TY - JOUR A1 - Leimkühler, Silke A1 - Iobbi-Nivol, Chantal T1 - Bacterial molybdoenzymes: old enzymes for new purposes JF - FEMS microbiology reviews N2 - Molybdoenzymes are widespread in eukaryotic and prokaryotic organisms where they play crucial functions in detoxification reactions in the metabolism of humans and bacteria, in nitrate assimilation in plants and in anaerobic respiration in bacteria. To be fully active, these enzymes require complex molybdenum-containing cofactors, which are inserted into the apoenzymes after folding. For almost all the bacterial molybdoenzymes, molybdenum cofactor insertion requires the involvement of specific chaperones. In this review, an overview on the molybdenum cofactor biosynthetic pathway is given together with the role of specific chaperones dedicated for molybdenum cofactor insertion and maturation. Many bacteria are involved in geochemical cycles on earth and therefore have an environmental impact. The roles of molybdoenzymes in bioremediation and for environmental applications are presented.This review gives an overview of the diverse mechanisms leading to the insertion of the different forms of the molybdenum cofactor into the respective target enzymes and summarizes the roles of different molybdoenzymes in the environment.This review gives an overview of the diverse mechanisms leading to the insertion of the different forms of the molybdenum cofactor into the respective target enzymes and summarizes the roles of different molybdoenzymes in the environment. KW - molybdenum cofactor KW - specific chaperons KW - TorD family KW - XdhC KW - molybdoenzyme maturation KW - bioremediation Y1 - 2016 U6 - https://doi.org/10.1093/femsre/fuv043 SN - 0168-6445 SN - 1574-6976 VL - 40 SP - 1 EP - 18 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Sarauli, David A1 - Borowski, Anja A1 - Peters, Kristina A1 - Schulz, Burkhard A1 - Fattakhova-Rohlfing, Dina A1 - Leimkühler, Silke A1 - Lisdat, Fred T1 - Investigation of the pH-Dependent Impact of Sulfonated Polyaniline on Bioelectrocatalytic Activity of Xanthine Dehydrogenase JF - ACS catalysis N2 - We report on the pH-dependent bioelectrocatalytic activity of the redox enzyme xanthine dehydrogenase (XDH) in the presence of sulfonated polyaniline PMSA1 (poly(2-methoxyaniline-5-sulfonic acid)-co-aniline). Ultraviolet-visible (UV-vis) spectroscopic measurements with both components in solution reveal electron transfer from the hypoxanthine (HX)-reduced enzyme to the polymer. The enzyme shows bioelectrocatalytic activity on indium tin oxide (ITO) electrodes, when the polymer is present. Depending on solution pH, different processes can be identified. It can be demonstrated that not only product-based communication with the electrode but also efficient polymer-supported bioelectrocatalysis occur. Interestingly, substrate dependent catalytic currents can be obtained in acidic and neutral solutions, although the highest activity of XDH with natural reaction partners is in the alkaline region. Furthermore, operation of the enzyme electrode without addition of the natural cofactor of XDH is feasible. Finally, macroporous ITO electrodes have been used as an immobilization platform for the fabrication of HX-sensitive electrodes. The study shows that the efficient polymer/enzyme interaction can be advantageously combined with the open structure of an electrode material of controlled pore size, resulting in good processability, stability, and defined signal transfer in the presence of a substrate. KW - enzyme bioelectrocatalysis KW - sulfonated polyanilines KW - xanthine dehydrogenase KW - pH-dependent electrochemistry KW - macroporous ITO electrodes Y1 - 2016 U6 - https://doi.org/10.1021/acscatal.6b02011 SN - 2155-5435 VL - 6 SP - 7152 EP - 7159 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Han, Xiao Xia A1 - Li, Junbo A1 - Öner, Ibrahim Halil A1 - Zhao, Bing A1 - Leimkühler, Silke A1 - Hildebrandt, Peter A1 - Weidinger, Inez M. T1 - Nickel electrodes as a cheap