@article{TeraoGarattiniRomaoetal.2020, author = {Terao, Mineko and Garattini, Enrico and Rom{\~a}o, Maria Jo{\~a}o and Leimk{\"u}hler, Silke}, title = {Evolution, expression, and substrate specificities of aldehyde oxidase enzymes in eukaryotes}, series = {The journal of biological chemistry}, volume = {295}, journal = {The journal of biological chemistry}, number = {16}, publisher = {American Society for Biochemistry and Molecular Biology}, address = {Rockville}, issn = {0021-9258}, doi = {10.1074/jbc.REV119.007741}, pages = {5377 -- 5389}, year = {2020}, abstract = {Aldehyde oxidases (AOXs) are a small group of enzymes belonging to the larger family of molybdo-flavoenzymes, along with the well-characterized xanthine oxidoreductase. The two major types of reactions that are catalyzed by AOXs are the hydroxylation of heterocycles and the oxidation of aldehydes to their corresponding carboxylic acids. Different animal species have different complements of AOX genes. The two extremes are represented in humans and rodents; whereas the human genome contains a single active gene (AOX1), those of rodents, such as mice, are endowed with four genes (Aox1-4), clustering on the same chromosome, each encoding a functionally distinct AOX enzyme. It still remains enigmatic why some species have numerous AOX enzymes, whereas others harbor only one functional enzyme. At present, little is known about the physiological relevance of AOX enzymes in humans and their additional forms in other mammals. These enzymes are expressed in the liver and play an important role in the metabolisms of drugs and other xenobiotics. In this review, we discuss the expression, tissue-specific roles, and substrate specificities of the different mammalian AOX enzymes and highlight insights into their physiological roles.}, language = {en} } @misc{Leimkuehler2020, author = {Leimk{\"u}hler, Silke}, title = {The biosynthesis of the molybdenum cofactors in Escherichia coli}, series = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {6}, issn = {1866-8372}, doi = {10.25932/publishup-51655}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-516559}, pages = {22}, year = {2020}, abstract = {The biosynthesis of the molybdenum cofactor (Moco) is highly conserved among all kingdoms of life. In all molybdoenzymes containing Moco, 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 four steps in in bacteria: (i) the starting point is the formation of the cyclic pyranopterin monophosphate (cPMP) from 5 '-GTP, (ii) in the second step the two sulfur atoms are inserted into cPMP leading to the formation of MPT, (iii) in the third step the molybdenum atom is inserted into MPT to form Moco and (iv) in the fourth step bis-Mo-MPT is formed and an additional modification of Moco is possible with the attachment of a nucleotide (CMP or GMP) to the phosphate group of MPT, forming the dinucleotide variants of Moco. This review presents an update on the well-characterized Moco biosynthesis in the model organism Escherichia coli including novel discoveries from the recent years.}, language = {en} } @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{LaunDuffusWahlefeldetal.2022, author = {Laun, Konstantin and Duffus, Benjamin R. and Wahlefeld, Stefan and Katz, Sagie and Belger, Dennis Heinz and Hildebrandt, Peter and Mroginski, Maria Andrea and Leimk{\"u}hler, Silke and Zebger, Ingo}, title = {Infrared spectroscopy flucidates the inhibitor binding sites in a metal-dependent formate dehydrogenase}, series = {Chemistry - a European journal}, journal = {Chemistry - a European journal}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {0947-6539}, doi = {10.1002/chem.202201091}, pages = {8}, year = {2022}, abstract = {Biological carbon dioxide (CO2) reduction is an important step by which organisms form valuable energy-richer molecules required for further metabolic processes. The Mo-dependent formate dehydrogenase (FDH) from Rhodobacter capsulatus catalyzes reversible formate oxidation to CO2 at a bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor. To elucidate potential substrate binding sites relevant for the mechanism, we studied herein the interaction with the inhibitory molecules azide and cyanate, which are isoelectronic to CO2 and charged as formate. We employed infrared (IR) spectroscopy in combination with density functional theory (DFT) and inhibition kinetics. One distinct inhibitory molecule was found to bind to either a non-competitive or a competitive binding site in the secondary coordination sphere of the active site. Site-directed mutagenesis of key amino acid residues in the vicinity of the bis-MGD cofactor revealed changes in both non-competitive and competitive binding, whereby the inhibitor is in case of the latter interaction presumably bound between the cofactor and the adjacent Arg587.}, language = {en} } @article{StrippDuffusFourmondetal.2022, author = {Stripp, Sven T. and Duffus, Benjamin R. and Fourmond, Vincent and Leger, Christophe and Leimk{\"u}hler, Silke and Hirota, Shun and Hu, Yilin and Jasniewski, Andrew and Ogata, Hideaki and Ribbe, Markus W.