@article{SarauliXuDietzeletal.2012,
  author    = {Sarauli, David and Xu, Chenggang and Dietzel, Birgit and Stiba, Konstanze and Leimk{\"u}hler, Silke and Schulz, Burkhard and Lisdat, Fred},
  title     = {Thin films of substituted polyanilines interactions with biomolecular systems},
  series = {Soft matter},
  volume    = {8},
  journal   = {Soft matter},
  number    = {14},
  publisher = {Royal Society of Chemistry},
  address   = {Cambridge},
  issn      = {1744-683X},
  doi       = {10.1039/c2sm07261k},
  pages     = {3848 -- 3855},
  year      = {2012},
  abstract  = {We use substituted polyanilines for the construction of new polymer electrodes for interaction studies with the redox protein cytochrome c (cyt c) and the enzyme sulfite oxidase (SO). For these purposes four different polyaniline copolymers are chemically synthesized. Three of them are copolymers, containing 2-methoxyaniline-5-sulfonic acid with variable ratios of aniline; the fourth copolymer consists of 3-amino-benzoic acid and aniline. The results show that all polymers are suitable for being immobilized as thin stable films on gold wire and indium tin oxide (ITO) electrode surfaces from DMSO solution. This can be demonstrated by cyclic voltammetry and UV-Vis spectroscopy measurements. Moreover, cyt c can be electrochemically detected not only in solution, but also immobilized on top of the polymer films. Furthermore, the appearance of a significant catalytic current has been demonstrated for the sulfonated polyanilines, when the polymer-coated protein electrode is being measured upon addition of sulfite oxidase, confirming the establishment of a bioanalytical signal chain. Best results have been obtained for the polymer with highest sulfonation grade. The redox switching of the polymer by the enzymatic reaction can also be analyzed by following the spectral properties of the polymer electrode.},
  language  = {en}
}
@article{HartmannTeraoGarattinietal.2012,
  author    = {Hartmann, Tobias and Terao, Mineko and Garattini, Enrico and Teutloff, Christian and Alfaro, Joshua F. and Jones, Jeffrey P. and Leimk{\"u}hler, Silke},
  title     = {The impact of single nucleotide polymorphisms on human aldehyde oxidase},
  series = {Drug metabolism and disposition : the biological fate of chemicals},
  volume    = {40},
  journal   = {Drug metabolism and disposition : the biological fate of chemicals},
  number    = {5},
  publisher = {American Society for Pharmacology and Experimental Therapeutics},
  address   = {Bethesda},
  issn      = {0090-9556},
  doi       = {10.1124/dmd.111.043828},
  pages     = {856 -- 864},
  year      = {2012},
  abstract  = {Aldehyde oxidase (AO) is a complex molybdo-flavoprotein that belongs to the xanthine oxidase family. AO is active as a homodimer, and each 150-kDa monomer binds two distinct [2Fe2S] clusters, FAD, and the molybdenum cofactor. AO has an important role in the metabolism of drugs based on its broad substrate specificity oxidizing aromatic aza-heterocycles, for example, N-1-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. Sequencing the 35 coding exons of the human AOX1 gene in a sample of 180 Italian individuals led to the identification of relatively frequent, synonymous, missense and nonsense single-nucleotide polymorphisms (SNPs). Human aldehyde oxidase (hAOX1) was purified after heterologous expression in Escherichia coli. The recombinant protein was obtained with a purity of 95\% and a yield of 50 mu g/l E. coli culture. Site-directed mutagenesis of the hAOX1 cDNA allowed the purification of protein variants bearing the amino acid changes R802C, R921H, N1135S, and H1297R, which correspond to some of the identified SNPs. The hAOX1 variants were purified and compared with the wild-type protein relative to activity, oligomerization state, and metal content. Our data show that the mutation of each amino acid residue has a variable impact on the ability of hAOX1 to metabolize selected substrates. Thus, the human population is characterized by the presence of functionally inactive hAOX1 allelic variants as well as variants encoding enzymes with different catalytic activities. Our results indicate that the presence of these allelic variants should be considered for the design of future drugs.},
  language  = {en}
}
@article{ChowdhuryDoscheLoehmannsroebenetal.2012,
  author    = {Chowdhury, Mita Mullick and Dosche, Carsten and Loehmannsr{\"o}ben, Hans-Gerd and Leimk{\"u}hler, Silke},
  title     = {Dual role of the molybdenum cofactor biosynthesis protein MOCS3 in tRNA thiolation and molybdenum cofactor biosynthesis in humans},
  series = {The journal of biological chemistry},
  volume    = {287},
  journal   = {The journal of biological chemistry},
  number    = {21},
  publisher = {American Society for Biochemistry and Molecular Biology},
  address   = {Bethesda},
  issn      = {0021-9258},
  doi       = {10.1074/jbc.M112.351429},
  pages     = {17297 -- 17307},
  year      = {2012},
  abstract  = {We studied two pathways that involve the transfer of persulfide sulfur in humans, molybdenum cofactor biosynthesis and tRNA thiolation. Investigations using human cells showed that the two-domain protein MOCS3 is shared between both pathways. MOCS3 has an N-terminal adenylation domain and a C-terminal rhodanese-like domain. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. MOCS2A and URM1 are beta-grasp fold proteins that contain a highly conserved C-terminal double glycine motif. The role of the terminal glycine of MOCS2A and URM1 was examined for the interaction and the cellular localization with MOCS3. Deletion of the C-terminal glycine of either MOCS2A or URM1 resulted in a loss of interaction with MOCS3. Enhanced cyan fluorescent protein and enhanced yellow fluorescent protein fusions of the proteins were constructed, and the fluorescence resonance energy transfer efficiency was determined by the decrease in the donor lifetime. The cellular localization results showed that extension of the C terminus with an additional glycine of MOCS2A and URM1 altered the localization of MOCS3 from the cytosol to the nucleus.},
  language  = {en}
}
@article{SivanesanKalaivaniFischeretal.2012,
  author    = {Sivanesan, Arumugam and Kalaivani, Govindasamy and Fischer, Anna and Stiba, Konstanze and Leimk{\"u}hler, Silke and Weidinger, Inez M.},
  title     = {Complementary surface-enhanced resonance raman Spectroscopic Biodetection of mixed protein solutions by Chitosan- and Silica-Coated Plasmon-Tuned Silver Nanoparticles},
  series = {Analytical chemistry},
  volume    = {84},
  journal   = {Analytical chemistry},
  number    = {13},
  publisher = {American Chemical Society},
  address   = {Washington},
  issn      = {0003-2700},
  doi       = {10.1021/ac301001a},
  pages     = {5759 -- 5764},
  year      = {2012},
