@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{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{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{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{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} } @misc{IobbiNivolLeimkuehler2013, author = {Iobbi-Nivol, Chantal and Leimk{\"u}hler, Silke}, title = {Molybdenum enzymes, their maturation and molybdenum cofactor biosynthesis in Escherichia coli}, series = {Biochimica et biophysica acta : Bioenergetics}, volume = {1827}, journal = {Biochimica et biophysica acta : Bioenergetics}, number = {8-9}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0005-2728}, doi = {10.1016/j.bbabio.2012.11.007}, pages = {1086 -- 1101}, year = {2013}, abstract = {Molybdenum cofactor (Moco) biosynthesis is an ancient, ubiquitous, and highly conserved pathway leading to the biochemical activation of molybdenum. Moco is the essential component of a group of redox enzymes, which are diverse in terms of their phylogenetic distribution and their architectures, both at the overall level and in their catalytic geometry. A wide variety of transformations are catalyzed by these enzymes at carbon, sulfur and nitrogen atoms, which include the transfer of an oxo group or two electrons to or from the substrate. More than 50 molybdoenzymes were identified in bacteria to date. In molybdoenzymes Mo is coordinated to a dithiolene group on the 6-alkyl side chain of a pterin called molybdopterin (MPT). The biosynthesis of Moco can be divided into four general steps in bacteria: I) formation of the cyclic pyranopterin monophosphate, 2) formation of MPT, 3) insertion of molybdenum into molybdopterin to form Moco, and 4) additional modification of Moco with the attachment of GMP or CMP to the phosphate group of MPT, forming the dinucleotide variant of Moco. This review will focus on molybdoenzymes, the biosynthesis of Moco, and its incorporation into specific target proteins focusing on Escherichia coli. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.}, 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} }