 4, 5-bisphosphate. Though binding is more efficient in the presence of the phosphoinositides, a much larger part of the dynamin molecule penetrates into PA-containing mixed-lipid systems. Thus, local lipid metabolism will dramatically influence dynamin-lipid interactions, and dynamin-lipid interactions are likely to play an important role in dynamin-dependent endocytosis. Our data suggest that dynamin is directly involved in membrane destabilization, a prerequisite to membrane fission.     

Oleosin is bifunctional enzyme that has both monoacylglycerol acyltransferase and phospholipase activities

In plants, fatty oils are generally stored in spherical intracellular organelles referred to as oleosomes that are covered by proteins such as oleosin. Seeds with high oil content have more oleosin than those with low oil content. However, the exact role of oleosin in oil accumulation is thus far unclear. Here, we report the isolation of a catalytically active 14 S multiprotein complex capable of acylating monoacylglycerol from the microsomal membranes of developing peanut cotyledons. Microsomal membranes from immature peanut seeds were solubilized using 8 m urea and 10 mm CHAPS. Using two-dimensional gel electrophoresis and mass spectrometry, we identified 27 proteins in the 14 S complex. The major proteins present in the 14 S complex are conarachin, the major allergen Ara h 1, and other seed storage proteins. We identified oleosin 3 as a part of the 14 S complex, which is capable of acylating monoacylglycerol. The recombinant OLE3 microsomes from Saccharomyces cerevisiae have been shown to have both a monoacylglycerol acyltransferase and a phospholipase A(2) activity. Overexpression of the oleosin 3 (OLE3) gene in S. cerevisiae resulted in an increased accumulation of diacylglycerols and triacylglycerols and decreased phospholipids. These findings provide a direct role for a structural protein (OLE3) in the biosynthesis and mobilization of plant oils.     

TGD4 involved in endoplasmic reticulum-to-chloroplast lipid trafficking is a phosphatidic acid binding protein

The synthesis of galactoglycerolipids, which are prevalent in photosynthetic membranes, involves enzymes at the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Genetic analysis of trigalactosyldiacylglycerol (TGD) proteins in Arabidopsis has demonstrated their role in polar lipid transfer from the ER to the chloroplast. The TGD1, 2, and 3 proteins resemble components of a bacterial-type ATP-binding cassette (ABC) transporter, with TGD1 representing the permease, TGD2 the substrate binding protein, and TGD3 the ATPase. However, the function of the TGD4 protein in this process is less clear and its location in plant cells remains to be firmly determined. The predicted C-terminal β-barrel structure of TGD4 is weakly similar to proteins of the outer cell membrane of Gram-negative bacteria. Here, we show that, like TGD2, the TGD4 protein when fused to DsRED specifically binds phosphatidic acid (PtdOH). As previously shown for tgd1 mutants, tgd4 mutants have elevated PtdOH content, probably in extraplastidic membranes. Using highly purified and specific antibodies to probe different cell fractions, we demonstrated that the TGD4 protein was present in the outer envelope membrane of chloroplasts, where it appeared to be deeply buried within the membrane except for the N-terminus, which was found to be exposed to the cytosol. It is proposed that TGD4 is either directly involved in the transfer of polar lipids, possibly PtdOH, from the ER to the outer chloroplast envelope membrane or in the transfer of PtdOH through the outer envelope membrane.     

Isolation of mutants and genes involved in cytochromes c biosynthesis in Rhodobacter capsulatus.

Mutants of the photosynthetic bacterium Rhodobacter capsulatus that have combined deficiencies in the cytochrome b/c1 complex and other c-type cytochromes have been isolated. These mutants were unable to grow anaerobically in the light or dark but could grow aerobically. Cosmids with R. capsulatus wild-type DNA that complement the mutants have been used to construct genetic and physical maps of the affected genes. Complementation profiles with Tn5 and mini-Mu insertions in these cosmids and subcloned fragments from them indicated that at least three genes (called helA, helB, and helC) are involved in the defects in cytochromes c biosynthesis. The genes are clustered, and helC is transcribed away from helA and helB. Stable insertion mutants in each gene were constructed. It is postulated that helA, helB, and helC are involved in posttranslational processing during cytochromes c synthesis.     