and versatile platform for studying structure and function of immobilized redox proteins JF - Analytica chimica acta : an international journal devoted to all branches of analytical chemistry N2 - Practical use of many bioelectronic and bioanalytical devices is limited by the need of expensive materials and time consuming fabrication. Here we demonstrate the use of nickel electrodes as a simple and cheap solid support material for bioelectronic applications. The naturally nanostructured electrodes showed a surprisingly high electromagnetic surface enhancement upon light illumination such that immobilization and electron transfer reactions of the model redox proteins cytochrome b(5) (Cyt b(5)) and cytochrome c (Cyt c) could be followed via surface enhanced resonance Raman spectroscopy. It could be shown that the nickel surface, when used as received, promotes a very efficient binding of the proteins upon preservation of their native structure. The immobilized redox proteins could efficiently exchange electrons with the electrode and could even act as an electron relay between the electrode and solubilized myoglobin. Our results open up new possibility for nickel electrodes as an exceptional good support for bioelectronic devices and biosensors on the one hand and for surface enhanced spectroscopic investigations on the other hand. (C) 2016 Elsevier B.V. All rights reserved. KW - Ni electrodes KW - Redox proteins KW - Surface enhanced Raman spectroscopy KW - Electron relay KW - Biocompatibility KW - Electron transfer Y1 - 2016 U6 - https://doi.org/10.1016/j.aca.2016.08.053 SN - 0003-2670 SN - 1873-4324 VL - 941 SP - 35 EP - 40 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Foti, Alessandro A1 - Hartmann, Tobias A1 - Coelho, Catarina A1 - Santos-Silva, Teresa A1 - Romao, Maria Joao A1 - Leimkühler, Silke T1 - Optimization of the Expression of Human Aldehyde Oxidase for Investigations of Single-Nucleotide Polymorphisms JF - Drug metabolism and disposition : the biological fate of chemicals N2 - Aldehyde oxidase (AOX1) is an enzyme with broad substrate specificity, catalyzing the oxidation of a wide range of endogenous and exogenous aldehydes as well as N-heterocyclic aromatic compounds. In humans, the enzyme’s role in phase I drug metabolism has been established and its importance is now emerging. However, the true physiologic function of AOX1 in mammals is still unknown. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified in human AOX1. SNPs are a major source of interindividual variability in the human population, and SNP-based amino acid exchanges in AOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. For the reliable analysis of the effect of amino acid exchanges in human proteins, the existence of reproducible expression systems for the production of active protein in ample amounts for kinetic, spectroscopic, and crystallographic studies is required. In our study we report an optimized expression system for hAOX1 in Escherichia coli using a codon-optimized construct. The codon-optimization resulted in an up to 15-fold increase of protein production and a simplified purification procedure. The optimized expression system was used to study three SNPs that result in amino acid changes C44W, G1269R, and S1271L. In addition, the crystal structure of the S1271L SNP was solved. We demonstrate that the recombinant enzyme can be used for future studies to exploit the role of AOX in drug metabolism, and for the identification and synthesis of new drugs targeting AOX when combined with crystallographic and modeling studies. Y1 - 2016 U6 - https://doi.org/10.1124/dmd.115.068395 SN - 0090-9556 SN - 1521-009X VL - 44 SP - 1277 EP - 1285 PB - American Society for Pharmacology and Experimental Therapeutics CY - Bethesda ER - TY - JOUR A1 - Cazelles, R. A1 - Lalaoui, N. A1 - Hartmann, Tobias A1 - Leimkühler, Silke A1 - Wollenberger, Ursula A1 - Antonietti, Markus A1 - Cosnier, S. T1 - Ready to use bioinformatics analysis as a tool to predict