@article{FotiDorendorfLeimkuehler2017, author = {Foti, Alessandro and Dorendorf, Frank and Leimk{\"u}hler, Silke}, title = {A single nucleotide polymorphism causes enhanced radical oxygen species production by human aldehyde oxidase}, series = {PLoS one}, volume = {12}, journal = {PLoS one}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0182061}, pages = {18338 -- 18347}, year = {2017}, abstract = {Aldehyde oxidases (AOXs) are molybdo-flavoenzymes characterized by broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into the corresponding carboxylic acids and hydroxylating various heteroaromatic rings. The enzymes use oxygen as the terminal electron acceptor and produce reduced oxygen species during turnover. The physiological function of mammalian AOX isoenzymes is still unclear, however, human AOX (hAOX1) is an emerging enzyme in phase-I drug metabolism. Indeed, the number of xenobiotics acting as hAOX1 substrates is increasing. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified within the hAOX1 gene. SNPs are a major source of inter-individual variability in the human population, and SNP-based amino acid exchanges in hAOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. In this report we selected ten novel SNPs resulting in amino acid exchanges in proximity to the FAD site of hAOX1 and characterized the purified enzymes after heterologous expression in Escherichia coli. The hAOX1 variants were characterized carefully by quantitative differences in their ability to produce superoxide radical. ROS represent prominent key molecules in physiological and pathological conditions in the cell. Our data reveal significant alterations in superoxide anion production among the variants. In particular the SNP-based amino acid exchange L438V in proximity to the isoalloxanzine ring of the FAD cofactor resulted in increased rate of superoxide radical production of 75\%. Considering the high toxicity of the superoxide in the cell, the hAOX1-L438V SNP variant is an eventual candidate for critical or pathological roles of this natural variant within the human population.}, language = {en} } @article{LeimkuehlerLemaireIobbiNivol2017, author = {Leimk{\"u}hler, Silke and Lemaire, Olivier N. and Iobbi-Nivol, Chantal}, title = {Bacterial Molybdoenzymes}, series = {Molybdenum and tungsten enzymes : biochemistry}, volume = {5}, journal = {Molybdenum and tungsten enzymes : biochemistry}, publisher = {Royal Society of Chemistry}, address = {Cambridge}, isbn = {978-1-78262-391-5}, doi = {10.1039/9781782623915-00117}, pages = {117 -- 142}, year = {2017}, abstract = {The biogenesis of molybdoenzymes is a cytoplasmic event requiring both the folded apoenzymes and the matured molybdenum cofactor. The structure and the complexity of the molybdenum cofactor varies in each molybdoenzyme family and consequently different accessory proteins are required for the maturation of the respective enzymes. Thus, for enzymes of both the DMSO reductase and xanthine oxidase families, specific chaperones exist which are dedicated to increase the stability and the folding of specific members of each family. In this review, we describe the role of these chaperones for molybdoenzyme maturation. We present a model which describes step by step the mechanism of the maturation of representative molybdoenzymes from each family.}, language = {en} } @article{KuecuekgoezeTeraoGarattinietal.2017, author = {Kuecuekgoeze, Goekhan and Terao, Mineko and Garattini, Enrico and Leimk{\"u}hler, Silke}, title = {Direct Comparison of the Enzymatic Characteristics and Superoxide Production of the Four Aldehyde Oxidase Enzymes Present in Mouse}, series = {Drug metabolism and disposition : the biological fate of chemicals}, volume = {45}, journal = {Drug metabolism and disposition : the biological fate of chemicals}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, address = {Bethesda}, issn = {0090-9556}, doi = {10.1124/dmd.117.075937}, pages = {947 -- 955}, year = {2017}, abstract = {Aldehyde oxidases (AOXs) are molybdoflavoenzymes with an important role in the metabolism and detoxification of heterocyclic compounds and aliphatic as well as aromatic aldehydes. The enzymes use oxygen as the terminal electron acceptor and produce reduced oxygen species during turnover. Four different enzymes, mAOX1, mAOX3, mAOX4, and mAOX2, which are the products of distinct genes, are present in the mouse. A direct and simultaneous comparison of the enzymatic properties and characteristics of the four enzymes has never been performed. In this report, the