@article{YanFrokjarEngelbrektetal.2021,
  author    = {Yan, Jiawei and Fr{\o}kj{\ae}r, Emil Egede and Engelbrekt, Christian and Leimk{\"u}hler, Silke and Ulstrup, Jens and Wollenberger, Ulla and Xiao, Xinxin and Zhang, Jingdong},
  title     = {Voltammetry and single-molecule in situ scanning tunnelling microscopy of the redox metalloenzyme human sulfite oxidase},
  series = {ChemElectroChem},
  volume    = {8},
  journal   = {ChemElectroChem},
  number    = {1},
  publisher = {Wiley-VCH},
  address   = {Weinheim},
  issn      = {2196-0216},
  doi       = {10.1002/celc.202001258},
  pages     = {164 -- 171},
  year      = {2021},
  abstract  = {Human sulfite oxidase (hSO) is a homodimeric two-domain enzyme central in the biological sulfur cycle. A pyranopterin molybdenum cofactor (Moco) is the catalytic site and a heme b(5) group located in the N-terminal domain. The two domains are connected by a flexible linker region. Electrons produced at the Moco in sulfite oxidation, are relayed via heme b(5) to electron acceptors or an electrode surface. Inter-domain conformational changes between an open and a closed enzyme conformation, allowing "gated" electron transfer has been suggested. We first recorded cyclic voltammetry (CV) of hSO on single-crystal Au(111)-electrode surfaces modified by self-assembled monolayers (SAMs) both of a short rigid thiol, cysteamine and of a longer structurally flexible thiol, omega-amino-octanethiol (AOT). hSO on cysteamine SAMs displays a well-defined pair of voltammetric peaks around -0.207 V vs. SCE in the absence of sulfite substrate, but no electrocatalysis. hSO on AOT SAMs displays well-defined electrocatalysis, but only "fair" quality voltammetry in the absence of sulfite. We recorded next in situ scanning tunnelling spectroscopy (STS) of hSO on AOT modified Au(111)-electrodes, disclosing, a 2-5 \% surface coverage of strong molecular scale contrasts, assigned to single hSO molecules, notably with no contrast difference in the absence and presence of sulfite. In situ STS corroborated this observation with a sigmoidal tunnelling current/overpotential correlation.},
  language  = {en}
}
@article{TadjoungWaffoMitrovaTiedemannetal.2021,
  author    = {Tadjoung Waffo, Armel Franklin and Mitrova, Biljana and Tiedemann, Kim and Iobbi-Nivol, Chantal and Leimk{\"u}hler, Silke and Wollenberger, Ulla},
  title     = {Electrochemical trimethylamine n-oxide biosensor with enzyme-based oxygen-scavenging membrane for long-term operation under ambient air},
  series = {Biosensors : open access journal},
  volume    = {11},
  journal   = {Biosensors : open access journal},
  number    = {4},
  publisher = {MDPI},
  address   = {Basel},
  issn      = {2079-6374},
  doi       = {10.3390/bios11040098},
  pages     = {17},
  year      = {2021},
  abstract  = {An amperometric trimethylamine N-oxide (TMAO) biosensor is reported, where TMAO reductase (TorA) and glucose oxidase (GOD) and catalase (Cat) were immobilized on the electrode surface, enabling measurements of mediated enzymatic TMAO reduction at low potential under ambient air conditions. The oxygen anti-interference membrane composed of GOD, Cat and polyvinyl alcohol (PVA) hydrogel, together with glucose concentration, was optimized until the O-2 reduction current of a Clark-type electrode was completely suppressed for at least 3 h. For the preparation of the TMAO biosensor, Escherichia coli TorA was purified under anaerobic conditions and immobilized on the surface of a carbon electrode and covered by the optimized O-2 scavenging membrane. The TMAO sensor operates at a potential of -0.8 V vs. Ag/AgCl (1 M KCl), where the reduction of methylviologen (MV) is recorded. The sensor signal depends linearly on TMAO concentrations between 2 mu M and 15 mM, with a sensitivity of 2.75 +/- 1.7 mu A/mM. The developed biosensor is characterized by a response time of about 33 s and an operational stability over 3 weeks. Furthermore, measurements of TMAO concentration were performed in 10\% human serum, where the lowest detectable concentration is of 10 mu M TMAO.},
  language  = {en}
}
@article{DeSousaMotaDinizCoelhoetal.2021,
  author    = {De Sousa Mota, Cristiano and Diniz, Ana and Coelho, Catarina and Santos-Silva, Teresa and Esmaeeli Moghaddam Tabalvandani, Mariam and Leimk{\"u}hler, Silke and Cabrita, Eurico J. and Marcelo, Filipa and Rom{\~a}o, Maria Jo{\~a}o},
  title     = {Interrogating the inhibition mechanisms of human aldehyde oxidase by X-ray crystallography and NMR spectroscopy},
  series = {Journal of medicinal chemistry / American Chemical Society},
  volume    = {64},
  journal   = {Journal of medicinal chemistry / American Chemical Society},
  number    = {17},
  publisher = {American Chemical Society},
  address   = {Washington},
