The effect of selenite on the growth rate and protein synthesis has been investigated in Rhodobacter sphaeroides. This photosynthetic bacterium efficiently reduces selenite with intracellular accumulation under both dark aerobic and anaerobic photosynthetic conditions. Addition of 1 mM selenite under these two growth conditions does not affect the final cell density, although a marked slowdown in growth rate is observed under aerobic growth. The proteome analysis of selenite response by two-dimensional gel electrophoresis shows an enhanced synthesis of some chaperones, an elongation factor, and enzymes associated to oxidative stress. The induction of these antioxidant proteins confirms that the major toxic effect of selenite is the formation of reactive oxygen species during its metabolism. In addition, we show that one mutant unable to precipitate selenite, selected from a transposon library, is affected in the smoK gene. This encodes a constituent of a putative ABC transporter implicated in the uptake of polyols. This mutant is less sensitive to selenite and does not express stress proteins identified in the wild type in response to selenite. This suggests that the entry of selenite into the cytoplasm is mediated by a polyol transporter in R. sphaeroides.Selenium, a naturally occurring element, is essential for biological systems at low concentrations but toxic at higher levels. In aerobic conditions, selenium is present predominantly in the high valence toxic and soluble forms selenate (SeO 4 2Ϫ , ϩVI) and selenite (SeO 3 2Ϫ , ϩIV), while the dominant species in anaerobic sediments is the elemental selenium (Se 0 ). In the environment, the reduction of these oxyanions occurs principally by biotic processes. The reduction of selenate or selenite into selenide is required, for example, for the synthesis of selenocysteine, an essential residue involved in the active site of various enzymes (12,38). For a few species of bacteria, selenate or selenite acts as electron acceptors in the first steps of an anaerobic respiratory process similar to denitrification (35). To date, only four species (Thauera selenatis, Sulfospirillum barnesii SES-3, Bacillus arsicoselenatis, and Bacillus selenitireducens) that present such a potential have been isolated (22,29,36). Reduction of selenate and selenite into elemental selenium, which is insoluble and nontoxic, is also used by various species of bacteria to overcome the toxic character of the oxyanions. Detoxification of the selenium oxyanions can also be achieved by methylation of these compounds. Both reduction and/or methylation of selenate and selenite have been demonstrated in the case of purple nonsulfur photosynthetic bacteria (25,39). Intracellular sequestration of the metal after reduction has been demonstrated for Rhodobacter sphaeroides cells in the case of tellurite (26). On the other hand, an extracellular reduction of selenite occurs for bacteria such as Rhodospirillum rubrum (17) or a marine photosynthetic bacterium (41). In addition to their tolerance to high co...

The periplasmic nitrate reductase (NapAB), a member of the DMSO reductase superfamily, catalyzes the first step of the denitrification process in bacteria. In this heterodimer, a di-heme NapB subunit is associated to the catalytic NapA subunit that binds a [4Fe-4S] cluster and a bis(molybdopterin guanine dinucleotide) cofactor. Here, we report the kinetic characterization of purified mutated heterodimers from Rhodobacter sphaeroides. By combining site-directed mutagenesis, redox potentiometry, EPR spectroscopy, and enzymatic characterization, we investigate the catalytic role of two conserved residues (M153 and R392) located in the vicinity of the molybdenum active site. We demonstrate that M153 and R392 are involved in nitrate binding: the Vm measured on the M153A and R392A mutants are similar to that measured on the wild-type enzyme, whereas the Km for nitrate is increased 10-fold and 200-fold, respectively. The use of an alternative enzymatic assay led us to discover that NapAB is uncompetitively inhibited by Zn2+ ions (Ki' = 1 microM). We used this property to further probe the active site access in the mutant enzymes. It is proposed that R392 acts as a filter by preventing a direct reduction of the Mo atom by small reducing molecules and partially protecting the active site against zinc inhibition. In addition, we show that M153 is a key residue mediating this inhibition likely by coordinating Zn2+ ions via its sulfur atom. This residue is not conserved in the DMSO reductase superfamily while it is conserved in the periplasmic nitrate reductase family. Zinc inhibition is therefore likely to be specific and restricted to periplasmic nitrate reductases.