four catalytically active mAOX enzymes were purified after heterologous expression in Escherichia coli. The kinetic parameters of the four mouse AOX enzymes were determined and compared with the use of six predicted substrates of physiologic and toxicological interest, i.e., retinaldehyde, N1-methylnicotinamide, pyridoxal, vanillin, 4-(dimethylamino) cinnamaldehyde (p-DMAC), and salicylaldehyde. While retinaldehyde, vanillin, p-DMAC, and salycilaldehyde are efficient substrates for the four mouse AOX enzymes, N1-methylnicotinamide is not a substrate of mAOX1 or mAOX4, and pyridoxal is notmetabolized by any of the purified enzymes. Overall, mAOX1, mAOX2, mAOX3, and mAOX4 are characterized by significantly different KM and kcat values for the active substrates. The four mouse AOXs are also characterized by quantitative differences in their ability to produce superoxide radicals. With respect to this last point, mAOX2 is the enzyme generating the largest rate of superoxide radicals of around 40\% in relation to moles of substrate converted, and mAOX1, the homolog to the human enzyme, produces a rate of approximately 30\% of superoxide radicals with the same substrate.}, language = {en} } @article{ParagasHumphreysMinetal.2017, author = {Paragas, Erickson M. and Humphreys, Sara C. and Min, Joshua and Joswig-Jones, Carolyn A. and Leimk{\"u}hler, Silke and Jones, Jeffrey P.}, title = {ecoAO}, series = {ACS OMEGA}, volume = {2}, journal = {ACS OMEGA}, publisher = {American Chemical Society}, address = {Washington}, issn = {2470-1343}, doi = {10.1021/acsomega.7b01054}, pages = {4820 -- 4827}, year = {2017}, abstract = {Although aldehyde oxidase (AO) is an important hepatic drug-metabolizing enzyme, it remains understudied and is consequently often overlooked in preclinical studies, an oversight that has resulted in the failure of multiple clinical trials. AO's preclusion to investigation stems from the following: (1) difficulties synthesizing metabolic standards due to the chemospecificity and regiospecificity of the enzyme and (2) significant inherent variability across existing in vitro systems including liver cytosol, S9 fractions, and primary hepatocytes, which lack specificity and generate discordant expression and activity profiles. Here, we describe a practical bacterial biotransformation system, ecoAO, addressing both issues simultaneously. ecoAO is a cell paste of MoCo-producing Escherichia coli strain TP1017 expressing human AO. It exhibits specific activity toward known substrates, zoniporide, 4-trans-(N,N-dimethylamino)cinnamaldehyde, O6-benzylguanine, and zaleplon; it also has utility as a biocatalyst, yielding milligram quantities of synthetically challenging metabolite standards such as 2-oxo-zoniporide. Moreover, ecoAO enables routine determination of kcat and V/K, which are essential parameters for accurate in vivo clearance predictions. Furthermore, ecoAO has potential as a preclinical in vitro screening tool for AO activity, as demonstrated by its metabolism of 3-aminoquinoline, a previously uncharacterized substrate. ecoAO promises to provide easy access to metabolites with the potential to improve pharmacokinetic clearance predictions and guide drug development.}, language = {en} } @article{NishinoOkamotoLeimkuehler2017, author = {Nishino, Takeshi and Okamoto, Ken and Leimk{\"u}hler, Silke}, title = {Enzymes of the Xanthine Oxidase Family}, series = {Molybdenum and tungsten enzymes : biochemistry}, volume = {5}, journal = {Molybdenum and tungsten enzymes : biochemistry}, publisher = {Royal Society of Chemistry}, address = {Cambridge}, isbn = {978-1-78262-391-5}, doi = {10.1039/9781782623915-00192}, pages = {192 -- 239}, year = {2017}, abstract = {Enzymes from the xanthine oxidase (XO) family of molybdenum enzymes are generally, with some exceptions, molybdenum iron-sulfur flavin hydroxylases. Mammalian xanthine oxidoreductase and aldehyde oxidase were among the first enzymes to be studied in detail more than 100 years ago and, surprisingly, they continue to be thoroughly studied in molecular detail with many open and unresolved questions remaining. Enzymes of the XO family are characterized by a molybdenum cofactor (Moco) active site with a MoVIOS(OH) ligand sphere where substrate hydroxylation of either aromatic or aliphatic carbon centers is catalyzed. During the reaction, electrons are transferred to the oxidizing substrate, most commonly O2 or NAD+, which react at the FAD site.