  abstract  = {Silver nanoparticles with identical plasmonic properties but different surface functionalities are synthesized and tested as chemically selective surface-enhanced resonance Raman (SERR) amplifiers in a two-component protein solution. The surface plasmon resonances of the particles are tuned to 413 nm to match the molecular resonance of protein heme cofactors. Biocompatible functionalization of the nanoparticles with a thin film of chitosan yields selective SERR enhancement of the anionic protein cytochrome b(5), whereas functionalization with SiO2 amplifies only the spectra of the cationic protein cytochrome c. As a result, subsequent addition of the two differently functionalized particles yields complementary information on the same mixed protein sample solution. Finally, the applicability of chitosan-coated Ag nanoparticles for protein separation was tested by in situ resonance Raman spectroscopy.},
  language  = {en}
}
@article{KalimuthuLeimkuehlerBernhardt2012,
  author    = {Kalimuthu, Palraj and Leimk{\"u}hler, Silke and Bernhardt, Paul V.},
  title     = {Catalytic electrochemistry of xanthine dehydrogenase},
  series = {The journal of physical chemistry : B, Condensed matter, materials, surfaces, interfaces \& biophysical chemistry},
  volume    = {116},
  journal   = {The journal of physical chemistry : B, Condensed matter, materials, surfaces, interfaces \& biophysical chemistry},
  number    = {38},
  publisher = {American Chemical Society},
  address   = {Washington},
  issn      = {1520-6106},
  doi       = {10.1021/jp307374z},
  pages     = {11600 -- 11607},
  year      = {2012},
  abstract  = {We report the mediated electrocatalytic voltammetry of the molybdoenzyme xanthine dehydrogenase (XDH) from Rhodobacter capsulatus at a thiol-modified Au electrode. The 2-electron acceptor N-methylphenazinium methanesulfonate (phenazine methosulfate, PMS) is an effective artificial electron transfer partner for XDH instead of its native electron acceptor NAD(+). XDH catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and xanthine to uric acid. Cyclic voltammetry was used to generate the active (oxidized) form of the mediator. Simulation of the catalytic voltammetry across a broad range of substrate and PMS concentrations at different sweep rates was achieved with the program DigiSim to yield a set of consistent rate and equilibrium constants that describe the catalytic system. This provides the first example of the mediated electrochemistry of a xanthine dehydrogenase (or oxidase) that is uncomplicated by interference from product oxidation. A remarkable two-step, sequential oxidation of hypoxanthine to uric acid via xanthine by XDH is observed.},
  language  = {en}
}
@article{RademacherHoffmannLackmannetal.2012,
  author    = {Rademacher, Corinna and Hoffmann, Marie-Christine and Lackmann, Jan-Wilm and Moser, Roman and Pf{\"a}nder, Yvonne and Leimk{\"u}hler, Silke and Narberhaus, Franz and Masepohl, Bernd},
  title     = {Tellurite resistance gene trgB confers copper tolerance to Rhodobacter capsulatus},
  series = {BioMetals : an international journal on the role of metal ions in biology, biochemistry and medicine},
  volume    = {25},
  journal   = {BioMetals : an international journal on the role of metal ions in biology, biochemistry and medicine},
  number    = {5},
  publisher = {Springer},
  address   = {Dordrecht},
  issn      = {0966-0844},
  doi       = {10.1007/s10534-012-9566-2},
  pages     = {995 -- 1008},
  year      = {2012},
  abstract  = {To identify copper homeostasis genes in Rhodobacter capsulatus, we performed random transposon Tn5 mutagenesis. Screening of more than 10,000 Tn5 mutants identified tellurite resistance gene trgB as a so far unrecognized major copper tolerance determinant. The trgB gene is flanked by tellurite resistance gene trgA and cysteine synthase gene cysK2. While growth of trgA mutants was only moderately restricted by tellurite, trgB and cysK2 mutants were severely affected by tellurite, which implies that viability under tellurite stress requires increased cysteine levels. Mutational analyses revealed that trgB was the only gene in this chromosomal region conferring cross-tolerance towards copper. Expression of the monocistronic trgB gene required promoter elements overlapping the trgA coding region as shown by nested deletions. Neither copper nor tellurite affected trgB transcription as demonstrated by reverse transcriptase PCR and trgB-lacZ fusions. Addition of tellurite or copper gave rise to increased cellular tellurium and copper concentrations, respectively, as determined by inductively coupled plasma-optical emission spectroscopy. By contrast, cellular iron concentrations remained fairly constant irrespective of tellurite or copper addition. This is the first study demonstrating a direct link between copper and tellurite response in bacteria.},
  language  = {en}
}
@article{FrascaRojasSalewskietal.2012,
  author    = {Frasca, Stefano and Rojas, Oscar and Salewski, Johannes and Neumann, Bettina and Stiba, Konstanze and Weidinger, Inez M. and Tiersch, Brigitte and Leimk{\"u}hler, Silke and Koetz, Joachim and Wollenberger, Ursula},
  title     = {Human sulfite oxidase electrochemistry on gold nanoparticles modified electrode},
  series = {Bioelectrochemistry : an international journal devoted to electrochemical aspects of biology and biological aspects of electrochemistry ; official journal of the Bioelectrochemical Society},
  volume    = {87},
  journal   = {Bioelectrochemistry : an international journal devoted to electrochemical aspects of biology and biological aspects of electrochemistry ; official journal of the Bioelectrochemical Society},
  publisher = {Elsevier},
  address   = {Lausanne},
  issn      = {1567-5394},
  doi       = {10.1016/j.bioelechem.2011.11.012},
  pages     = {33 -- 41},
  year      = {2012},
  abstract  = {The present study reports a facile approach for sulfite biosensing, based on enhanced direct electron transfer of a human sulfite oxidase (hSO) immobilized on a gold nanoparticles modified electrode. The spherical core shell AuNPs were prepared via a new method by reduction of HAuCl4 with branched poly(ethyleneimine) in an ionic liquids resulting particles with a diameter less than 10 nm. These nanoparticles were covalently attached to a mercaptoundecanoic acid modified Au-electrode where then hSO was adsorbed and an enhanced interfacial electron transfer and electrocatalysis was achieved. UV/Vis and resonance Raman spectroscopy, in combination with direct protein voltammetry, are employed for the characterization of the system and reveal no perturbation of the structural integrity of the redox protein. The proposed biosensor exhibited a quick steady-state current response, within 2 s, a linear detection range between 0.5 and 5.4 mu M with a high sensitivity (1.85 nA mu M-1). The investigated system provides remarkable advantages in the possibility to work at low applied potential and at very high ionic strength. Therefore these properties could make the proposed system useful in the development of bioelectronic devices and its application in real samples.},
  language  = {en}
}
@article{CoelhoMahroTrincaoetal.2012,
  author    = {Coelho, Catarina and Mahro, Martin and Trincao, Jose and Carvalho, Alexandra T. P. and Ramos, Maria Joao and Terao, Mineko and Garattini, Enrico and Leimk{\"u}hler, Silke and Romao, Maria Joao},
  title     = {The first mammalian aldehyde oxidase crystal structure insights into substrate specificity},