  issn      = {0022-2623},
  doi       = {10.1021/acs.jmedchem.1c01125},
  pages     = {13025 -- 13037},
  year      = {2021},
  abstract  = {Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitors-thioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition. Additionally, we describe the first crystal structure of hAOX1 in complex with raloxifene. Raloxifene binds tightly at the entrance of the substrate tunnel, stabilizing the flexible entrance gates and elucidating an unusual substrate-dependent mechanism of inhibition with potential impact on drug-drug interactions. This study can be considered as a proof-of-concept for an efficient experimental screening of prospective substrates and inhibitors of hAOX1 relevant in drug discovery.},
  language  = {en}
}
@article{Leimkuehler2021,
  author    = {Leimk{\"u}hler, Silke},
  title     = {Transition metals in catalysis},
  series = {Inorganics : open access journal},
  volume    = {9},
  journal   = {Inorganics : open access journal},
  number    = {1},
  publisher = {MDPI},
  address   = {Basel},
  issn      = {2304-6740},
  doi       = {10.3390/inorganics9010006},
  pages     = {2},
  year      = {2021},
  language  = {en}
}
@article{GarridoLeimkuehler2021,
  author    = {Garrido, Claudia and Leimk{\"u}hler, Silke},
  title     = {The inactivation of human aldehyde oxidase 1 by hydrogen peroxide and superoxide},
  series = {Drug metabolism and disposition / American Society for Pharmacology and Experimental Therapeutics},
  volume    = {49},
  journal   = {Drug metabolism and disposition / American Society for Pharmacology and Experimental Therapeutics},
  number    = {9},
  publisher = {American Society for Pharmacology and Experimental Therapeutics},
  address   = {Bethesda},
  issn      = {1521-009X},
  doi       = {10.1124/dmd.121.000549},
  pages     = {729 -- 735},
  year      = {2021},
  abstract  = {Mammalian aldehyde oxidases (AOX) are molybdo-flavoenzymes of pharmacological and pathophysiologic relevance that are involved in phase I drug metabolism and, as a product of their enzymatic activity, are also involved in the generation of reactive oxygen species. So far, the physiologic role of aldehyde oxidase 1 in the human body remains unknown. The human enzyme hAOX1 is characterized by a broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into their corresponding carboxylic acids, and hydroxylating various heteroaromatic rings. The enzyme uses oxygen as terminal electron acceptor to produce hydrogen peroxide and superoxide during turnover. Since hAOX1 and, in particular, some natural variants produce not only H2O2 but also high amounts of superoxide, we investigated the effect of both ROS molecules on the enzymatic activity of hAOX1 in more detail. We compared hAOX1 to the high-O-2(.-)-producing natural variant L438V for their time-dependent inactivation with H2O2/O-2(.-) during substrate turnover. We show that the inactivation of the hAOX1 wild-type enzyme is mainly based on the production of hydrogen peroxide, whereas for the variant L438V, both hydrogen peroxide and superoxide contribute to the time-dependent inactivation of the enzyme during turnover. Further, the level of inactivation was revealed to be substrate-dependent: using substrates with higher turnover numbers resulted in a faster inactivation of the enzymes. Analysis of the inactivation site of the enzyme identified a loss of the terminal sulfido ligand at the molybdenum active site by the produced ROS during turnover.},
  language  = {en}
}
@article{YildizLeimkuehler2021,
  author    = {Yildiz, Tugba and Leimk{\"u}hler, Silke},
  title     = {TusA is a versatile protein that links translation efficiency to cell division in Escherichia coli},
  series = {Journal of bacteriology},
  volume    = {203},
  journal   = {Journal of bacteriology},
  number    = {7},
  publisher = {American Society for Microbiology},
  address   = {Washington},
  issn      = {1098-5530},
  doi       = {10.1128/JB.00659-20},
  pages     = {20},
  year      = {2021},
  abstract  = {To enable accurate and efficient translation, sulfur modifications are introduced posttranscriptionally into nucleosides in tRNAs. The biosynthesis of tRNA sulfur modifications involves unique sulfur trafficking systems for the incorporation of sulfur atoms in different nucleosides of tRNA. One of the proteins that is involved in inserting the sulfur for 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U34) modifications in tRNAs is the TusA protein. TusA, however, is a versatile protein that is also involved in numerous other cellular pathways. Despite its role as a sulfur transfer protein for the 2-thiouridine formation in tRNA, a fundamental role of TusA in the general physiology of Escherichia