}, title = {Second and outer coordination sphere effects in nitrogenase, hydrogenase, formate dehydrogenase, and CO dehydrogenase}, series = {Chemical reviews : CR}, volume = {122}, journal = {Chemical reviews : CR}, number = {14}, publisher = {American Chemical Society}, address = {Washington, DC}, issn = {0009-2665}, doi = {10.1021/acs.chemrev.1c00914}, pages = {11900 -- 11973}, year = {2022}, abstract = {Gases like H-2, N-2, CO2, and CO are increasingly recognized as critical feedstock in "green" energy conversion and as sources of nitrogen and carbon for the agricultural and chemical sectors. However, the industrial transformation of N-2, CO2, and CO and the production of H-2 require significant energy input, which renders processes like steam reforming and the Haber-Bosch reaction economically and environmentally unviable. Nature, on the other hand, performs similar tasks efficiently at ambient temperature and pressure, exploiting gas-processing metalloenzymes (GPMs) that bind low-valent metal cofactors based on iron, nickel, molybdenum, tungsten, and sulfur. Such systems are studied to understand the biocatalytic principles of gas conversion including N-2 fixation by nitrogenase and H-2 production by hydrogenase as well as CO2 and CO conversion by formate dehydrogenase, carbon monoxide dehydrogenase, and nitrogenase. In this review, we emphasize the importance of the cofactor/protein interface, discussing how second and outer coordination sphere effects determine, modulate, and optimize the catalytic activity of GPMs. These may comprise ionic interactions in the second coordination sphere that shape the electron density distribution across the cofactor, hydrogen bonding changes, and allosteric effects. In the outer coordination sphere, proton transfer and electron transfer are discussed, alongside the role of hydrophobic substrate channels and protein structural changes. Combining the information gained from structural biology, enzyme kinetics, and various spectroscopic techniques, we aim toward a comprehensive understanding of catalysis beyond the first coordination sphere.}, language = {en} } @article{FujiharaZhangJacksonetal.2022, author = {Fujihara, Kenji M. and Zhang, Bonnie Z. and Jackson, Thomas D. and Ogunkola, Moses and Nijagal, Brunda and Milne, Julia V. and Sallman, David A. and Ang, Ching-Seng and Nikolic, Iva and Kearney, Conor J. and Hogg, Simon J. and Cabalag, Carlos S. and Sutton, Vivien R. and Watt, Sally and Fujihara, Asuka T. and Trapani, Joseph A. and Simpson, Kaylene J. and Stojanovski, Diana and Leimk{\"u}hler, Silke and Haupt, Sue and Phillips, Wayne A. and Clemons, Nicholas J.}, title = {Eprenetapopt triggers ferroptosis, inhibits NFS1 cysteine desulfurase, and synergizes with serine and glycine dietary restriction}, series = {Science Advances}, volume = {8}, journal = {Science Advances}, number = {37}, publisher = {American Assoc. for the Advancement of Science}, address = {Washington}, issn = {2375-2548}, doi = {10.1126/sciadv.abm9427}, pages = {13}, year = {2022}, abstract = {The mechanism of action of eprenetapopt (APR-246, PRIMA-1MET) as an anticancer agent remains unresolved, al-though the clinical development of eprenetapopt focuses on its reported mechanism of action as a mutant-p53 reactivator. Using unbiased approaches, this study demonstrates that eprenetapopt depletes cellular antioxidant glutathione levels by increasing its turnover, triggering a nonapoptotic, iron-dependent form of cell death known as ferroptosis. Deficiency in genes responsible for supplying cancer cells with the substrates for de novo glutathione synthesis (SLC7A11, SHMT2, and MTHFD1L), as well as the enzymes required to synthesize glutathione (GCLC and GCLM), augments the activity of eprenetapopt. Eprenetapopt also inhibits iron-sulfur cluster biogenesis by limit-ing the cysteine desulfurase activity of NFS1, which potentiates ferroptosis and may restrict cellular proliferation. The combination of eprenetapopt with dietary serine and glycine restriction synergizes to inhibit esophageal xenograft tumor growth. These findings reframe the canonical view of eprenetapopt from a mutant-p53 reactivator to a ferroptosis inducer.}, 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{DuffusSchrapersSchuthetal.2020, author = {Duffus, Benjamin R. and Schrapers, Peer and Schuth, Nils and Mebs, Stefan and Dau, Holger and Leimk{\"u}hler, Silke and Haumann, Michael}, title = {Anion binding and oxidative modification at the molybdenum cofactor of formate dehydrogenase from Rhodobacter capsulatus studied by X-ray absorption spectroscopy}, series = {Inorganic chemistry}, volume = {59}, journal = {Inorganic chemistry}, number = {1}, publisher = {American Chemical Society}, address = {Washington, DC}, issn = {0020-1669}, doi = {10.1021/acs.inorgchem.9b01613}, pages = {214 -- 225}, year = {2020}, abstract = {Formate dehydrogenase (FDH) enzymes are versatile catalysts for CO2 conversion. The FDH from Rhodobacter capsulatus contains a molybdenum cofactor with the dithiolene functions of two pyranopterin guanine dinucleotide molecules, a conserved cysteine, and a sulfido group bound at Mo(VI). In this study, we focused on metal oxidation state and coordination changes in response to exposure to O-2, inhibitory anions, and redox agents using X-ray absorption spectroscopy (XAS) at the Mo K-edge. Differences in the oxidative modification of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor relative to samples prepared aerobically without inhibitor, such as variations in the relative numbers of sulfido (Mo=S) and oxo (Mo=O) bonds, were observed in the presence of azide (N-3(-)) or cyanate (OCN-). Azide provided best protection against O-2, resulting in a quantitatively sulfurated cofactor with a displaced cysteine ligand and optimized formate oxidation activity. Replacement of the cysteine ligand by a formate (HCO2-) ligand at the molybdenum in active enzyme is compatible with our XAS data. Cyanide (CN-) inactivated the enzyme by replacing the sulfido ligand at Mo(VI) with an oxo ligand. Evidence that the sulfido group may become protonated upon molybdenum reduction was obtained. Our results emphasize the role of coordination flexibility at the molybdenum center during inhibitory and catalytic processes of FDH enzymes.}, language = {en} }