  series = {The journal of biological chemistry},
  volume    = {287},
  journal   = {The journal of biological chemistry},
  number    = {48},
  publisher = {American Society for Biochemistry and Molecular Biology},
  address   = {Bethesda},
  issn      = {0021-9258},
  doi       = {10.1074/jbc.M112.390419},
  pages     = {40690 -- 40702},
  year      = {2012},
  abstract  = {Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 angstrom. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOX and xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity.},
  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{DahlRadonBuehningetal.2013,
  author    = {Dahl, Jan-Ulrik and Radon, Christin and B{\"u}hning, Martin and Nimtz, Manfred and Leichert, Lars I. and Denis, Yann and Jourlin-Castelli, Cecile and Iobbi-Nivol, Chantal and Mejean, Vincent and Leimk{\"u}hler, Silke},
  title     = {The Sulfur Carrier Protein TusA Has a Pleiotropic Role in Escherichia coli That Also Affects Molybdenum Cofactor Biosynthesis},
  series = {JOURNAL OF BIOLOGICAL CHEMISTRY},
  volume    = {288},
  journal   = {JOURNAL OF BIOLOGICAL CHEMISTRY},
  number    = {8},
  publisher = {AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC},
  address   = {BETHESDA},
  issn      = {0021-9258},
  doi       = {10.1074/jbc.M112.431569},
  pages     = {5426 -- 5442},
  year      = {2013},
  abstract  = {The Escherichia coli L-cysteine desulfurase IscS mobilizes sulfur from L-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a Delta tusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the Delta tusA mutant. Characterization of the Delta tusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50\%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes.},
  language  = {en}
}
@article{MareljaChowdhuryDoscheetal.2013,
  author    = {Marelja, Zvonimir and Chowdhury, Mita Mullick and Dosche, Carsten and Hille, Carsten and Baumann, Otto and L{\"o}hmannsr{\"o}ben, Hans-Gerd and Leimk{\"u}hler, Silke},
  title     = {The L-cysteine desulfurase NFS1 is localized in the cytosol where it provides the sulfur for molybdenum cofactor biosynthesis in humans},
  series = {PLoS one},
  volume    = {8},
  journal   = {PLoS one},
  number    = {4},
  publisher = {PLoS},
  address   = {San Fransisco},
  issn      = {1932-6203},
  doi       = {10.1371/journal.pone.0060869},
  pages     = {13},
  year      = {2013},
  abstract  = {In humans, the L-cysteine desulfurase NFS1 plays a crucial role in the mitochondrial iron-sulfur cluster biosynthesis and in the thiomodification of mitochondrial and cytosolic tRNAs. We have previously demonstrated that purified NFS1 is able to transfer sulfur to the C-terminal domain of MOCS3, a cytosolic protein involved in molybdenum cofactor biosynthesis and tRNA thiolation. However, no direct evidence existed so far for the interaction of NFS1 and MOCS3 in the cytosol of human cells. Here, we present direct data to show the interaction of NFS1 and MOCS3 in the cytosol of human cells using Forster resonance energy transfer and a split-EGFP system. The colocalization of NFS1 and MOCS3 in the cytosol was confirmed by immunodetection of fractionated cells and localization studies using confocal fluorescence microscopy. Purified NFS1 was used to reconstitute the lacking molybdoenzyme activity of the Neurospora crassa nit-1 mutant, giving additional evidence that NFS1 is the sulfur donor for Moco biosynthesis in eukaryotes in general.},
  language  = {en}
}
@article{SivanesanLyAdamkiewiczetal.2013,
  author    = {Sivanesan, Arumugam and Ly, Khoa H. and Adamkiewicz, Witold and Stiba, Konstanze and Leimk{\"u}hler, Silke and Weidinger, Inez M.},
  title     = {Tunable electric field enhancement and redox chemistry on TiO2 Island films via covalent attachment to Ag or Au nanostructures},
  series = {The journal of physical chemistry : C, Nanomaterials and interfaces},
  volume    = {117},
  journal   = {The journal of physical chemistry : C, Nanomaterials and interfaces},
  number    = {22},
  publisher = {American Chemical Society},
  address   = {Washington},
  issn      = {1932-7447},
  doi       = {10.1021/jp4032578},
  pages     = {11866 -- 11872},
  year      = {2013},
  abstract  = {Ag-TiO2 and Au-TiO2 hybrid electrodes were designed by covalent attachment of TiO2 nanoparticles to Ag or Au electrodes via an organic linker. The optical and electronic properties of these systems were investigated using the cytochrome b(5) (Cyt b(5)) domain of sulfite oxidase, exclusively attached to the TiO2 surface, as a Raman marker and model redox enzyme. Very strong SERR signals of Cyt b(5) were obtained for Ag-supported systems due to plasmonic field enhancement of Ag. Time-resolved surface-enhanced resonance Raman spectroscopic measurements yielded a remarkably fast electron transfer kinetic (k = 60 s(-1)) of Cyt b(5) to Ag. A much lower Raman intensity was observed for Au-supported systems with undefined and slow redox behavior. We explain this phenomenon on the basis of the different potential of zero charge of the two metals that largely influence the electronic properties of the TiO2 island film.},
  language  = {en}
}
@article{ReschkeSigfridssonKaufmannetal.2013,
  author    = {Reschke, Stefan and Sigfridsson, Kajsa G. V. and Kaufmann, Paul and Leidel, Nils and Horn, Sebastian and Gast, Klaus and Schulzke, Carola and Haumann, Michael and Leimk{\"u}hler, Silke},
  title     = {Identification of a bis-molybdopterin intermediate in molybdenum cofactor biosynthesis in escherichia coli},
  series = {The journal of biological chemistry},
  volume    = {288},
  journal   = {The journal of biological chemistry},
  number    = {41},
  publisher = {American Society for Biochemistry and Molecular Biology},
  address   = {Bethesda},
  issn      = {0021-9258},
  doi       = {10.1074/jbc.M113.497453},
  pages     = {29736 -- 29745},
  year      = {2013},
  abstract  = {The molybdenum cofactor is an important cofactor, and its biosynthesis is essential for many organisms, including humans. Its basic form comprises a single molybdopterin (MPT) unit, which binds a molybdenum ion bearing three oxygen ligands via a dithiolene function, thus forming Mo-MPT. In bacteria, this form is modified to form the bis-MPT guanine dinucleotide cofactor with two MPT units coordinated at one molybdenum atom, which additionally contains GMPs bound to the terminal phosphate group of the MPTs (bis-MGD). The MobA protein catalyzes the nucleotide addition to MPT, but the mechanism of the biosynthesis of the bis-MGD cofactor has remained enigmatic. We have established an in vitro system for studying bis-MGD assembly using purified compounds. Quantification of the MPT/molybdenum and molybdenum/phosphorus ratios, time-dependent assays for MPT and MGD detection, and determination of the numbers and lengths of Mo-S and Mo-O bonds by X-ray absorption spectroscopy enabled identification of a novel bis-Mo-MPT intermediate on MobA prior to nucleotide attachment. The addition of Mg-GTP to MobA loaded with bis-Mo-MPT resulted in formation and release of the final bis-MGD product. This cofactor was fully functional and reconstituted the catalytic activity of apo-TMAO reductase (TorA). We propose a reaction sequence for bis-MGD formation, which involves 1) the formation of bis-Mo-MPT, 2) the addition of two GMP units to form bis-MGD on MobA, and 3) the release and transfer of the mature cofactor to the target protein TorA, in a reaction that is supported by the specific chaperone TorD, resulting in an active molybdoenzyme.},
