@article{BadalyanDierichStibaetal.2014, author = {Badalyan, Artavazd and Dierich, Marlen and Stiba, Konstanze and Schwuchow, Viola and Leimk{\"u}hler, Silke and Wollenberger, Ulla}, title = {Electrical wiring of the aldehyde oxidoreductase PaoABC with a polymer containing osmium redox centers}, series = {Biosensors}, volume = {4}, journal = {Biosensors}, number = {4}, publisher = {MDPI}, address = {Basel}, doi = {10.3390/bios4040403}, pages = {403 -- 421}, year = {2014}, abstract = {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.}, language = {en} } @article{BadalyanNeumannSchaalLeimkuehleretal.2013, author = {Badalyan, Artavazd and Neumann-Schaal, Meina and Leimk{\"u}hler, Silke and Wollenberger, Ursula}, title = {A Biosensor for aromatic aldehydes comprising the mediator dependent PaoABC-Aldehyde oxidoreductase}, series = {Electroanalysis : an international journal devoted to fundamental and practical aspects of electroanalysis}, volume = {25}, journal = {Electroanalysis : an international journal devoted to fundamental and practical aspects of electroanalysis}, number = {1}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1040-0397}, doi = {10.1002/elan.201200362}, pages = {101 -- 108}, year = {2013}, abstract = {A novel aldehyde oxidoreductase (PaoABC) from Escherichia coli was utilized for the development of an oxygen insensitive biosensor for benzaldehyde. The enzyme was immobilized in polyvinyl alcohol and currents were measured for aldehyde oxidation with different one and two electron mediators with the highest sensitivity for benzaldehyde in the presence of hexacyanoferrate(III). The benzaldehyde biosensor was optimized with respect to mediator concentration, enzyme loading and pH using potassium hexacyanoferrate(III). The linear measuring range is between 0.5200 mu M benzaldehyde. In correspondence with the substrate selectivity of the enzyme in solution the biosensor revealed a preference for aromatic aldehydes and less effective conversion of aliphatic aldehydes. The biosensor is oxygen independent, which is a particularly attractive feature for application. The biosensor can be applied to detect contaminations with benzaldehyde in solvents such as benzyl alcohol, where traces of benzaldehyde in benzyl alcohol down to 0.0042?\% can be detected.}, language = {en} } @article{BadalyanYogaSchwuchowetal.2013, author = {Badalyan, Artavazd and Yoga, Etienne Galemou and Schwuchow, Viola and P{\"o}ller, Sascha and Schuhmann, Wolfgang and Leimk{\"u}hler, Silke and Wollenberger, Ursula}, title = {Analysis of the interaction of the molybdenum hydroxylase PaoABC from Escherichia coli with positively and negatively charged metal complexes}, series = {Electrochemistry communications : an international journal dedicated to rapid publications in electrochemistry}, volume = {37}, journal = {Electrochemistry communications : an international journal dedicated to rapid publications in electrochemistry}, publisher = {Elsevier}, address = {New York}, issn = {1388-2481}, doi = {10.1016/j.elecom.2013.09.017}, pages = {5 -- 7}, year = {2013}, abstract = {An unusual behavior of the periplasmic aldehyde oxidoreductase (PaoABC) from Escherichia coil has been observed from electrochemical investigations of the enzyme catalyzed oxidation of aromatic aldehydes with different mediators under different conditions of ionic strength. The enzyme has similarity to other molybdoenzymes of the xanthine oxidase family, but the catalytic behavior turned out to be very different. Under steady state conditions the turnover of PaoABC is maximal at pH 4 for the negatively charged ferricyanide and at pH 9 for a positively charged osmium complex. Stopped-flow kinetic measurements of the catalytic half reaction showed that oxidation of benzaldehyde proceeds also above pH 7. Thus, benzaldehyde oxidation can proceed under acidic and basic conditions using this enzyme, a property which has not been described before for molybdenum hydroxylases. It is also suggested that the electron transfer with artificial electron acceptors and PaoABC can proceed at different protein sites and depends on the nature of the electron acceptor in addition to the ionic strength. (C) 2013 Elsevier B.V. All rights reserved.}, language = {en} } @article{BechiHerterMcKennaetal.2014, author = {Bechi, Beatrice and Herter, Susanne and McKenna, Shane and Riley, Christopher and Leimk{\"u}hler, Silke and Turner, Nicholas J. and Carnell, Andrew J.}, title = {Catalytic bio-chemo and bio-bio tandem oxidation reactions for amide and carboxylic acid synthesis}, series = {Green chemistry : an international journal and green chemistry resource}, volume = {16}, journal = {Green chemistry : an international journal and green chemistry resource}, number = {10}, publisher = {Royal Society of Chemistry}, address = {Cambridge}, issn = {1463-9262}, doi = {10.1039/c4gc01321b}, pages = {4524 -- 4529}, year = {2014}, abstract = {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.