TY - JOUR A1 - Badalyan, Artavazd A1 - Dierich, Marlen A1 - Stiba, Konstanze A1 - Schwuchow, Viola A1 - Leimkühler, Silke A1 - Wollenberger, Ulla T1 - Electrical wiring of the aldehyde oxidoreductase PaoABC with a polymer containing osmium redox centers BT - biosensors for benzaldehyde and GABA JF - Biosensors N2 - Biosensors for the detection of benzaldehyde and g-aminobutyric acid (GABA) are reported using aldehyde oxidoreductase PaoABC from Escherichia coli immobilized in a polymer containing bound low potential osmium redox complexes. The electrically connected enzyme already electrooxidizes benzaldehyde at potentials below −0.15 V (vs. Ag|AgCl, 1 M KCl). The pH-dependence of benzaldehyde oxidation can be strongly influenced by the ionic strength. The effect is similar with the soluble osmium redox complex and therefore indicates a clear electrostatic effect on the bioelectrocatalytic efficiency of PaoABC in the osmium containing redox polymer. At lower ionic strength, the pH-optimum is high and can be switched to low pH-values at high ionic strength. This offers biosensing at high and low pH-values. A “reagentless” biosensor has been formed with enzyme wired onto a screen-printed electrode in a flow cell device. The response time to addition of benzaldehyde is 30 s, and the measuring range is between 10–150 µM and the detection limit of 5 µM (signal to noise ratio 3:1) of benzaldehyde. The relative standard deviation in a series (n = 13) for 200 µM benzaldehyde is 1.9%. For the biosensor, a response to succinic semialdehyde was also identified. Based on this response and the ability to work at high pH a biosensor for GABA is proposed by coimmobilizing GABA-aminotransferase (GABA-T) and PaoABC in the osmium containing redox polymer. KW - redox polymer KW - aldehyde oxidoreductase KW - ionic strength KW - benzaldehyde KW - GABA KW - biosensor Y1 - 2014 U6 - https://doi.org/10.3390/bios4040403 VL - 4 IS - 4 SP - 403 EP - 421 PB - MDPI CY - Basel ER - TY - GEN A1 - Badalyan, Artavazd A1 - Dierich, Marlen A1 - Stiba, Konstanze A1 - Schwuchow, Viola A1 - Leimkühler, Silke A1 - Wollenberger, Ulla T1 - Electrical wiring of the aldehyde oxidoreductase PaoABC with a polymer containing osmium redox centers BT - biosensors for benzaldehyde and GABA T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Biosensors for the detection of benzaldehyde and g-aminobutyric acid (GABA) are reported using aldehyde oxidoreductase PaoABC from Escherichia coli immobilized in a polymer containing bound low potential osmium redox complexes. The electrically connected enzyme already electrooxidizes benzaldehyde at potentials below −0.15 V (vs. Ag|AgCl, 1 M KCl). The pH-dependence of benzaldehyde oxidation can be strongly influenced by the ionic strength. The effect is similar with the soluble osmium redox complex and therefore indicates a clear electrostatic effect on the bioelectrocatalytic efficiency of PaoABC in the osmium containing redox polymer. At lower ionic strength, the pH-optimum is high and can be switched to low pH-values at high ionic strength. This offers biosensing at high and low pH-values. A “reagentless” biosensor has been formed with enzyme wired onto a screen-printed electrode in a flow cell device. The response time to addition of benzaldehyde is 30 s, and the measuring range is between 10–150 µM and the detection limit of 5 µM (signal to noise ratio 3:1) of benzaldehyde. The relative standard deviation in a series (n = 13) for 200 µM benzaldehyde is 1.9%. For the biosensor, a response to succinic semialdehyde was also identified. Based on this response and the ability to work at high pH a biosensor for GABA is proposed by coimmobilizing GABA-aminotransferase (GABA-T) and PaoABC in the osmium containing redox polymer. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1082 KW - redox polymer KW - aldehyde oxidoreductase KW - ionic strength KW - benzaldehyde KW - GABA KW - biosensor Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-475070 SN - 1866-8372 IS - 1082 ER - TY - JOUR A1 - Otrelo-Cardoso, Ana Rita A1 - Schwuchow, Viola A1 - Rodrigues, David A1 - Cabrita, Eurico J. A1 - Leimkühler, Silke A1 - Romao, Maria Joao A1 - Santos-Silva, Teresa T1 - Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes JF - PLoS one N2 - Molybdenum and tungsten enzymes require specific chaperones for folding and cofactor insertion. PaoD is the chaperone of the periplasmic aldehyde oxidoreductase PaoABC. It is the last gene in the paoABCD operon in Escherichia coli and its presence is crucial for obtaining mature enzyme. PaoD is an unstable, 35 kDa, protein. Our biochemical studies showed that it is a dimer in solution with a tendency to form large aggregates, especially after freezing/thawing cycles. In order to improve stability, PaoD was thawed in the presence of two ionic liquids [C(4)mim]Cl and [C(2)OHmim]PF6 and no protein precipitation was observed. This allowed protein concentration and crystallization using polyethylene glycol or ammonium sulfate as precipitating agents. Saturation transfer difference - nuclear magnetic resonance (STD-NMR) experiments have also been performed in order to investigate the effect of the ionic liquids in the stabilization process, showing a clear interaction between the acidic ring protons of the cation and, most likely, negatively charged residues at the protein surface. DLS assays also show a reduction of the overall size of the protein aggregates in presence of ionic liquids. Furthermore, cofactor binding studies on PaoD showed that the protein is able to discriminate between molybdenum and tungsten bound to the molybdenum cofactor, since only a Mo-MPT form of the cofactor remained bound to PaoD. Y1 - 2014 U6 - https://doi.org/10.1371/journal.pone.0087295 SN - 1932-6203 VL - 9 IS - 1 PB - PLoS CY - San Fransisco ER - TY - JOUR A1 - Otrelo-Cardoso, Ana Rita A1 - da Silva Correia, Marcia Alexandra A1 - Schwuchow, Viola A1 - Svergun, Dmitri I. A1 - Romao, Maria Joao A1 - Leimkühler, Silke A1 - Santos-Silva, Teresa T1 - Structural Data on the Periplasmic Aldehyde Oxidoreductase PaoABC from Escherichia coli: SAXS and Preliminary X-ray Crystallography Analysis JF - International journal of molecular sciences N2 - The periplasmic aldehyde oxidoreductase PaoABC from Escherichia coli is a molybdenum enzyme involved in detoxification of aldehydes in the cell. It is an example of an heterotrimeric enzyme of the xanthine oxidase family of enzymes which does not dimerize via its molybdenum cofactor binding domain. In order to structurally characterize PaoABC, X-ray crystallography and small angle X-ray scattering (SAXS) have been carried out. The protein crystallizes in the presence of 20% (w/v) polyethylene glycol 3350 using the hanging-drop vapour diffusion method. Although crystals were initially twinned, several experiments were done to overcome twinning and lowering the crystallization temperature (293 K to 277 K) was the solution to the problem. The non-twinned crystals used to solve the structure diffract X-rays to beyond 1.80 angstrom and belong to the C2 space group, with cell parameters a = 109.42 angstrom, b = 78.08 angstrom, c = 151.77 angstrom, = 99.77 degrees, and one molecule in the asymmetric unit. A molecular replacement solution was found for each subunit separately, using several proteins as search models. SAXS data of PaoABC were also collected showing that, in solution, the protein is also an heterotrimer. KW - periplasmic aldehyde oxidoreductase KW - X-ray crystallography KW - small angle X-ray scattering KW - crystal twinning Y1 - 2014 U6 - https://doi.org/10.3390/ijms15022223 SN - 1422-0067 VL - 15 IS - 2 SP - 2223 EP - 2236 PB - MDPI CY - Basel ER - TY - JOUR A1 - Boehmer, Nadine A1 - Hartmann, Tobias A1 - Leimkühler, Silke T1 - The chaperone FdsC for Rhodobacter capsulatus formate dehydrogenase binds the bis-molybdopterin guanine dinucleotide cofactor JF - FEBS letters : the journal for rapid publication of short reports in molecular biosciences N2 - Molybdoenzymes are complex enzymes in which the molybdenum cofactor (Moco) is deeply buried in the enzyme. Most molybdoenzymes contain a specific chaperone for the insertion of Moco. For the formate dehydrogenase FdsGBA from Rhodobacter capsulatus the two chaperones FdsC and FdsD were identified to be essential for enzyme activity, but are not a subunit of the mature enzyme. Here, we purified and characterized the FdsC protein after heterologous expression in Escherichia coli. We were able to copurify FdsC with the bound Moco derivate bis-molybdopterin guanine dinucleotide. This cofactor successfully was used as a source to reconstitute the activity of molybdoenzymes. Structured summary of protein interactions: FdsC and FdsC bind by molecular sieving (View interaction) FdsD binds to RcMobA by surface plasmon resonance (View interaction) FdsC binds to RcMobA by surface plasmon resonance (View interaction) FdsC binds to FdsA by surface plasmon resonance (View interaction) KW - Molybdenum cofactor KW - L-cysteine desulfurase KW - Formate dehydrogenase KW - Chaperone KW - bis-MGD Y1 - 2014 U6 - https://doi.org/10.1016/j.febslet.2013.12.033 SN - 0014-5793 SN - 1873-3468 VL - 588 IS - 4 SP - 531 EP - 537 PB - Elsevier CY - Amsterdam ER - TY - CHAP A1 - Leimkühler, Silke T1 - Studies on the Oxygen tolerant formate deyhdrogenase from rhodobacter capsulatus T2 - Journal of biological inorganic chemistry Y1 - 2014 SN - 0949-8257 SN - 1432-1327 VL - 19 SP - S72 EP - S72 PB - Springer CY - New York ER - TY - JOUR A1 - Marelja, Zvonimir A1 - Dambowsky, Miriam A1 - Bolis, Marco A1 - Georgiou, Marina L. A1 - Garattini, Enrico A1 - Missirlis, Fanis A1 - Leimkühler, Silke T1 - The four aldehyde oxidases of Drosophila melanogaster have different gene expression patterns and enzyme substrate specificities JF - The journal of experimental biology N2 - In the genome of Drosophila melanogaster, four genes coding for aldehyde oxidases (AOX1-4) were identified on chromosome 3. Phylogenetic analysis showed that the AOX gene cluster evolved via independent duplication events in the vertebrate and invertebrate lineages. The functional role and the substrate specificity of the distinct Drosophila AOX enzymes is unknown. Two loss-of-function mutant alleles in this gene region, low pyridoxal oxidase (Po-lpo) and aldehyde oxidase-1 (Aldox-1(n1)) are associated with a phenotype characterized by undetectable AOX enzymatic activity. However, the genes involved and the corresponding mutations have not yet been identified. In this study we characterized the activities, substrate specificities and expression profiles of the four AOX enzymes in D. melanogaster. We show that the Po-lpo-associated phenotype is the consequence of a structural alteration of the AOX1 gene. We identified an 11-bp deletion in the Po-lpo allele, resulting in a frame-shift event, which removes the molybdenum cofactor domain of the encoded enzyme. Furthermore, we show that AOX2 activity is detectable only during metamorphosis and characterize a Minos-AOX2 insertion in this developmental gene that disrupts its activity. We demonstrate that the Aldox-1(n1) phenotype maps to the AOX3 gene and AOX4 activity is not detectable in our assays. KW - Aldehyde oxidase KW - Molybdoenzymes KW - Drosophila melanogaster KW - Gene duplication KW - Substrate specificities Y1 - 2014 U6 - https://doi.org/10.1242/jeb.102129 SN - 0022-0949 SN - 1477-9145 VL - 217 IS - 12 SP - 2201 EP - 2211 PB - Company of Biologists Limited CY - Cambridge ER - TY - JOUR A1 - Dey, Pradip A1 - Adamovski, Miriam A1 - Friebe, Simon