  language  = {en}
}
@article{LimFriemelMarumetal.2013,
  author    = {Lim, Sze Chern and Friemel, Martin and Marum, Justine E. and Tucker, Elena J. and Bruno, Damien L. and Riley, Lisa G. and Christodoulou, John and Kirk, Edwin P. and Boneh, Avihu and DeGennaro, Christine M. and Springer, Michael and Mootha, Vamsi K. and Rouault, Tracey A. and Leimk{\"u}hler, Silke and Thorburn, David R. and Compton, Alison G.},
  title     = {Mutations in LYRM4, encoding ironsulfur cluster biogenesis factor ISD11, cause deficiency of multiple respiratory chain complexes},
  series = {Human molecular genetics},
  volume    = {22},
  journal   = {Human molecular genetics},
  number    = {22},
  publisher = {Oxford Univ. Press},
  address   = {Oxford},
  issn      = {0964-6906},
  doi       = {10.1093/hmg/ddt295},
  pages     = {4460 -- 4473},
  year      = {2013},
  abstract  = {Ironsulfur clusters (ISCs) are important prosthetic groups that define the functions of many proteins. Proteins with ISCs (called ironsulfur or FeS proteins) are present in mitochondria, the cytosol, the endoplasmic reticulum and the nucleus. They participate in various biological pathways including oxidative phosphorylation (OXPHOS), the citric acid cycle, iron homeostasis, heme biosynthesis and DNA repair. Here, we report a homozygous mutation in LYRM4 in two patients with combined OXPHOS deficiency. LYRM4 encodes the ISD11 protein, which forms a complex with, and stabilizes, the sulfur donor NFS1. The homozygous mutation (c.203GT, p.R68L) was identified via massively parallel sequencing of 1000 mitochondrial genes (MitoExome sequencing) in a patient with deficiency of complexes I, II and III in muscle and liver. These three complexes contain ISCs. Sanger sequencing identified the same mutation in his similarly affected cousin, who had a more severe phenotype and died while a neonate. Complex IV was also deficient in her skeletal muscle. Several other FeS proteins were also affected in both patients, including the aconitases and ferrochelatase. Mutant ISD11 only partially complemented for an ISD11 deletion in yeast. Our in vitro studies showed that the l-cysteine desulfurase activity of NFS1 was barely present when co-expressed with mutant ISD11. Our findings are consistent with a defect in the early step of ISC assembly affecting a broad variety of FeS proteins. The differences in biochemical and clinical features between the two patients may relate to limited availability of cysteine in the newborn period and suggest a potential approach to therapy.},
  language  = {en}
}
@article{ReschkeNiksWilsonetal.2013,
  author    = {Reschke, Stefan and Niks, Dimitri and Wilson, Heather and Sigfridsson, Kajsa G. V. and Haumann, Michael and Rajagopalan, K. V. and Hine, Russ and Leimk{\"u}hler, Silke},
  title     = {Effect of exchange of the cysteine molybdenum ligand with selenocysteine on the structure and function of the active site in human sulfite oxidase},
  series = {Biochemistry},
  volume    = {52},
  journal   = {Biochemistry},
  number    = {46},
  publisher = {American Chemical Society},
  address   = {Washington},
  issn      = {0006-2960},
  doi       = {10.1021/bi4008512},
  pages     = {8295 -- 8303},
  year      = {2013},
  abstract  = {Sulfite oxidase (SO) is an essential molybdoenzyme for humans, catalyzing the final step in the degradation of sulfur-containing amino acids and lipids, which is the oxidation of sulfite to sulfate. The catalytic site of SO consists of a molybdenum ion bound to the dithiolene sulfurs of one molybdopterin (MPT) molecule, carrying two oxygen ligands, and is further coordinated by the thiol sulfur of a conserved cysteine residue. We have exchanged four non-active site cysteines in the molybdenum cofactor (Moco) binding domain of human SO (SOMD) with serine using site-directed mutagenesis. This facilitated the specific replacement of the active site Cys207 with selenocysteine during protein expression in Escherichia coli. The sulfite oxidizing activity (k(cat)/K-M) of SeSOMD4Ser was increased at least 1.5-fold, and the pH optimum was shifted to a more acidic value compared to those of SOMD4Ser and SOMD4Cys(wt) X-ray absorption spectroscopy revealed a Mow Se bond length of 2.51 A, likely caused by the specific binding of Sec207 to the molybdenum, and otherwise rather similar square-pyramidal S/Se(Cys)(O2MoS2)-S-VI(MPT) site structures in the three constructs. The low-pH form of the Mo(V) electron paramagnetic resonance (EPR) signal of SeSOM4Ser was altered compared to those of SOMD4Ser and SOMD4cy,(,), with g, in particular shifted to a lower magnetic field, due to the Se ligation at the molybdenum. In contrast, the Mo(V) EPR signal of the high-pH form was unchanged. The substantially stronger effect of substituting selenocysteine for cysteine at low pH as compared to high pH is most likely due to the decreased covalency of the Mo Se bond.},
  language  = {en}
}
@article{HartmannLeimkuehler2013,
  author    = {Hartmann, Tobias and Leimk{\"u}hler, Silke},
  title     = {The oxygen-tolerant and NAD+-dependent formate dehydrogenase from Rhodobacter capsulatus is able to catalyze the reduction of CO2 to formate},
  series = {The FEBS journal},
  volume    = {280},
  journal   = {The FEBS journal},
  number    = {23},
  publisher = {Wiley-Blackwell},
  address   = {Hoboken},
  issn      = {1742-464X},
  doi       = {10.1111/febs.12528},
  pages     = {6083 -- 6096},
  year      = {2013},
  abstract  = {The formate dehydrogenase from Rhodobactercapsulatus (RcFDH) is an oxygen-tolerant protein with an ()(2) subunit composition that is localized in the cytoplasm. It belongs to the group of metal and NAD(+)-dependent FDHs with the coordination of a molybdenum cofactor, four [Fe4S4] clusters and one [Fe2S2] cluster associated with the -subunit, one [Fe4S4] cluster and one FMN bound to the -subunit, and one [Fe2S2] cluster bound to the -subunit. RcFDH was heterologously expressed in Escherichiacoli and characterized. Cofactor analysis showed that the bis-molybdopterin guanine dinucleotide cofactor is bound to the FdsA subunit containing a cysteine ligand at the active site. A turnover rate of 2189min(-1) with formate as substrate was determined. The back reaction for the reduction of CO2 was catalyzed with a k(cat) of 89min(-1). The preference for formate oxidation shows an energy barrier for CO2 reduction of the enzyme. Furthermore, the FMN-containing and [Fe4S4]-containing -subunit together with the [Fe2S2]-containing -subunit forms a diaphorase unit with activities for both NAD(+) reduction and NADH oxidation. In addition to the structural genes fdsG, fdsB, and fdsA, the fds operon in R.capsulatus contains the fdsC and fdsD genes. Expression studies showed that RcFDH is only active when both FdsC and FdsD are present. Both proteins are proposed to be involved in bis-molybdopterin guanine dinucleotide modification and insertion into RcFDH.},