Discovery of a bifunctional acyltransferase responsible for ornithine lipid synthesis in Serratia proteamaculans

Ornithine lipids (OLs) are phosphorus‐free membrane lipids that can be formed by many bacteria but that are absent from archaea and eukaryotes. A function for OLs in stress conditions and in host–bacteria interactions has been shown in some bacteria. Some bacterial species have been described that can form OLs, but lack the known genes (olsBA) involved in its biosynthesis, which implied the existence of a second pathway. Here we describe the bifunctional protein OlsF from Serratia proteamaculans involved in OL formation. Expression of OlsF and its homologue from Flavobacterium johnsoniae in Escherichia coli causes OL formation. Deletion of OlsF in S. proteamaculans caused the absence of OL formation. Homologues of OlsF are widely distributed among γ‐, δ‐ and ε‐Proteobacteria and in the Cytophaga‐Flavobacterium‐Bacteroidetes group of bacteria, including several well‐studied pathogens for which the presence of OLs has not been suspected, such as for example Vibrio cholerae and Klebsiella pneumonia. Using genomic data, we predict that about 50% of bacterial species can form OLs.     

Crystal structure of a bifunctional transformylase and cyclohydrolase enzyme in purine biosynthesis

ATIC, the product of the purH gene, is a 64 kDa bifunctional enzyme that possesses the final two activities in de novo purine biosynthesis, AICAR transformylase and IMP cyclohydrolase. The crystal structure of avian ATIC has been determined to 1.75 A resolution by the MAD method using a Se-methionine modified enzyme. ATIC forms an intertwined dimer with an extensive interface of approximately 5,000 A(2) per monomer. Each monomer is composed of two novel, separate functional domains. The N-terminal domain (up to residue 199) is responsible for the IMPCH activity, whereas the AICAR Tfase activity resides in the C-terminal domain (200-593). The active sites of the IMPCH and AICAR Tfase domains are approximately 50 A apart, with no structural evidence of a tunnel connecting the two active sites. The crystal structure of ATIC provides a framework to probe both catalytic mechanisms and to design specific inhibitors for use in cancer chemotherapy and inflammation.     

Carotenoid biosynthesis in photosynthetic bacteria. Genetic characterization of the Rhodobacter capsulatus CrtI protein

Carotenoids are photoprotective pigments present in many photosynthetic and nonphotosynthetic organisms. The desaturation of phytoene into phytofluene is an early step in the biosynthetic pathway that in the photosynthetic bacterium Rhodobacter capsulatus is mediated by the product of the crtI gene. Here we report the sequence of this gene and the identification of CrtI as a membrane protein of approximate Mr 60,000. Mutant strains with 5-fold lower or 10-fold higher levels of CrtI with respect to wild type have only small differences in their carotenoid content, indicating that the cellular concentration of CrtI is not a limiting factor in carotenoid biosynthesis. However, a correlation was found between the levels of CrtI and the formation of a photosynthetic antenna system.     

Sulfide-quinone and sulfide-cytochrome reduction in Rhodobacter capsulatus

The reduction by sulfide of exogenous ubiquinone is compared to the reduction of cytochromes in chromatophores of Rhodobacter capsulatus. From titrations with sulfide values for V_max of 300 and 10 moles reduced/mg bacteriochlorophyll a·h, and for K_m of 5 and 3 M were estimated, for decyl-ubiquinone-and cytochrome c-reduction, respectively. Both reactions are sensitive to KCN, as has been found for sulfide-quinone reductase (SQR) in Oscillatoria limnetica, which is a flavoprotein. Effects of inhibitors interfering with quinone binding sites suggest that at least part of the electron transport from sulfide in R. capsulatus employs the cytochrome bc ₁-complex via the ubiquinone pool.Abbreviations BChl a bacteriochlorophyll a - DAD diaminodurene - decyl-UQ decyl-ubiquinone - LED light emitting diode - NQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide - PQ-1 plastoquinone 1 - SQR sulfide-quinone reductase (E.C. 1.8.5.'.) - UQ ubiquinone 10 - Q_c the quinone reduction site on the cytochrome b ₆ f/bc ₁, complex (also termed Q_i or Q_r or Q_n) - Q_s the quinone reduction site on SQR - Q_z quinol oxidation site on the b ₆ f/bc ₁, complex (also termed Q_o or Q_p)     