}, language = {en} } @article{BoehmerHartmannLeimkuehler2014, author = {Boehmer, Nadine and Hartmann, Tobias and Leimk{\"u}hler, Silke}, title = {The chaperone FdsC for Rhodobacter capsulatus formate dehydrogenase binds the bis-molybdopterin guanine dinucleotide cofactor}, series = {FEBS letters : the journal for rapid publication of short reports in molecular biosciences}, volume = {588}, journal = {FEBS letters : the journal for rapid publication of short reports in molecular biosciences}, number = {4}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0014-5793}, doi = {10.1016/j.febslet.2013.12.033}, pages = {531 -- 537}, year = {2014}, abstract = {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)}, language = {en} } @article{BrietzkeDietzKellingetal.2017, author = {Brietzke, Thomas Martin and Dietz, Thomas and Kelling, Alexandra and Schilde, Uwe and Bois, Juliana and Kelm, Harald and Reh, Manuel and Schmitz, Markus and Koerzdoerfer, Thomas and Leimk{\"u}hler, Silke and Wollenberger, Ulla and Krueger, Hans-Joerg and Holdt, Hans-J{\"u}rgen}, title = {The 1,6,7,12-Tetraazaperylene Bridging Ligand as an Electron Reservoir and Its Disulfonato Derivative as Redox Mediator in an Enzyme-Electrode Process}, series = {Chemistry - a European journal}, volume = {23}, journal = {Chemistry - a European journal}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {0947-6539}, doi = {10.1002/chem.201703639}, pages = {15583 -- 15587}, year = {2017}, abstract = {The homodinuclear ruthenium(II) complex [{Ru(l-N4Me2)}(2)(-tape)](PF6)(4) {[1](PF6)(4)} (l-N4Me2=N,N-dimethyl-2,11-diaza[3.3](2,6)-pyridinophane, tape=1,6,7,12-tetraazaperylene) can store one or two electrons in the energetically low-lying * orbital of the bridging ligand tape. The corresponding singly and doubly reduced complexes [{Ru(l-N4Me2)}(2)(-tape(.-))](PF6)(3) {[2](PF6)(3)} and [{Ru(l-N4Me2)}(2)(-tape(2-))](PF6)(2) {[3](PF6)(2)}, respectively, were electrochemically generated, successfully isolated and fully characterized by single-crystal X-ray crystallography, spectroscopic methods and magnetic susceptibility measurements. The singly reduced complex [2](PF6)(3) contains the -radical tape(.-) and the doubly reduced [3](PF6)(2) the diamagnetic dianion tape(2-) as bridging ligand, respectively. Nucleophilic aromatic substitution at the bridging tape in [1](4+) by two sulfite units gave the complex [{Ru(l-N4Me2)}(2){-tape-(SO3)(2)}](2+) ([4](2+)). Complex dication [4](2+) was exploited as a redox mediator between an anaerobic homogenous reaction solution of an enzyme system (sulfite/sulfite oxidase) and the electrode via participation of the low-energy *-orbital of the disulfonato-substituted bridging ligand tape-(SO3)(2)(2-) (E-red1=-0.1V versus Ag/AgCl/1m KCl in water).}, language = {en} } @article{BurschelDecovicNuberetal.2018, author = {Burschel, Sabrina and Decovic, Doris Kreuzer and Nuber, Franziska and Stiller, Marie and Hofmann, Maud and Zupok, Arkadiusz and Siemiatkowska, Beata and Gorka, Michal Jakub and Leimk{\"u}hler, Silke and Friedrich, Thorsten}, title = {Iron-sulfur cluster carrier proteins involved in the assembly of Escherichia coli NADH}, series = {Molecular microbiology}, volume = {111}, journal = {Molecular microbiology}, number = {1}, publisher = {Wiley}, address = {Hoboken}, issn = {0950-382X}, doi = {10.1111/mmi.14137}, pages = {31 -- 45}, year = {2018}, abstract = {The NADH:ubiquinone oxidoreductase (respiratory complex I) is the main entry point for electrons into the Escherichia coli aerobic respiratory chain. With its sophisticated setup of 13 different subunits and 10 cofactors, it is anticipated that various chaperones are needed for its proper maturation. However, very little is known about the assembly of E. coli complex I, especially concerning the incorporation of the iron-sulfur clusters. To identify iron-sulfur cluster carrier proteins possibly involved in the process, we generated knockout strains of NfuA, BolA, YajL, Mrp, GrxD and IbaG that have been reported either to be involved in the maturation of mitochondrial complex I or to exert influence on the clusters of bacterial complex. We determined the NADH and succinate oxidase activities of membranes from the mutant strains to monitor the specificity of the individual mutations for complex I. The deletion of NfuA, BolA and Mrp led to a decreased stability and partially disturbed assembly of the complex as determined by sucrose gradient centrifugation and native PAGE. EPR spectroscopy of cytoplasmic membranes revealed that the BolA deletion results in the loss of the binuclear Fe/S cluster N1b.