  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{MahroBrasCerqueiraetal.2013,
  author    = {Mahro, Martin and Bras, Natercia F. and Cerqueira, Nuno M. F. S. A. and Teutloff, Christian and Coelho, Catarina and Romao, Maria Joao and Leimk{\"u}hler, Silke},
  title     = {Identification of crucial amino acids in mouse aldehyde oxidase 3 that determine substrate specificity},
  series = {PLoS one},
  volume    = {8},
  journal   = {PLoS one},
  number    = {12},
  publisher = {PLoS},
  address   = {San Fransisco},
  issn      = {1932-6203},
  doi       = {10.1371/journal.pone.0082285},
  pages     = {12},
  year      = {2013},
  abstract  = {In order to elucidate factors that determine substrate specificity and activity of mammalian molybdo-flavoproteins we performed site directed mutagenesis of mouse aldehyde oxidase 3 (mAOX3). The sequence alignment of different aldehyde oxidase (AOX) isoforms identified variations in the active site of mAOX3 in comparison to other AOX proteins and xanthine oxidoreductases (XOR). Based on the structural alignment of mAOX3 and bovine XOR, differences in amino acid residues involved in substrate binding in XORs in comparison to AOXs were identified. We exchanged several residues in the active site to the ones found in other AOX homologues in mouse or to residues present in bovine XOR in order to examine their influence on substrate selectivity and catalytic activity. Additionally we analyzed the influence of the [2Fe-2S] domains of mAOX3 on its kinetic properties and cofactor saturation. We applied UV-VIS and EPR monitored redox-titrations to determine the redox potentials of wild type mAOX3 and mAOX3 variants containing the iron-sulfur centers of mAOX1. In addition, a combination of molecular docking and molecular dynamic simulations (MD) was used to investigate factors that modulate the substrate specificity and activity of wild type and AOX variants. The successful conversion of an AOX enzyme to an XOR enzyme was achieved exchanging eight residues in the active site of mAOX3. It was observed that the absence of the K889H exchange substantially decreased the activity of the enzyme towards all substrates analyzed, revealing that this residue has an important role in catalysis.},
  language  = {en}
}
@article{GisinMuellerPfaenderetal.2010,
  author    = {Gisin, Jonathan and Mueller, Alexandra and Pfaender, Yvonne and Leimk{\"u}hler, Silke and Narberhaus, Franz and Masepohl, Bernd},
  title     = {A Rhodobacter capsulatus member of a universal permease family imports molybdate and other oxyanions},
  issn      = {0021-9193},
  doi       = {10.1128/Jb.00742-10},
  year      = {2010},
  abstract  = {Molybdenum (Mo) is an important trace element that is toxic at high concentrations. To resolve the mechanisms underlying Mo toxicity, Rhodobacter capsulatus mutants tolerant to high Mo concentrations were isolated by random transposon Tn5 mutagenesis. The insertion sites of six independent isolates mapped within the same gene predicted to code for a permease of unknown function located in the cytoplasmic membrane. During growth under Mo-replete conditions, the wild-type strain accumulated considerably more Mo than the permease mutant. For mutants defective for the permease, the high-affinity molybdate importer ModABC, or both transporters, in vivo Mo-dependent nitrogenase (Mo-nitrogenase) activities at different Mo concentrations suggested that ModABC and the permease import molybdate in nanomolar and micromolar ranges, respectively. Like the permease mutants, a mutant defective for ATP sulfurylase tolerated high Mo concentrations, suggesting that ATP sulfurylase is the main target of Mo inhibition in R. capsulatus. Sulfate-dependent growth of a double mutant defective for the permease and the high-affinity sulfate importer CysTWA was reduced compared to those of the single mutants, implying that the permease plays an important role in sulfate uptake. In addition, permease mutants tolerated higher tungstate and vanadate concentrations than the wild type, suggesting that the permease acts as a general oxyanion importer. We propose to call this permease PerO (for oxyanion permease). It is the first reported bacterial molybdate transporter outside the ABC transporter family.},
  language  = {en}
}
@article{WiethausMuellerNeumannetal.2009,
  author    = {Wiethaus, Jessica and Mueller, Alexandra and Neumann, Meina and Neumann, Sandra and Leimk{\"u}hler, Silke and Narberhaus, Franz and Masepohl, Bernd},
  title     = {Specific interactions between four Molybdenum-binding proteins contribute to Mo-dependent gene regulation in Rhodobacter capsulatus},
  issn      = {0021-9193},
  doi       = {10.1128/Jb.00526-09},
  year      = {2009},
  abstract  = {The phototrophic purple bacterium Rhodobacter capsulatus encodes two transcriptional regulators, MopA and MopB, with partially overlapping and specific functions in molybdate-dependent gene regulation. Both MopA and MopB consist of an N-terminal DNA-binding helix-turn-helix domain and a C-terminal molybdate-binding di-MOP domain. They formed homodimers as apo-proteins and in the molybdate-bound state as shown by yeast two-hybrid (Y2H) studies, glutaraldehyde cross-linking, gel filtration chromatography, and copurification experiments. Y2H studies suggested that both the DNA- binding and the molybdate-binding domains contribute to dimer formation. Analysis of molybdate binding to MopA and MopB revealed a binding stoichiometry of four molybdate oxyanions per homodimer. Specific interaction partners of MopA and MopB were the molybdate transporter ATPase ModC and the molbindin-like Mop protein, respectively. Like other molbindins, the R. capsulatus Mop protein formed hexamers, which were stabilized by binding of six molybdate oxyanions per hexamer. Heteromer formation of MopA and MopB was shown by Y2H studies and copurification experiments. Reporter gene activity of a strictly MopA-dependent mop-lacZ fusion in mutant strains defective for either mopA, mopB, or both suggested that MopB negatively modulates expression of the mop promoter. We propose that depletion of the active MopA homodimer pool by formation of MopA-MopB heteromers might represent a fine-tuning mechanism controlling mop gene expression.},
  language  = {en}
}
@article{SpricigoDronovLisdatetal.2009,
  author    = {Spricigo, Roberto and Dronov, Roman and Lisdat, Fred and Leimk{\"u}hler, Silke and Scheller, Frieder W. and Wollenberger, Ursula},
  title     = {Electrocatalytic sulfite biosensor with human sulfite oxidase co-immobilized with cytochrome c in a polyelectrolyte-containing multilayer},
  issn      = {1618-2642},
  doi       = {10.1007/s00216-008-2432-y},
  year      = {2009},