The biosynthesis of phosphatidic acid and phosphatidylinositol in mammalian pancreas

1. The labelling of guinea-pig pancreas phospholipids in vivo after intraperitoneal injection of [(32)P]orthophosphate is described. 2. Acyl-CoA synthetase activity in pancreas homogenates has been studied. There is no absolute requirement for added fatty acids, indicating an adequate supply of endogenous fatty acids in these preparations. 3. Phosphatidic acid is formed in guinea-pig pancreas preparations by two distinct routes, namely the acylation of l-3-glycerophosphate and the phosphorylation of 1,2-diglyceride. Phosphatidic acid formed by either mechanism is converted into phosphatidylinositol by guinea-pig pancreas in vitro. 4. The enzymes of pancreas that convert phosphatidic acid into phosphatidylinositol via CDP-diglyceride have been characterized. 5. Addition of bovine serum albumin is necessary in assaying certain of these enzymes.     

Pulmonary phosphatidic acid phosphatase and lipid phosphate phosphohydrolase

The lung contains two distinct forms of phosphatidic acid phosphatase (PAP). PAP1 is a cytosolic enzyme that is activated through fatty acid-induced translocation to the endoplasmic reticulum, where it converts phosphatidic acid (PA) to diacylglycerol (DAG) for the biosynthesis of phospholipids and neutral lipids. PAP1 is Mg(2+) dependent and sulfhydryl reagent sensitive. PAP2 is a six-transmembrane-domain integral protein localized to the plasma membrane. Because PAP2 degrades sphingosine-1-phosphate (S1P) and ceramide-1-phosphate in addition to PA and lyso-PA, it has been renamed lipid phosphate phosphohydrolase (LPP). LPP is Mg(2+) independent and sulfhydryl reagent insensitive. This review describes LPP isoforms found in the lung and their location in signaling platforms (rafts/caveolae). Pulmonary LPPs likely function in the phospholipase D pathway, thereby controlling surfactant secretion. Through lowering the levels of lyso-PA and S1P, which serve as agonists for endothelial differentiation gene receptors, LPPs regulate cell division, differentiation, apoptosis, and mobility. LPP activity could also influence transdifferentiation of alveolar type II to type I cells. It is considered likely that these lipid phosphohydrolases have critical roles in lung morphogenesis and in acute lung injury and repair.     

Intracytoplasmic membrane vesiculation in light-harvesting mutants of Rhodobacter sphaeroides and Rhodobacter capsulatus

Abstract In Chlamydomonas reinhardtii there are three glutamate dehydrogenase isozymes which can use both NADH and NADPH as cofactors and respond differently to different nitrogen sources and several stress conditions. From data of induction of isozymes in different metabolic situations, we propose a possible physiological role for each of them in algal carbon and nitrogen metabolism.     

The role of active site glutamate residues in catalysis of Rhodobacter capsulatus xanthine dehydrogenase

Xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus catalyzes the hydroxylation of xanthine to uric acid with NAD+ as the electron acceptor. R. capsulatus XDH forms an (alphabeta)2 heterotetramer and is highly homologous to homodimeric eukaryotic xanthine oxidoreductases. Here we first describe reductive titration and steady state kinetics on recombinant wild-type R. capsulatus XDH purified from Escherichia coli, and we then proceed to evaluate the catalytic importance of the active site residues Glu-232 and Glu-730. The steady state and rapid reaction kinetics of an E232A variant exhibited a significant decrease in both kcat and kred as well as increased Km and Kd values as compared with the wild-type protein. No activity was determined for the E730A, E730Q, E730R, and E730D variants in either the steady state or rapid reaction experiments, indicating at least a 10(7) decrease in catalytic effectiveness for this variant. This result is fully consistent with the proposed role of this residue as an active site base that initiates catalysis.