coli has also been discovered. Poor viability, a defect in cell division, and a filamentous cell morphology have been described previously for tusA-deficient cells. In this report, we aimed to dissect the role of TusA for cell viability. We were able to show that the lack of the thiolation status of wobble uridine (U-34) nucleotides present on Lys, Gln, or Glu in tRNAs has a major consequence on the translation efficiency of proteins; among the affected targets are the proteins RpoS and Fis. Both proteins are major regulatory factors, and the deregulation of their abundance consequently has a major effect on the cellular regulatory network, with one consequence being a defect in cell division by regulating the FtsZ ring formation. <br /> IMPORTANCE More than 100 different modifications are found in RNAs. One of these modifications is the mnm(5)s(2)U modification at the wobble position 34 of tRNAs for Lys, Gln, and Glu. The functional significance of U34 modifications is substantial since it restricts the conformational flexibility of the anticodon, thus providing translational fidelity. We show that in an Escherichia coli TusA mutant strain, involved in sulfur transfer for the mnm(5)s(2)U34 thio modifications, the translation efficiency of RpoS and Fis, two major cellular regulatory proteins, is altered. Therefore, in addition to the transcriptional regulation and the factors that influence protein stability, tRNA modifications that ensure the translational efficiency provide an additional crucial regulatory factor for protein synthesis.},
  language  = {en}
}
@article{HasnatZupokOlasApeltetal.2021,
  author    = {Hasnat, Muhammad Abrar and Zupok, Arkadiusz and Olas-Apelt, Justyna Jadwiga and M{\"u}ller-R{\"o}ber, Bernd and Leimk{\"u}hler, Silke},
  title     = {A-type carrier proteins are involved in [4Fe-4S] cluster insertion into the radical S-adenosylmethionine protein MoaA for the synthesis of active molybdoenzymes},
  series = {Journal of bacteriology},
  volume    = {203},
  journal   = {Journal of bacteriology},
  number    = {12},
  publisher = {American Society for Microbiology},
  address   = {Washington},
  issn      = {1098-5530},
  doi       = {10.1128/JB.00086-21},
  pages     = {20},
  year      = {2021},
  abstract  = {Iron sulfur (Fe-S) clusters are important biological cofactors present in proteins with crucial biological functions, from photosynthesis to DNA repair, gene expression, and bioenergetic processes. For the insertion of Fe-S clusters into proteins, A-type carrier proteins have been identified. So far, three of them have been characterized in detail in Escherichia coli, namely, IscA, SufA, and ErpA, which were shown to partially replace each other in their roles in [4Fe-4S] cluster insertion into specific target proteins. To further expand the knowledge of [4Fe-4S] cluster insertion into proteins, we analyzed the complex Fe-S cluster-dependent network for the synthesis of the molybdenum cofactor (Moco) and the expression of genes encoding nitrate reductase in E. coli. Our studies include the identification of the A-type carrier proteins ErpA and IscA, involved in [4Fe-4S] cluster insertion into the radical Sadenosyl-methionine (SAM) enzyme MoaA. We show that ErpA and IscA can partially replace each other in their role to provide [4Fe-4S] clusters for MoaA. Since most genes expressing molybdoenzymes are regulated by the transcriptional regulator for fumarate and nitrate reduction (FNR) under anaerobic conditions, we also identified the proteins that are crucial to obtain an active FNR under conditions of nitrate respiration. We show that ErpA is essential for the FNR-dependent expression of the narGHJI operon, a role that cannot be compensated by IscA under the growth conditions tested. SufA does not appear to have a role in Fe-S cluster insertion into MoaA or FNR under anaerobic growth employing nitrate respiration, based on the low level of gene expression. <br /> IMPORTANCE Understanding the assembly of iron-sulfur (Fe-S) proteins is relevant to many fields, including nitrogen fixation, photosynthesis, bioenergetics, and gene regulation. Remaining critical gaps in our knowledge include how Fe-S clusters are transferred to their target proteins and how the specificity in this process is achieved, since different forms of Fe-S clusters need to be delivered to structurally highly diverse target proteins. Numerous Fe-S carrier proteins have been identified in prokaryotes like Escherichia coli, including ErpA, IscA, SufA, and NfuA. In addition, the diverse Fe-S cluster delivery proteins and their target proteins underlie a complex regulatory network of expression, to ensure that both proteins are synthesized under particular growth conditions.},
  language  = {en}
}