  abstract  = {An efficient electrocatalytic biosensor for sulfite detection was developed by co-immobilizing sulfite oxidase and cytochrome c with polyaniline sulfonic acid in a layer-by-layer assembly. QCM, UV-Vis spectroscopy and cyclic voltammetry revealed increasing loading of electrochemically active protein with the formation of multilayers. The sensor operates reagentless at low working potential. A catalytic oxidation current was detected in the presence of sulfite at the modified gold electrode, polarized at +0.1 V ( vs. Ag/AgCl 1 M KCl). The stability of the biosensor performance was characterized and optimized. A 17-bilayer electrode has a linear range between 1 and 60 mu M sulfite with a sensitivity of 2.19 mA M-1 sulfite and a response time of 2 min. The electrode retained a stable response for 3 days with a serial reproducibility of 3.8\% and lost 20\% of sensitivity after 5 days of operation. It is possible to store the sensor in a dry state for more than 2 months. The multilayer electrode was used for determination of sulfite in unspiked and spiked samples of red and white wine. The recovery and the specificity of the signals were evaluated for each sample.},
  language  = {en}
}
@article{NeumannMittelstaedtSeduketal.2009,
  author    = {Neumann, Meina and Mittelstaedt, Gerd and Seduk, Farida and Iobbi-Nivol, Chantal and Leimk{\"u}hler, Silke},
  title     = {MocA is a specific cytidylyltransferase involved in molybdopterin cytosine dinucleotide biosynthesis in Escherichia coli},
  issn      = {0021-9258},
  doi       = {10.1074/jbc.M109.008565},
  year      = {2009},
  abstract  = {We have purified and characterized a specific CTP: molybdopterin cytidylyltransferase for the biosynthesis of the molybdopterin (MPT) cytosine dinucleotide (MCD) cofactor in Escherichia coli. The protein, named MocA, shows 22\% amino acid sequence identity to E. coli MobA, the specific GTP: molybdopterin guanylyltransferase for molybdopterin guanine dinucleotide biosynthesis. MocA is essential for the activity of the MCD-containing enzymes aldehyde oxidoreductase Yag-TSR and the xanthine dehydrogenases XdhABC and XdhD. Using a fully defined in vitro assay, we showed that MocA, Mo-MPT, CTP, and MgCl2 are required and sufficient for MCD biosynthesis in vitro. The activity of MocA is specific for CTP; other nucleotides such as ATP and GTP were not utilized. In the defined in vitro system a turnover number of 0.37 +/- 0.01 min(-1) was obtained. A1:1 binding ratio of MocA to Mo-MPT and CTP was determined to monomeric MocA with dissociation constants of 0.23 +/- 0.02 mu M for CTP and 1.17 +/- 0.18 mu M for Mo-MPT. We showed that MocA was also able to convert MPT to MCD in the absence of molybdate, however, with only one catalytic turnover. The addition of molybdate after one turnover gave rise to a higher MCD production, revealing that MCD remains bound to MocA in the absence of molybdate. This work presents the first characterization of a specific enzyme involved in MCD biosynthesis in bacteria.},
  language  = {en}
}
@article{NeumannMittelstaedtIobbiNivoletal.2009,
  author    = {Neumann, Meina and Mittelstaedt, Gerd and Iobbi-Nivol, Chantal and Saggu, Miguel and Lendzian, Friedhelm and Hildebrandt, Peter and Leimk{\"u}hler, Silke},
  title     = {A periplasmic aldehyde oxidoreductase represents the first molybdopterin cytosine dinucleotide cofactor containing molybdo-flavoenzyme from Escherichia coli},
  issn      = {1742-464X},
  doi       = {10.1111/j.1742-4658.2009.07000.x},
  year      = {2009},
  abstract  = {Three DNA regions carrying genes encoding putative homologs of xanthine dehydrogenases were identified in Escherichia coli, named xdhABC, xdhD, and yagTSRQ. Here, we describe the purification and characterization of gene products of the yagTSRQ operon, a molybdenum-containing iron-sulfur flavoprotein from E. coli, which is located in the periplasm. The 135 kDa enzyme comprised a noncovalent (alpha beta gamma) heterotrimer with a large (78.1 kDa) molybdenum cofactor (Moco)-containing YagR subunit, a medium (33.9 kDa) FAD-containing YagS subunit, and a small (21.0 kDa) 2 x [2Fe2S]-containing YagT subunit. YagQ is not a subunit of the mature enzyme, and the protein is expected to be involved in Moco modification and insertion into YagTSR. Analysis of the form of Moco present in YagTSR revealed the presence of the molybdopterin cytosine dinucleotide cofactor. Two different [2Fe2S] clusters, typical for this class of enzyme, were identified by EPR. YagTSR represents the first example of a molybdopterin cytosine dinucleotide-containing protein in E. coli. Kinetic characterization of the enzyme revealed that YagTSR converts a broad spectrum of aldehydes, with a preference for aromatic aldehydes. Ferredoxin instead of NAD(+) or molecular oxygen was used as terminal electron acceptor. Complete growth inhibition of E. coli cells devoid of genes from the yagTSRQ operon was observed by the addition of cinnamaldehyde to a low-pH medium. This finding shows that YagTSR might have a role in the detoxification of aromatic aldehydes for E. coli under certain growth conditions.},
  language  = {en}
}
@article{HaenzelmannDahlKuperetal.2009,
  author    = {Haenzelmann, Petra and Dahl, Jan U. and Kuper, Jochen and Urban, Alexander and Mueller-Theissen, Ursula and Leimk{\"u}hler, Silke and Schindelin, Hermann},
  title     = {Crystal structure of YnjE from Escherichia coli, a sulfurtransferase with three rhodanese domains},
  issn      = {0961-8368},
  doi       = {10.1002/pro.260},
  year      = {2009},
  abstract  = {Rhodaneses/sulfurtransferases are ubiquitous enzymes that catalyze the transfer of sulfane sulfur from a donor molecule to a thiophilic acceptor via an active site cysteine that is modified to a persulfide during the reaction. Here, we present the first crystal structure of a triple-domain rhodanese-like protein, namely YnjE from Escherichia coli, in two states where its active site cysteine is either unmodified or present as a persulfide. Compared to well- characterized tandem domain rhodaneses, which are composed of one inactive and one active domain, YnjE contains an extra N-terminal inactive rhodanese-like domain. Phylogenetic analysis reveals that YnjE triple-domain homologs can be found in a variety of other gamma-proteobacteria, in addition, some single-, tandem-, four and even six-domain variants exist. All YnjE rhodaneses are characterized by a highly conserved active site loop (CGTGWR) and evolved independently from other rhodaneses, thus forming their own subfamily. On the basis of structural comparisons with other rhodaneses and kinetic studies, YnjE, which is more similar to thiosulfate:cyanide sulfurtransferases than to 3- mercaptopyruvate:cyanide sulfurtransferases, has a different substrate specificity that depends not only on the composition of the active site loop with the catalytic cysteine at the first position but also on the surrounding residues. In vitro YnjE can be efficiently persulfurated by the cysteine desulfurase IscS. The catalytic site is located within an elongated cleft, formed by the central and C-terminal domain and is lined by bulky hydrophobic residues with the catalytic active cysteine largely shielded from the solvent.},
  language  = {en}
}
@article{DietzelKuperDoebbleretal.2009,
  author    = {Dietzel, Uwe and Kuper, Jochen and Doebbler, Jennifer A. and Schulte, Antje and Truglio, James J. and Leimk{\"u}hler, Silke and Kisker, Caroline},