}, language = {en} } @article{BuehningFriemelLeimkuehler2017, author = {B{\"u}hning, Martin and Friemel, Martin and Leimk{\"u}hler, Silke}, title = {Functional Complementation Studies Reveal Different Interaction Partners of Escherichia coil IscS and Human NFS1}, series = {Biochemistry}, volume = {56}, journal = {Biochemistry}, publisher = {American Chemical Society}, address = {Washington}, issn = {0006-2960}, doi = {10.1021/acs.biochem.7b00627}, pages = {4592 -- 4605}, year = {2017}, abstract = {The trafficking and delivery of sulfur to cofactors and nucleosides is a highly regulated and conserved process among all organisms. All sulfur transfer pathways generally have an L-cysteine desulfurase as an initial sulfur mobilizing enzyme in common, which serves as a sulfur donor for the biosynthesis of sulfur-containing biomolecules like iron sulfur (Fe-S) clusters, thiamine, biotin, lipoic acid, the molybdenum cofactor (Moco), and thiolated nucleosides in tRNA. The human L-cysteine desulfurase NFS1 and the Escherichia coli homologue IscS share a level of amino acid sequence identity of similar to 60\%. While E. coli IscS has a versatile role in the cell and was shown to have numerous interaction partners, NFS1 is mainly localized in mitochondria with a crucial role in the biosynthesis of Fe-S clusters. Additionally, NFS1 is also located in smaller amounts in the cytosol with a role in Moco biosynthesis and mcm(5)s(2)U34 thio modifications of nucleosides in tRNA. NFS1 and IscS were conclusively shown to have different interaction partners in their respective organisms. Here, we used functional complementation studies of an E. coli iscS deletion strain with human NFS1 to dissect their conserved roles in the transfer of sulfur to a specific target protein. Our results show that human NFS1 and E. coli IscS share conserved binding sites for proteins involved in Fe-S cluster assembly like IscU, but not with proteins for tRNA thio modifications or Moco biosynthesis. In addition, we show that human NFS1 was almost fully able to complement the role of IscS in Moco biosynthesis when its specific interaction partner protein MOCS3 from humans was also present.}, language = {en} } @article{BuehningVallerianiLeimkuehler2017, author = {B{\"u}hning, Martin and Valleriani, Angelo and Leimk{\"u}hler, Silke}, title = {The role of SufS is restricted to Fe-S cluster biosynthesis in escherichia coli}, series = {Biochemistry}, volume = {56}, journal = {Biochemistry}, publisher = {American Chemical Society}, address = {Washington}, issn = {0006-2960}, doi = {10.1021/acs.biochem.7b00040}, pages = {1987 -- 2000}, year = {2017}, abstract = {In Escherichia coli, two different systems that are important for the coordinate formation of Fe-S clusters have been identified, namely, the ISC and SUF systems. The ISC system is the housekeeping Fe-S machinery, which provides Fe-S clusters for numerous cellular proteins. The IscS protein of this system was additionally revealed to be the primary sulfur donor for several sulfur-containing molecules with important biological functions, among which are the molybdenum cofactor (Moco) and thiolated nucleosides in tRNA. Here, we show that deletion of central components of the ISC system in addition to IscS leads to an overall decrease in Fe-S cluster enzyme and molybdoenzyme activity in addition to a decrease in the number of Fe-S-dependent thiomodifications of tRNA, based on the fact that some proteins involved in Moco biosynthesis and tRNA thiolation are Fe-S-dependent. Complementation of the ISC deficient strains with the suf operon restored the activity of Fe-S-containing proteins, including the MoaA protein, which is involved in the conversion of 5′GTP to cyclic pyranopterin monophosphate in the fist step of Moco biosynthesis. While both systems share a high degree of similarity, we show that the function of their respective l-cysteine desulfurase IscS or SufS is specific for each cellular pathway. It is revealed that SufS cannot play the role of IscS in sulfur transfer for the formation of 2-thiouridine, 4-thiouridine, or the dithiolene group of molybdopterin, being unable to interact with TusA or ThiI. The results demonstrate that the role of the SUF system is exclusively restricted to Fe-S cluster assembly in the cell.}, language = {en} } @article{CazellesLalaouiHartmannetal.2016, author = {Cazelles, R. and Lalaoui, N. and Hartmann, Tobias and Leimk{\"u}hler, Silke and Wollenberger, Ursula and Antonietti, Markus and Cosnier, S.}, title = {Ready to use bioinformatics analysis as a tool to predict immobilisation strategies for protein direct electron transfer (DET)}, series = {Polymer : the international journal for the science and technology of polymers}, volume = {85}, journal = {Polymer : the international journal for the science and technology of polymers}, publisher = {Elsevier}, address = {Oxford}, issn = {0956-5663}, doi = {10.1016/j.bios.2016.04.078}, pages = {90 -- 95}, year = {2016}, language = {en} }