immobilisation strategies for protein direct electron transfer (DET) JF - Polymer : the international journal for the science and technology of polymers KW - Bioinformatic KW - Bioelectrocatalysis KW - Electron transfer KW - Dehydrogenase KW - Nicotinamide Y1 - 2016 U6 - https://doi.org/10.1016/j.bios.2016.04.078 SN - 0956-5663 SN - 1873-4235 VL - 85 SP - 90 EP - 95 PB - Elsevier CY - Oxford ER - TY - JOUR A1 - Zeng, Ting A1 - Frasca, Stefano A1 - Rumschöttel, Jens A1 - Koetz, Joachim A1 - Leimkühler, Silke A1 - Wollenberger, Ursula T1 - Role of Conductive Nanoparticles in the Direct Unmediated Bioelectrocatalysis of Immobilized Sulfite Oxidase JF - Electroanalysis : an international journal devoted to fundamental and practical aspects of electroanalysis KW - Direct electron transfer KW - Protein voltammetry KW - Human sulfite oxidase KW - Bioelectrocatalysis KW - Nanoparticles Y1 - 2016 U6 - https://doi.org/10.1002/elan.201600246 SN - 1040-0397 SN - 1521-4109 VL - 28 SP - 2303 EP - 2310 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Terao, Mineko A1 - Romao, Maria Joao A1 - Leimkühler, Silke A1 - Bolis, Marco A1 - Fratelli, Maddalena A1 - Coelho, Catarina A1 - Santos-Silva, Teresa A1 - Garattini, Enrico T1 - Structure and function of mammalian aldehyde oxidases JF - Archives of toxicology : official journal of EUROTOX N2 - Mammalian aldehyde oxidases (AOXs; EC1.2.3.1) are a group of conserved proteins belonging to the family of molybdo-flavoenzymes along with the structurally related xanthine dehydrogenase enzyme. AOXs are characterized by broad substrate specificity, oxidizing not only aromatic and aliphatic aldehydes into the corresponding carboxylic acids, but also hydroxylating a series of heteroaromatic rings. The number of AOX isoenzymes expressed in different vertebrate species is variable. The two extremes are represented by humans, which express a single enzyme (AOX1) in many organs and mice or rats which are characterized by tissue-specific expression of four isoforms (AOX1, AOX2, AOX3, and AOX4). In vertebrates each AOX isoenzyme is the product of a distinct gene consisting of 35 highly conserved exons. The extant species-specific complement of AOX isoenzymes is the result of a complex evolutionary process consisting of a first phase characterized by a series of asynchronous gene duplications and a second phase where the pseudogenization and gene deletion events prevail. In the last few years remarkable advances in the elucidation of the structural characteristics and the catalytic mechanisms of mammalian AOXs have been made thanks to the successful crystallization of human AOX1 and mouse AOX3. Much less is known about the physiological function and physiological substrates of human AOX1 and other mammalian AOX isoenzymes, although the importance of these proteins in xenobiotic metabolism is fairly well established and their relevance in drug development is increasing. This review article provides an overview and a discussion of the current knowledge on mammalian AOX. KW - Aldehyde oxidase KW - Molybdo-flavoenzymes KW - Xanthine oxidoreductase KW - Drug metabolism Y1 - 2016 U6 - https://doi.org/10.1007/s00204-016-1683-1 SN - 0340-5761 SN - 1432-0738 VL - 90 SP - 753 EP - 780 PB - Springer CY - Heidelberg ER - TY - JOUR A1 - Correia, Marcia A. S. A1 - Otrelo-Cardoso, Ana Rita A1 - Schwuchow, Viola A1 - Clauss, Kajsa G. V. Sigfridsson A1 - Haumann, Michael A1 - Romao, Maria Joao A1 - Leimkühler, Silke A1 - Santos-Silva, Teresa T1 - The Escherichia coli Periplasmic Aldehyde Oxidoreductase Is an Exceptional Member of the Xanthine Oxidase Family of Molybdoenzymes JF - ACS chemical biology N2 - The xanthine oxidase (XO) family comprises molybdenum-dependent enzymes that usually form homodimers (or dimers of heterodimers/trimers) organized in three domains that harbor two [2Fe-2S] clusters, one FAD, and a Mo cofactor. In this work, we crystallized an unusual member of the family, the periplasmic aldehyde oxidoreductase PaoABC from