  title     = {Mechanism of substrate and inhibitor binding of Rhodobacter capsulatus xanthine dehydrogenase},
  issn      = {0021-9258},
  doi       = {10.1074/jbc.M808114200},
  year      = {2009},
  abstract  = {Rhodobacter capsulatus xanthine dehydrogenase (XDH) is an (alpha beta)(2) heterotetrameric cytoplasmic enzyme that resembles eukaryotic xanthine oxidoreductases in respect to both amino acid sequence and structural fold. To obtain a detailed understanding of the mechanism of substrate and inhibitor binding at the active site, we solved crystal structures of R. capsulatus XDH in the presence of its substrates hypoxanthine, xanthine, and the inhibitor pterin-6- aldehyde using either the inactive desulfo form of the enzyme or an active site mutant (E(B)232Q) to prevent substrate turnover. The hypoxanthine-and xanthine-bound structures reveal the orientation of both substrates at the active site and show the importance of residue GluB-232 for substrate positioning. The oxygen atom at the C-6 position of both substrates is oriented toward Arg(B)-310 in the active site. Thus the substrates bind in an orientation opposite to the one seen in the structure of the reduced enzyme with the inhibitor oxypurinol. The tightness of the substrates in the active site suggests that the intermediate products must exit the binding pocket to allow first the attack of the C-2, followed by oxidation of the C-8 atom to form the final product uric acid. Structural studies of pterin-6-aldehyde, a potent inhibitor of R. capsulatus XDH, contribute further to the understanding of the relative positioning of inhibitors and substrates in the binding pocket. Steady state kinetics reveal a competitive inhibition pattern with a K-i of 103.57 +/- 18.96 nM for pterin-6-aldehyde.},
  language  = {en}
}
@article{SpricigoRichterLeimkuehleretal.2010,
  author    = {Spricigo, Roberto and Richter, Claudia and Leimk{\"u}hler, Silke and Gorton, Lo and Scheller, Frieder W. and Wollenberger, Ursula},
  title     = {Sulfite biosensor based on osmium redox polymer wired sulfite oxidase},
  issn      = {0927-7757},
  doi       = {10.1016/j.colsurfa.2009.09.001},
  year      = {2010},
  abstract  = {A biosensor, based on a redoxactive osmium polymer and sulfite oxidase on screen-printed electrodes, is presented here as a promising method for the detection of sulfite. A catalytic oxidative current was generated when a sample containing sulfite was pumped over the carbon screen-printed electrode modified with osmium redox polymer wired sulfite oxidase. A stationary value was reached after approximately 50 s and a complete measurement lasted no more than 3 min. The electrode polarized at -0.1 V (vs. Ag vertical bar AgCl 1M KCl) permits minimizing the influence of interfering substances, since these compounds can be unspecific oxidized at higher potentials. Because of the good stability of the protein film on the electrode surface, a well functioning biosensor-flow system was possible to construct. The working stability and reproducibility were further enhanced by the addition of bovine serum albumin generating a more long-term stable and biocompatible protein environment. The optimized biosensor showed a stable signal for more than a week of operation and a coefficient of variation of 4.8\% for 12 successive measurements. The lower limit of detection of the sensor was 0.5 mu M sulfite and the response was linear until 100 mu M. The high sensitivity permitted a 1:500 dilution of wine samples. The immobilization procedure and the operational conditions granted minimized interferences. Additionally, repeating the immobilization procedure to form several layers of wired SO further increased the sensitivity of such a sensor. Finally. the applicability of the developed sulfite biosensor was tested on real samples, such as white and red wines.},
  language  = {en}
}
@article{ZhangUrbanMiharaetal.2010,
  author    = {Zhang, Wanjiao and Urban, Alexander and Mihara, Hisaaki and Leimk{\"u}hler, Silke and Kurihara, Tatsuo and Esaki, Nobuyoshi},
  title     = {IscS functions as a primary sulfur-donating enzyme by interacting specifically with MoeB and MoaD in the biosynthesis of molybdopterin in escherichia coli},
  issn      = {0021-9258},
  doi       = {10.1074/jbc.M109.082172},
  year      = {2010},
  abstract  = {The persulfide sulfur formed on an active site cysteine residue of pyridoxal 5'-phosphate-dependent cysteine desulfurases is subsequently incorporated into the biosynthetic pathways of a variety of sulfur-containing cofactors and thionucleosides. In molybdenum cofactor biosynthesis, MoeB activates the C terminus of the MoaD subunit of molybdopterin (MPT) synthase to form MoaD-adenylate, which is subsequently converted to a thiocarboxylate for the generation of the dithiolene group of MPT. It has been shown that three cysteine desulfurases (CsdA, SufS, and IscS) of Escherichia coli can transfer sulfur from L-cysteine to the thiocarboxylate of MoaD in vitro. Here, we demonstrate by surface plasmon resonance analyses that IscS, but not CsdA or SufS, interacts with MoeB and MoaD. MoeB and MoaD can stimulate the IscS activity up to 1.6-fold. Analysis of the sulfuration level of MoaD isolated from strains defective in cysteine desulfurases shows a largely decreased sulfuration level of the protein in an iscS deletion strain but not in a csdA/sufS deletion strain. We also show that another iscS deletion strain of E. coli accumulates compound Z, a direct oxidation product of the immediate precursor of MPT, to the same extent as an MPT synthase-deficient strain. In contrast, analysis of the content of compound Z in Delta csdA and Delta sufS strains revealed no such accumulation. These findings indicate that IscS is the primary physiological sulfur-donating enzyme for the generation of the thiocarboxylate of MPT synthase in MPT biosynthesis.},
  language  = {en}
}
@article{MatthiesNimtzLeimkuehler2005,
  author    = {Matthies, A. and Nimtz, M. and Leimk{\"u}hler, Silke},
  title     = {Molybdenum cofactor biosynthesis in humans : Identification of a persulfide group in the rhodanese-like domain of MOCS3 by mass spectrometry},
  issn      = {0006-2960},
  year      = {2005},
  abstract  = {The human MOCS3 protein contains an N-terminal domain similar to the Escherichia coli MoeB protein and a C- terminal segment displaying similarities to the sulfurtransferase rhodanese. MOCS3 is proposed to catalyze both the adenylation and the subsequent generation of a thiocarboxylate group at the C-terminus of the smaller subunit of molybdopterin (MPT) synthase during Moco biosynthesis in humans. Recent studies have shown that the MOCS3 rhodanese-like domain (MOCS3-RLD) catalyzes the transfer of sulfur from thiosulfate to cyanide and is also able to provide the sulfur for the thiocarboxylation of MOCS2A in a defined in vitro system for the generation of MPT from precursor Z. MOCS3-RLD contains four cysteine residues of which only C412 in the six amino acid active loop is conserved in homologous proteins from other organisms. ESI-MS/MS studies gave direct evidence for the formation of a persulfide group that is exclusively formed on C412. Simultaneous mutagenesis of the remaining three cysteine residues showed that none of them is involved in the sulfur transfer reaction in vitro. A disulfide bridge was identified to be formed between C316 and C324, and possible roles of the three noncatalytic cysteine residues are discussed. By ESI-MS/MS a partially gluconoylated N- terminus of the His(6)-tagged MOCS3-RLD was identified (mass increment of 178 Da) which resulted in a heterogeneity of the protein but did not influence sulfurtransferase activity},
