Development of a NanoBRET assay to validate inhibitors of Sirt2-mediated lysine deacetylation and defatty-acylation that block prostate cancer cell migration.

Sirtuin2 (Sirt2) with its NAD+-dependent deacetylase and defatty-acylase activities plays a central role in the regulation of specific cellular functions. Dysregulation of Sirt2 activity has been associated with the pathogenesis of many diseases, thus making Sirt2 a promising target for pharmaceutical intervention. Herein, we present new high affinity Sirt2 selective Sirtuin-Rearranging Ligands (SirReals) that inhibit both Sirt2-dependent deacetylation and defatty-acylation in vitro and in cells. We show that simultaneous inhibition of both Sirt2 activities results in strongly reduced levels of the oncoprotein c-Myc and an inhibition of cancer cell migration. Furthermore, we describe the development of a NanoBRET-based assay for Sirt2, thereby providing a method to study cellular target engagement for Sirt2 in a straightforward and accurately quantifiable manner. Applying this assay, we could confirm cellular Sirt2 binding of our new Sirt2 inhibitors and correlate their anticancer effects with their cellular target engagement.

How Thermophilic Gram-Positive Organisms Perform Extracellular Electron Transfer: Characterization of the Cell Surface Terminal Reductase OcwA.

Extracellular electron transfer is the key process underpinning the development of bioelectrochemical systems for the production of energy or added-value compounds. Thermincola potens JR is a promising Gram-positive bacterium to be used in these systems because it is thermophilic. In this paper, we describe the structural and functional properties of the nonaheme cytochrome OcwA, which is the terminal reductase of this organism. The structure of OcwA, determined at 2.2-Å resolution, shows that the overall fold and organization of the hemes are not related to other metal reductases and instead are similar to those of multiheme cytochromes involved in the biogeochemical cycles of nitrogen and sulfur. We show that, in addition to solid electron acceptors, OcwA can also reduce soluble electron shuttles and oxyanions. These data reveal that OcwA can work as a multipurpose respiratory enzyme allowing this organism to grow in environments with rapidly changing availability of terminal electron acceptors without the need for transcriptional regulation and protein synthesis.IMPORTANCE Thermophilic Gram-positive organisms were recently shown to be a promising class of organisms to be used in bioelectrochemical systems for the production of electrical energy. These organisms present a thick peptidoglycan layer that was thought to preclude them to perform extracellular electron transfer (i.e., exchange catabolic electrons with solid electron acceptors outside the cell). In this paper, we describe the structure and functional mechanisms of the multiheme cytochrome OcwA, the terminal reductase of the Gram-positive bacterium Thermincola potens JR found at the cell surface of this organism. The results presented here show that this protein can take the role of a respiratory "Swiss Army knife," allowing this organism to grow in environments with soluble and insoluble substrates. Moreover, it is shown that it is unrelated to terminal reductases found at the cell surface of other electroactive organisms. Instead, OcwA is similar to terminal reductases of soluble electron acceptors. Our data reveal that terminal oxidoreductases of soluble and insoluble substrates are evolutionarily related, providing novel insights into the evolutionary pathway of multiheme cytochromes.

Key bacterial multi-centered metal enzymes involved in nitrate and sulfate respiration.

Many essential life processes, such as photosynthesis, respiration, nitrogen fixation, depend on transition metal ions and their ability to catalyze multi-electron redox and hydrolytic transformations. Here we review some recent structural studies on three multi-site metal enzymes involved in respiratory processes which represent important branches within the global cycles of nitrogen and sulfur: (i) the multi-heme enzyme cytochrome c nitrite reductase, (ii) the FAD, FeS-enzyme adenosine-5'-phosphosulfate reductase, and (iii) the siroheme, FeS-enzyme sulfite reductase. Structural information comes from X-ray crystallography and spectroscopical techniques, in special cases catalytically competent intermediates could be trapped and characterized by X-ray crystallography.

Pentahaem cytochrome c nitrite reductase: reaction with hydroxylamine, a potential reaction intermediate and substrate.

The pentahaem enzyme cytochrome c nitrite reductase catalyses the reduction of nitrite to ammonia, a key reaction in the biological nitrogen cycle. The enzyme can also transform nitrogen monoxide and hydroxylamine, two potential bound reaction intermediates, into ammonia. Structural and mechanistic aspects of the multihaem enzyme are discussed in comparison with hydroxylamine oxidoreductase, a trimeric protein with eight haem molecules per subunit.

Effects of dimerization on protein electron transfer.