BackgroundYedY, a molybdoenzyme belonging to the sulfite oxidase family, is found in most Gram-negative bacteria. It contains a twin-arginine signal sequence that is cleaved after its translocation into the periplasm. Despite a weak reductase activity with substrates such as dimethyl sulfoxide or trimethylamine N-oxide, its natural substrate and its role in the cell remain unknown. Although sequence conservation of the YedY family displays a strictly conserved hydrophobic C-terminal residue, all known studies on Escherichia coli YedY have been performed with an enzyme containing a 6 histidine-tag at the C-terminus which could hamper enzyme activity.ResultsIn this study, we demonstrate that the tag fused to the C-terminus of Rhodobacter sphaeroides YedY is detrimental to the enzyme’s reductase activity and results in an eight-fold decrease in catalytic efficiency. Nonetheless this C-terminal tag does not influence the properties of the molybdenum active site, as assayed by EPR spectroscopy. When a cleavable His-tag was fused to the N-terminus of the mature enzyme in the absence of the signal sequence, YedY was expressed and folded with its cofactor. However, when the signal sequence was added upstream of the N-ter tag, the amount of enzyme produced was approximately ten-fold higher.ConclusionOur study thus underscores the risk of using a C-terminus tagged enzyme while studying YedY, and presents an alternative strategy to express signal sequence-containing enzymes with an N-terminal tag. It brings new insights into molybdoenzyme maturation in R. sphaeroides showing that for some enzymes, maturation can occur in the absence of the signal sequence but that its presence is required for high expression of active enzyme.

In Rhodobacter sphaeroides periplasmic nitrate reductase NapAB, the major Mo(V) form (the "high g" species) in air-purified samples is inactive and requires reduction to irreversibly convert into a catalytically competent form (Fourmond et al., J. Phys. Chem., 2008). In the present work, we study the kinetics of the activation process by combining EPR spectroscopy and direct electrochemistry. Upon reduction, the Mo (V) "high g" resting EPR signal slowly decays while the other redox centers of the protein are rapidly reduced, which we interpret as a slow and gated (or coupled) intramolecular electron transfer between the [4Fe-4S] center and the Mo cofactor in the inactive enzyme. Besides, we detect spin-spin interactions between the Mo(V) ion and the [4Fe-4S](1+) cluster which are modified upon activation of the enzyme, while the EPR signatures associated to the Mo cofactor remain almost unchanged. This shows that the activation process, which modifies the exchange coupling pathway between the Mo and the [4Fe-4S](1+) centers, occurs further away than in the first coordination sphere of the Mo ion. Relying on structural data and studies on Mo-pyranopterin and models, we propose a molecular mechanism of activation which involves the pyranopterin moiety of the molybdenum cofactor that is proximal to the [4Fe-4S] cluster. The mechanism implies both the cyclization of the pyran ring and the reduction of the oxidized pterin to give the competent tricyclic tetrahydropyranopterin form.

Methionine (Met) is prone to oxidation and can be converted to Met sulfoxide (MetO), which exists as R- and S-diastereomers. MetO can be reduced back to Met by the ubiquitous methionine sulfoxide reductase (Msr) enzymes. Canonical MsrA and MsrB were shown to be absolutely stereospecific for the reduction of S-diastereomer and R-diastereomer, respectively. Recently, a new enzymatic system, MsrQ/MsrP which is conserved in all gram-negative bacteria, was identified as a key actor for the reduction of oxidized periplasmic proteins. The haem-binding membrane protein MsrQ transmits reducing power from the electron transport chains to the molybdoenzyme MsrP, which acts as a protein-MetO reductase. The MsrQ/MsrP function was well established genetically, but the identity and biochemical properties of MsrP substrates remain unknown. In this work, using the purified MsrP enzyme from the photosynthetic bacteria Rhodobacter sphaeroides as a model, we show that it can reduce a broad spectrum of protein substrates. The most efficiently reduced MetO is found in clusters, in amino acid sequences devoid of threonine and proline on the C-terminal side. Moreover, R. sphaeroides MsrP lacks stereospecificity as it can reduce both R- and S-diastereomers of MetO, similarly to its Escherichia coli homolog, and preferentially acts on unfolded oxidized proteins. Overall, these results provide important insights into the function of a bacterial envelop protecting system, which should help understand how bacteria cope in harmful environments.

No abstract

Highly regioselective: An efficient synthesis of the imidazo[1,2-b]pyrazole core has been developed, and the first regioselective palladium-catalyzed direct arylation of the C-3 position is described (see scheme). Good to excellent yields were obtained for a wide range of aryl partners with electron-rich and electron-poor substituents. This methodology allows rapid access to a large variety of imidazo[1,2-b]pyrazole products and could open the way to the design of new biologically active compounds.

No abstract