  language  = {en}
}
@article{LeimkuehlerCharcossetLatouretal.2005,
  author    = {Leimk{\"u}hler, Silke and Charcosset, M. and Latour, P. and Dorche, C. and Kleppe, S. and Scaglia, F. and Szymczak, I. and Schupp, P. and Hahnewald, Rita and Reiss, J.},
  title     = {Ten novel mutations in the molybdenum cofactor genes MOCS1 and MOCS2 and in vitro characterization of a MOCS2 mutation that abolishes the binding ability of molybdopterin synthase},
  issn      = {0340-6717},
  year      = {2005},
  abstract  = {Molybdenum cofactor deficiency (MIM\#252150) is a severe autosomal- recessive disorder with a devastating outcome. The cofactor is the product of a complex biosynthetic pathway involving four different genes (MOCS1, MOCS2, MOCS3 and GEPH). This disorder is caused almost exclusively by mutations in the MOCS1 or MOCS2 genes. Mutations affecting this biosynthetic pathway result in a lethal phenotype manifested by progressive neurological damage via the inactivation of the molybdenum cofactor-dependent enzyme, sulphite oxidase. Here we describe a total of ten novel disease-causing mutations in the MOCS1 and MOCS2 genes. Nine out of these ten mutations were classified as pathogenic in nature, since they create a stop codon, affect constitutive splice site positions, or change strictly conserved motifs. The tenth mutation abolishes the stop codon of the MOCS2B gene, thus elongating the corresponding protein. The mutation was expressed in vitro and was found to abolish the binding affinities of the large subunit of molybdopterin synthase (MOCS2B) for both precursor Z and the small subunit of molybdopterin synthase (MOCS2A)},
  language  = {en}
}
@article{ForlaniCeredaFreueretal.2005,
  author    = {Forlani, Fabio and Cereda, Angelo and Freuer, Andrea and Nimtz, Manfred and Leimk{\"u}hler, Silke and Pagani, Silvia},
  title     = {The cysteine-desulfurase IscS promotes the production of the rhodanese RhdA in the persulfurated form},
  issn      = {0014-5793},
  year      = {2005},
  abstract  = {After heterologous expression in Escherichia coli, the Azotobacter vinelandii rhodanese RhdA is purified in a persulfurated form (RhdA-SSH). We identified L-cysteine as the most effective sulfur source in producing RhdA-SSH. An E. coli soluble extract was required for in vitro persulfuration of RhdA, and the addition of pyridoxal-5'-phosphate increased RhdA-SSH production, indicating a likely involvement of a cysteine desulfurase. We were able to show the formation of a covalent complex between IscS and RhdA. By combining a time-course fluorescence assay and mass spectrometry analysis, we demonstrated the transfer of sulfur from E. coli IscS to RhdA. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved},
  language  = {en}
}
@article{SezerSpricigoUteschetal.2010,
  author    = {Sezer, Murat and Spricigo, Roberto and Utesch, Tillmann and Millo, Diego and Leimk{\"u}hler, Silke and Mroginski, Maria A. and Wollenberger, Ursula and Hildebrandt, Peter and Weidinger, Inez M.},
  title     = {Redox properties and catalytic activity of surface-bound human sulfite oxidase studied by a combined surface enhanced resonance Raman spectroscopic and electrochemical approach},
  issn      = {1463-9076},
  doi       = {10.1039/B927226g},
  year      = {2010},
  abstract  = {Human sulfite oxidase (hSO) was immobilised on SAM-coated silver electrodes under preservation of the native heme pocket structure of the cytochrome b5 (Cyt b5) domain and the functionality of the enzyme. The redox properties and catalytic activity of the entire enzyme were studied by surface enhanced resonance Raman (SERR) spectroscopy and cyclic voltammetry (CV) and compared to the isolated heme domain when possible. It is shown that heterogeneous electron transfer and catalytic activity of hSO sensitively depend on the local environment of the enzyme. Increasing the ionic strength of the buffer solution leads to an increase of the heterogeneous electron transfer rate from 17 s(-1) to 440 s(- 1) for hSO as determined by SERR spectroscopy. CV measurements demonstrate an increase of the apparent turnover rate for the immobilised hSO from 0.85 s(-1) in 100 mM buffer to 5.26 s(-1) in 750 mM buffer. We suggest that both effects originate from the increased mobility of the surface-bound enzyme with increasing ionic strength. In agreement with surface potential calculations we propose that at high ionic strength the enzyme is immobilised via the dimerisation domain to the SAM surface. The flexible loop region connecting the Moco and the Cyt b5 domain allows alternating contact with the Moco interaction site and the SAM surface, thereby promoting the sequential intramolecular and heterogeneous electron transfer from Moco via Cyt b5 to the electrode. At lower ionic strength, the contact time of the Cyt b5 domain with the SAM surface is longer, corresponding to a slower overall electron transfer process.},
  language  = {en}
}
@article{NeumannSchulteJuenemannetal.2006,
  author    = {Neumann, Meina and Schulte, Marc and J{\"u}nemann, Nora and St{\"o}cklein, Walter F. M. and Leimk{\"u}hler, Silke},
  title     = {Rhodobacter capsulatus XdhC is involved in molybdenum cofactor binding and insertion into xanthine dehydrogenase},
  doi       = {10.1074/jbc.M601617200},
  year      = {2006},
  abstract  = {Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a cytoplasmic enzyme with an (alpha beta) 2 heterodimeric structure that is highly identical to homodimeric eukaryotic xanthine oxidoreductases. The crystal structure revealed that the molybdenum cofactor (Moco) is deeply buried within the protein. A protein involved in Moco insertion and XDH maturation has been identified, which was designated XdhC. XdhC was shown to be essential for the production of active XDH but is not a subunit of the purified enzyme. Here we describe the purification of XdhC and the detailed characterization of its role for XDH maturation. We could show that XdhC binds Moco in stoichiometric amounts, which subsequently can be inserted into Moco-free apo-XDH. A specific interaction between XdhC and XdhB was identified. We show that XdhC is required for the stabilization of the sulfurated form of Moco present in enzymes of the xanthine oxidase family. Our findings imply that enzyme-specific proteins exist for the biogenesis of molybdoenzymes, coordinating Moco binding and insertion into their respective target proteins. So far, the requirement of such proteins for molybdoenzyme maturation has been described only for prokaryotes},
  language  = {en}
}