Escherichia coli. This is the first example of an E. coli protein containing a molybdopterin-cytosine-dinucleotide cofactor and is the only heterotrimer of the XO family so far structurally characterized. The crystal structure revealed the presence of an unexpected [4Fe-4S] cluster, anchored to an additional 40 residues subdomain. According to phylogenetic analysis, proteins containing this cluster are widely spread in many bacteria phyla, putatively through repeated gene transfer events. The active site of PaoABC is highly exposed to the surface with no aromatic residues and an arginine (PaoC-R440) making a direct interaction with PaoC-E692, which acts as a base catalyst. In order to understand the importance of R440, kinetic assays were carried out, and the crystal structure of the PaoC-R440H variant was also determined. Y1 - 2016 U6 - https://doi.org/10.1021/acschembio.6b00572 SN - 1554-8929 SN - 1554-8937 VL - 11 SP - 2923 EP - 2935 PB - American Chemical Society CY - Washington ER - TY - GEN A1 - Yan, Robert A1 - Friemel, Martin A1 - Aloisi, Claudia A1 - Huynen, Martijn A1 - Taylor, Ian A. A1 - Leimkühler, Silke A1 - Pastore, Annalisa T1 - The eukaryotic-specific Isd11 is a complex- orphan protein with ability to bind the prokaryotic IscS T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The eukaryotic protein Isd11 is a chaperone that binds and stabilizes the central component of the essential metabolic pathway responsible for formation of iron-sulfur clusters in mitochondria, the desulfurase Nfs1. Little is known about the exact role of Isd11. Here, we show that human Isd11 (ISD11) is a helical protein which exists in solution as an equilibrium between monomer, dimeric and tetrameric species when in the absence of human Nfs1 (NFS1). We also show that, surprisingly, recombinant ISD11 expressed in E. coli co-purifies with the bacterial orthologue of NFS1, IscS. Binding is weak but specific suggesting that, despite the absence of Isd11 sequences in bacteria, there is enough conservation between the two desulfurases to retain a similar mode of interaction. This knowledge may inform us on the conservation of the mode of binding of Isd11 to the desulfurase. We used evolutionary evidence to suggest Isd11 residues involved in the interaction. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 551 KW - sulfur cluster formation KW - Escherichia coli KW - cysteine desulfurase KW - interacting protein KW - bacterial frataxin KW - statistical-model KW - biogenesis KW - biosynthesis KW - NFS1 KW - deficiency Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-411906 SN - 1866-8372 IS - 551 ER - TY - JOUR A1 - Yan, Robert A1 - Friemel, Martin A1 - Aloisi, Claudia A1 - Huynen, Martijn A1 - Taylor, Ian A. A1 - Leimkühler, Silke A1 - Pastore, Annalisa T1 - The Eukaryotic-Specific ISD11 Is a Complex-Orphan Protein with Ability to Bind the Prokaryotic IscS JF - PLoS one N2 - The eukaryotic protein Isd11 is a chaperone that binds and stabilizes the central component of the essential metabolic pathway responsible for formation of iron-sulfur clusters in mitochondria, the desulfurase Nfs1. Little is known about the exact role of Isd11. Here, we show that human Isd11 (ISD11) is a helical protein which exists in solution as an equilibrium between monomer, dimeric and tetrameric species when in the absence of human Nfs1 (NFS1). We also show that, surprisingly, recombinant ISD11 expressed in E. coli co-purifies with the bacterial orthologue of NFS1, IscS. Binding is weak but specific suggesting that, despite the absence of Isd11 sequences in bacteria, there is enough conservation between the two desulfurases to retain a similar mode of interaction. This knowledge may inform us on the conservation of the mode of binding of Isd11 to the desulfurase. We used evolutionary evidence to suggest Isd11 residues involved in the interaction. Y1 - 2016 U6 - https://doi.org/10.1371/journal.pone.0157895 SN - 1932-6203 VL - 11 SP - 383 EP - 395 PB - PLoS CY - San Fransisco ER -