In order to investigate the relationship between the rate of protein-protein electron transfer and the structure of the association complex, a dimer of the blue copper protein azurin was constructed and its electron exchange properties were determined. For this purpose, a site for covalent cross-linking was engineered by replacing the surface-exposed asparagine 42 with a cysteine. This mutation enabled the formation of disulfide-linked homo-dimers of azurin. Based on NMR line-broadening experiments, the electron self-exchange (e.s.e.) rate constant for this dimer was determined to be 4.2(+/-0.7) x 10(5)M(-1)s(-1), which is a seven-fold decrease relative to wild-type azurin. This difference is ascribed to a less accessible hydrophobic patch in the dimer. To discriminate between intramolecular electron transfer within a dimer and intermolecular electron transfer between two dimers, the e.s.e. rate constant of (Cu-Cu)-N42C dimers was compared with that of (Zn-Cu)- and (Ag-Cu)-N42C dimers. As Zn and Ag are redox inactive, the intramolecular electron transfer reaction in these latter dimers can be eliminated. The e.s.e. rate constants of the three dimers are the same and an upper limit for the intramolecular electron transfer rate of 10 s(-1) could be determined. This rate is compatible with a Cu-Cu distance of 18 A or more, which is larger than the Cu - Cu distance of 15 A observed in the wild-type crystal structure that shows two monomers that face each other with opposing hydrophobic patches. Modelling of the dimer shows that the Cu-Cu distance should be in the range of 17 A < rCu-Cu < 28 A, which is in agreement with the experimental findings. For efficient electron transfer, it appears crucial that the two molecules interact in the proper orientation. Direct cross-linking may disturb the formation of such an optimal electron transfer complex.

Spectroscopic investigation and determination of reactivity and structure of the tetraheme cytochrome c3 from Desulfovibrio desulfuricans Essex 6.

Cytochrome c3, a small (14-kDa) soluble tetraheme protein was isolated from the periplasmic fraction of Desulfovibrio desulfuricans strain Essex 6. Its major physiological function appears to be that of an electron carrier for the periplasmic hydrogenase. It has been also shown to interact with the high-molecular-mass cytochrome complex in the cytoplasmic membrane, which eventually feeds electrons into the membraneous quinone pool, as well as with the membrane-associated dissimilatory sulfite reductase. The EPR spectra show features of four different low-spin Fe(III) hemes. Orthorhombic crystals of cytochrome c3 were obtained and X-ray diffraction data were collected to below 2 A resolution. The structure was solved by molecular replacement using cytochrome c3 from D. desulfuricans ATCC 27774 as a search model.

Crystal structure of plantacyanin, a basic blue cupredoxin from spinach.

The crystal structure of the basic blue protein (plantacyanin) from spinach (SBP) has been solved to a resolution of 2.05 A by molecular replacement using the homologous protein from cucumber (CBP) as a model. Although the sequence identity of 58% between both proteins is only moderate, the three-dimensional structures turned out to be highly similar and the buried residues, which form the hydrophobic core of the protein, are almost completely conserved. However, the redox potentials of both proteins differ by 40 mV, and a comparison of the two structures leads to a single lysine replacing a proline in the cucumber sequence, which causes a shift of the peptide chain and thus a subtle distortion of the copper ligand geometry in respect to CBP. The crystal contained three monomers of SBP in the asymmetric unit which show considerable variations in outer loop regions owing to crystal packing, but not in the regions presumed to be essential for redox partner recognition and redox potential fine tuning of the copper centers. Still, bond length variations at the copper site are at the same scale between the monomers of SBP as they are in respect to CBP, indicating that in the oxidized state the protein does not impose a high conformational strain on the copper.

Cytochrome c nitrite reductase from Wolinella succinogenes. Structure at 1.6 A resolution, inhibitor binding, and heme-packing motifs.

Cytochrome c nitrite reductase catalyzes the 6-electron reduction of nitrite to ammonia. This second part of the respiratory pathway of nitrate ammonification is a key step in the biological nitrogen cycle. The x-ray structure of the enzyme from the epsilon-proteobacterium Wolinella succinogenes has been solved to a resolution of 1.6 A. It is a pentaheme c-type cytochrome whose heme groups are packed in characteristic motifs that also occur in other multiheme cytochromes. Structures of W. succinogenes nitrite reductase have been obtained with water bound to the active site heme iron as well as complexes with two inhibitors, sulfate and azide, whose binding modes and inhibitory functions differ significantly. Cytochrome c nitrite reductase is part of a highly optimized respiratory system found in a wide range of Gram-negative bacteria. It reduces both anionic and neutral substrates at the distal side of a lysine-coordinated high-spin heme group, which is accessible through two different channels, allowing for a guided flow of reaction educt and product. Based on sequence comparison and secondary structure prediction, we have demonstrated that cytochrome c nitrite reductases constitute a protein family of high structural similarity.

Bacterial cytochrome c nitrite reductase: new structural and functional aspects.