A1 - Badalyan, Artavazd A1 - Mutihac, Radu-Cristian A1 - Paulus, Florian A1 - Leimkühler, Silke A1 - Wollenberger, Ursula A1 - Haag, Rainer T1 - Dendritic polyglycerol-poly(ethylene glycol)-based polymer networks for biosensing application JF - ACS applied materials & interfaces N2 - This work describes the formation of a new dendritic polyglycerol-poly(ethylene glycol)-based 3D polymer network as a matrix for immobilization of the redox enzyme periplasmatic aldehyde oxidoreductase to create an electrochemical biosensor. The novel network is built directly on the gold surface, where it simultaneously stabilizes the enzyme for up to 4 days. The prepared biosensors can be used for amperometric detection of benzaldehyde in the range of 0.8-400 mu M. KW - biosensors KW - hydrogel KW - amperometry KW - dendritic Y1 - 2014 U6 - https://doi.org/10.1021/am502018x SN - 1944-8244 VL - 6 IS - 12 SP - 8937 EP - 8941 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Hahn, Aaron A1 - Reschke, Stefan A1 - Leimkühler, Silke A1 - Risse, Thomas T1 - Ketoxime coupling of p-Acetylphenylalanine at neutral pH for site-directed spin labeling of human sulfite oxidase JF - The journal of physical chemistry : B, Condensed matter, materials, surfaces, interfaces & biophysical chemistry N2 - Site-directed spin labeling of the unnatural amino acid p-acetylphenylalanine (p-AcPhe) using oxime based coupling chemistry is successfully applied to investigate human sulfite oxidase (hSO), a protein containing an essential cysteine residue, which impedes the use of thiol based coupling chemistry. The protein was found to be sensitive toward typical reaction conditions of oxime coupling, namely, acidic reaction conditions and elevated temperatures. Thus, coupling at neutral pH and room temperature is mandatory. Three catalysts described in the literature to accelerate the reaction rate have been tested. Best spin labeling efficiencies were observed for p-methoxyaniline, while the other catalysts described in the literature to have even better performance for oxime coupling at neutral pH were substantially less active or led to precipitation of the protein. A clear correlation of spin labeling efficiency with the local environment of the residue is found, shedding some light on the importance of the sterically demanding reaction complex between p-AcPhe, the aniline catalyst, and the spin label for the reaction rate. The analysis of the line shape has shown that its interpretation in terms of local environment is more challenging as compared to the well-established spin labels based on cysteine chemistry. To this end the results presented here indicate that the larger steric demand of the spin labeled p-AcPhe can induce structural effects instead of reporting on them. Y1 - 2014 U6 - https://doi.org/10.1021/jp503471j SN - 1520-6106 VL - 118 IS - 25 SP - 7077 EP - 7084 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Bechi, Beatrice A1 - Herter, Susanne A1 - McKenna, Shane A1 - Riley, Christopher A1 - Leimkühler, Silke A1 - Turner, Nicholas J. A1 - Carnell, Andrew J. T1 - Catalytic bio-chemo and bio-bio tandem oxidation reactions for amide and carboxylic acid synthesis JF - Green chemistry : an international journal and green chemistry resource N2 - A catalytic toolbox for three different water-based one-pot cascades to convert aryl alcohols to amides and acids and cyclic amines to lactams, involving combination of oxidative enzymes (monoamine oxidase, xanthine dehydrogenase, galactose oxidase and laccase) and chemical oxidants (TBHP or Cul(cat)/H2O2) at mild temperatures, is presented. Mutually compatible conditions were found to afford products in good to excellent yields. Y1 - 2014 U6 - https://doi.org/10.1039/c4gc01321b SN - 1463-9262 SN - 1463-9270 VL - 16 IS - 10 SP - 4524 EP - 4529 PB - Royal Society of Chemistry CY - Cambridge ER -