}, language = {en} } @article{BuehningFriemelLeimkuehler2017, author = {B{\"u}hning, Martin and Friemel, Martin and Leimk{\"u}hler, Silke}, title = {Functional Complementation Studies Reveal Different Interaction Partners of Escherichia coil IscS and Human NFS1}, series = {Biochemistry}, volume = {56}, journal = {Biochemistry}, publisher = {American Chemical Society}, address = {Washington}, issn = {0006-2960}, doi = {10.1021/acs.biochem.7b00627}, pages = {4592 -- 4605}, year = {2017}, abstract = {The trafficking and delivery of sulfur to cofactors and nucleosides is a highly regulated and conserved process among all organisms. All sulfur transfer pathways generally have an L-cysteine desulfurase as an initial sulfur mobilizing enzyme in common, which serves as a sulfur donor for the biosynthesis of sulfur-containing biomolecules like iron sulfur (Fe-S) clusters, thiamine, biotin, lipoic acid, the molybdenum cofactor (Moco), and thiolated nucleosides in tRNA. The human L-cysteine desulfurase NFS1 and the Escherichia coli homologue IscS share a level of amino acid sequence identity of similar to 60\%. While E. coli IscS has a versatile role in the cell and was shown to have numerous interaction partners, NFS1 is mainly localized in mitochondria with a crucial role in the biosynthesis of Fe-S clusters. Additionally, NFS1 is also located in smaller amounts in the cytosol with a role in Moco biosynthesis and mcm(5)s(2)U34 thio modifications of nucleosides in tRNA. NFS1 and IscS were conclusively shown to have different interaction partners in their respective organisms. Here, we used functional complementation studies of an E. coli iscS deletion strain with human NFS1 to dissect their conserved roles in the transfer of sulfur to a specific target protein. Our results show that human NFS1 and E. coli IscS share conserved binding sites for proteins involved in Fe-S cluster assembly like IscU, but not with proteins for tRNA thio modifications or Moco biosynthesis. In addition, we show that human NFS1 was almost fully able to complement the role of IscS in Moco biosynthesis when its specific interaction partner protein MOCS3 from humans was also present.}, language = {en} } @article{LeimkuehlerMendel2017, author = {Leimk{\"u}hler, Silke and Mendel, Ralf-Rainer}, title = {Molybdenum Cofactor Biosynthesis}, series = {Molybdenum and tungsten enzymes: biochemistry}, volume = {5}, journal = {Molybdenum and tungsten enzymes: biochemistry}, publisher = {Royal Society of Chemistry}, address = {Cambridge}, isbn = {978-1-78262-391-5}, doi = {10.1039/9781782623915}, pages = {100 -- 116}, year = {2017}, abstract = {The biosynthesis of the molybdenum cofactor (Moco) is highly conserved among all kingdoms of life. In all molybdoenzymes with the exception of nitrogenase, the molybdenum atom is coordinated to a dithiolene group present in the pterin-based 6-alkyl side chain of molybdopterin (MPT). In general, the biosynthesis of Moco can be divided into three steps in eukaryotes, and four steps in bacteria and archaea: (i) the starting point is the formation of the cyclic pyranopterin monophosphate (cPMP) from 5′GTP, (ii) in the second step the two sulfur molecules are inserted into cPMP leading to the formation of MPT, (iii) in the third step the molybdenum atom is inserted into molybdopterin to form Moco and (iv) additional modification of Moco occurs in bacteria and archaea with the attachment of a nucleotide (CMP or GMP) to the phosphate group of MPT, forming the dinucleotide variants of Moco. This review will focus on the biosynthesis of Moco in bacteria, humans and plants.}, language = {en} } @article{ReschkeMebsSigfridssonClaussetal.2017, author = {Reschke, Stefan and Mebs, Stefan and Sigfridsson-Clauss, Kajsa G. V. and Kositzki, Ramona and Leimk{\"u}hler, Silke and Haumann, Michael}, title = {Protonation and Sulfido versus Oxo Ligation Changes at the Molybdenum Cofactor in Xanthine Dehydrogenase (XDH) Variants Studied by X-ray Absorption Spectroscopy}, series = {Inorganic chemistry}, volume = {56}, journal = {Inorganic chemistry}, number = {4}, publisher = {American Chemical Society}, address = {Washington}, issn = {0020-1669}, doi = {10.1021/acs.inorgchem.6b02846}, pages = {2165 -- 2176}, year = {2017}, abstract = {Enzymes of the xanthine oxidase family are among the best characterized mononuclear molybdenum enzymes. Open questions about their mechanism of transfer of an oxygen atom to the substrate remain. The enzymes share a molybdenum cofactor (Moco) with the metal ion binding a molybdopterin (MPT) molecule via its dithiolene function and terminal sulfur and oxygen groups. For xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus, we used X-ray absorption spectroscopy to determine the Mo site structure, its changes in a pH range of 5-10, and the influence of amino acids (Glu730 and Gln179) close to Moco in wild-type (WT), Q179A, and E730A variants, complemented by enzyme kinetics and quantum chemical studies. Oxidized WT and Q179A revealed a similar Mo (VI) ion with each one MPT, Mo=O, Mo-O-, and Mo=S ligand, and a weak Mo-O(E730) bond at alkaline pH. Protonation of an oxo to a hydroxo (OH) ligand (pK similar to 6.8) causes inhibition of XDH at acidic pH, whereas deprotonated xanthine (pK similar to 8.8) is an inhibitor at alkaline pH. A similar acidic pK for the WT and Q179A. variants, as well as the metrical parameters of the Mo site and density functional theory calculations, suggested protonation at the equatorial oxo group. The sulfido was replaced with an oxo ligand in the inactive E730A variant, further showing another oxo and one Mo OH ligand at Mo, which are independent of pH. Our findings suggest a reaction mechanism for XDH in which an initial oxo rather than a hydroxo group and the sulfido ligand are essential for xanthine oxidation.}, language = {en} } @misc{Leimkuehler2017, author = {Leimk{\"u}hler, Silke}, title = {Shared function and moonlighting proteins in molybdenum cofactor biosynthesis}, series = {Biological chemistry}, volume = {398}, journal = {Biological chemistry}, publisher = {De Gruyter}, address = {Berlin}, issn = {1431-6730}, doi = {10.1515/hsz-2017-0110}, pages = {1009 -- 1026}, year = {2017}, abstract = {The biosynthesis of the molybdenum cofactor (Moco) is a highly conserved pathway in bacteria, archaea and eukaryotes. The molybdenum atom in Moco-containing enzymes is coordinated to the dithiolene group of a tricyclic pyranopterin monophosphate cofactor. The biosynthesis of Moco can be divided into three conserved steps, with a fourth present only in bacteria and archaea: (1) formation of cyclic pyranopterin monophosphate, (2) formation of molybdopterin (MPT), (3) insertion of molybdenum into MPT to form Mo-MPT, and (4) additional modification of Mo-MPT in bacteria with the attachment of a GMP or CMP nucleotide, forming the dinucleotide variants of Moco. While the proteins involved in the catalytic reaction of each step of Moco biosynthesis are highly conserved among the Phyla, a surprising link to other cellular pathways has been identified by recent discoveries. In particular, the pathways for FeS cluster assembly and thio-modifications of tRNA are connected to Moco biosynthesis by sharing the same protein components. Further, proteins involved in Moco biosynthesis are not only shared with other pathways, but additionally have moonlighting roles. This review gives an overview of Moco biosynthesis in bacteria and humans and highlights the shared function and moonlighting roles of the participating proteins.}, language = {en} } @misc{LeimkuehlerBuehningBeilschmidt2017, author = {Leimk{\"u}hler, Silke and B{\"u}hning, Martin and Beilschmidt, Lena}, title = {Shared sulfur mobilization routes for tRNA thiolation and molybdenum cofactor biosynthesis in prokaryotes and eukaryotes}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch Naturwissenschaftliche Reihe}, number = {1015}, issn = {1866-8372}, doi = {10.25932/publishup-47501}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-475011}, pages = {22}, year = {2017}, abstract = {Modifications of transfer RNA (tRNA) have been shown to play critical roles in the biogenesis, metabolism, structural stability and function of RNA molecules, and the specific modifications of nucleobases with sulfur atoms in tRNA are present in pro- and eukaryotes. Here, especially the thiomodifications xm(5)s(2)U at the wobble position 34 in tRNAs for Lys, Gln and Glu, were suggested to have an important role during the translation process by ensuring accurate deciphering of the genetic code and by stabilization of the tRNA structure. The trafficking and delivery of sulfur nucleosides is a complex process carried out by sulfur relay systems involving numerous proteins, which not only deliver sulfur to the specific tRNAs but also to other sulfur-containing molecules including iron-sulfur clusters, thiamin, biotin, lipoic acid and molybdopterin (MPT). Among the biosynthesis of these sulfur-containing molecules, the biosynthesis of the molybdenum cofactor (Moco) and the synthesis of thio-modified tRNAs in particular show a surprising link by sharing protein components for sulfur mobilization in pro- and eukaryotes.}, language = {en} }