Cytochrome c nitrite reductase catalyzes the six-electron reduction of nitrite to ammonia as a key step within the biological nitrogen cycle. Most recently, the crystal structure of the soluble enzyme from Sulfurospirillum deleyianum could be solved to 1.9 A resolution. This set the basis for new experiments on structural and functional aspects of the pentaheme protein which carries a Ca(2+) ion close to the active site heme. In the crystal, the protein was a homodimer with ten hemes in very close packing. The strong interaction between the nitrite reductase monomers also occurred in solution according to the dependence of the activity on the protein concentration. Addition of Ca(2+) to the enzyme as isolated had a stimulating effect on the activity. Ca(2+) could be removed from the enzyme by treatment with chelating agents such as EGTA or EDTA which led to a decrease in activity. In addition to nitrite, the enzyme converted NO, hydroxylamine and O-methyl hydroxylamine to ammonia at considerable rates. With N2O the activity was much lower; most likely dinitrogen was the product in this case. Cytochrome c nitrite reductase exhibited a remarkably high sulfite reductase activity, with hydrogen sulfide as the product. A paramagnetic Fe(II)-NO, S = 1/2 adduct was identified by rapid freeze EPR spectroscopy under turnover conditions with nitrite. This potential reaction intermediate of the reduction of nitrite to ammonia was also observed with PAPA NONOate and Spermine NONOate.

A NapC/NirT-type cytochrome c (NrfH) is the mediator between the quinone pool and the cytochrome c nitrite reductase of Wolinella succinogenes.

Wolinella succinogenes can grow by anaerobic respiration with nitrate or nitrite using formate as electron donor. Two forms of nitrite reductase were isolated from the membrane fraction of W. succinogenes. One form consisted of a 58 kDa polypeptide (NrfA) that was identical to the periplasmic nitrite reductase. The other form consisted of NrfA and a 22 kDa polypeptide (NrfH). Both forms catalysed nitrite reduction by reduced benzyl viologen, but only the dimeric form catalysed nitrite reduction by dimethylnaphthoquinol. Liposomes containing heterodimeric nitrite reductase, formate dehydrogenase and menaquinone catalysed the electron transport from formate to nitrite; this was coupled to the generation of an electrochemical proton potential (positive outside) across the liposomal membrane. It is concluded that the electron transfer from menaquinol to the catalytic subunit (NrfA) of W. succinogenes nitrite reductase is mediated by NrfH. The structural genes nrfA and nrfH were identified in an apparent operon (nrfHAIJ) with two additional genes. The gene nrfA encodes the precursor of NrfA carrying an N-terminal signal peptide (22 residues). NrfA (485 residues) is predicted to be a hydrophilic protein that is similar to the NrfA proteins of Sulfurospirillum deleyianum and of Escherichia coli. NrfH (177 residues) is predicted to be a membrane-bound tetrahaem cytochrome c belonging to the NapC/NirT family. The products of nrfI and nrfJ resemble proteins involved in cytochrome c biogenesis. The C-terminal third of NrfI (902 amino acid residues) is similar to CcsA proteins from Gram-positive bacteria, cyanobacteria and chloroplasts. The residual N-terminal part of NrfI resembles Ccs1 proteins. The deduced NrfJ protein resembles the thioredoxin-like proteins (ResA) of Helicobacter pylori and of Bacillus subtilis, but lacks the common motif CxxC of ResA. The properties of three deletion mutants of W. succinogenes (DeltanrfJ, DeltanrfIJ and DeltanrfAIJ) were studied. Mutants DeltanrfAIJ and DeltanrfIJ did not grow with nitrite as terminal electron acceptor or with nitrate in the absence of NH4+ and lacked nitrite reductase activity, whereas mutant DeltanrfJ showed wild-type properties. The NrfA protein formed by mutant DeltanrfIJ seemed to lack part of the haem C, suggesting that NrfI is involved in NrfA maturation.

Structure of cytochrome c nitrite reductase.

The enzyme cytochrome c nitrite reductase catalyses the six-electron reduction of nitrite to ammonia as one of the key steps in the biological nitrogen cycle, where it participates in the anaerobic energy metabolism of dissimilatory nitrate ammonification. Here we report on the crystal structure of this enzyme from the microorganism Sulfurospirillum deleyianum, which we solved by multiwavelength anomalous dispersion methods. We propose a reaction scheme for the transformation of nitrite based on structural and spectroscopic information. Cytochrome c nitrite reductase is a functional dimer, with 10 close-packed haem groups of type c and an unusual lysine-coordinated high-spin haem at the active site. By comparing the haem arrangement of this nitrite reductase with that of other multihaem cytochromes, we have been able to identify a family of proteins in which the orientation of haem groups is conserved whereas structure and function are not.