@inproceedings{DuffusHartmannTeutloffetal.2019, author = {Duffus, Benjamin R. and Hartmann, Tobias and Teutloff, Christian and Leimk{\"u}hler, Silke}, title = {Refining catalytic insights toward the chemical mechanism of R. capsulatus formate dehydrogenase via EPR spectroscopy}, series = {Abstracts of papers : joint conference / The Chemical Institute of Cananda, CIC, American Chemical Society, ACS}, volume = {257}, booktitle = {Abstracts of papers : joint conference / The Chemical Institute of Cananda, CIC, American Chemical Society, ACS}, publisher = {American Chemical Society}, address = {Washington}, issn = {0065-7727}, pages = {1}, year = {2019}, language = {en} } @article{SendvonKozierowskiPanzneretal.2009, author = {Send, Sebastian and von Kozierowski, Marc and Panzner, Tobias and Gorfman, Semen and Nurdan, Kivanc and Walenta, Albert H. and Pietsch, Ullrich and Leitenberger, Wolfram and Hartmann, Robert and Str{\"u}der, Lothar}, title = {Energy-dispersive Laue diffraction by means of a frame-store pnCCD}, issn = {0021-8898}, doi = {10.1107/S0021889809039867}, year = {2009}, 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{SchrapersHartmannKositzkietal.2015, author = {Schrapers, Peer and Hartmann, Tobias and Kositzki, Ramona and Dau, Holger and Reschke, Stefan and Schulzke, Carola and Leimk{\"u}hler, Silke and Haumann, Michael}, title = {'Sulfido and Cysteine Ligation Changes at the Molybdenum Cofactor during Substrate Conversion by Formate Dehydrogenase (FDH) from Rhodobacter capsulatus}, series = {Inorganic chemistry}, volume = {54}, journal = {Inorganic chemistry}, number = {7}, publisher = {American Chemical Society}, address = {Washington}, issn = {0020-1669}, doi = {10.1021/ic502880y}, pages = {3260 -- 3271}, year = {2015}, abstract = {Formate dehydrogenase (FDH) enzymes are attractive catalysts for potential carbon dioxide conversion applications. The FDH from Rhodobacter capsulatus (RcFDH) binds a bis-molybdopterin-guanine-dinucleotide (bis-MGD) cofactor, facilitating reversible formate (HCOO-) to CO2 oxidation. We characterized the molecular structure of the active site of wildtype RcFDH and protein variants using X-ray absorption spectroscopy (XAS) at the Mo K-edge. This approach has revealed concomitant binding of a sulfido ligand (Mo=S) and a conserved cysteine residue (S(Cys386)) to Mo(VI) in the active oxidized molybdenum cofactor (Moco), retention of such a coordination motif at Mo(V) in a chemically reduced enzyme, and replacement of only the S(Cys386) ligand by an oxygen of formate upon Mo(IV) formation. The lack of a Mo=S bond in RcFDH expressed in the absence of FdsC implies specific metal sulfuration by this bis-MGD binding chaperone. This process still functioned in the Cys386Ser variant, showing no Mo-S(Cys386) ligand, but retaining a Mo=S bond. The C386S variant and the protein expressed without FdsC were inactive in formate oxidation, supporting that both Moligands are essential for catalysis. Low-pH inhibition of RcFDH was attributed to protonation at the conserved His387, supported by the enhanced activity of the His387Met variant at low pH, whereas inactive cofactor species showed sulfido-to-oxo group exchange at the Mo ion. Our results support that the sulfido and S(Cys386) ligands at Mo and a hydrogen-bonded network including His387 are crucial for positioning, deprotonation, and oxidation of formate during the reaction cycle of RcFDH.}, language = {en} } @article{HartmannTeraoGarattinietal.2012, author = {Hartmann, Tobias and Terao, Mineko and Garattini, Enrico and Teutloff, Christian and Alfaro, Joshua F. and Jones, Jeffrey P. and Leimk{\"u}hler, Silke}, title = {The impact of single nucleotide polymorphisms on human aldehyde oxidase}, series = {Drug metabolism and disposition : the biological fate of chemicals}, volume = {40}, journal = {Drug metabolism and disposition : the biological fate of chemicals}, number = {5}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, address = {Bethesda}, issn = {0090-9556}, doi = {10.1124/dmd.111.043828}, pages = {856 -- 864}, year = {2012}, abstract = {Aldehyde oxidase (AO) is a complex molybdo-flavoprotein that belongs to the xanthine oxidase family. AO is active as a homodimer, and each 150-kDa monomer binds two distinct [2Fe2S] clusters, FAD, and the molybdenum cofactor. AO has an important role in the metabolism of drugs based on its broad substrate specificity oxidizing aromatic aza-heterocycles, for example, N-1-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. Sequencing the 35 coding exons of the human AOX1 gene in a sample of 180 Italian individuals led to the identification of relatively frequent, synonymous, missense and nonsense single-nucleotide polymorphisms (SNPs). Human aldehyde oxidase (hAOX1) was purified after heterologous expression in Escherichia coli. The recombinant protein was obtained with a purity of 95\% and a yield of 50 mu g/l E. coli culture. Site-directed mutagenesis of the hAOX1 cDNA allowed the purification of protein variants bearing the amino acid changes R802C, R921H, N1135S, and H1297R, which correspond to some of the identified SNPs. The hAOX1 variants were purified and compared with the wild-type protein relative to activity, oligomerization state, and metal content. Our data show that the mutation of each amino acid residue has a variable impact on the ability of hAOX1 to metabolize selected substrates. Thus, the human population is characterized by the presence of functionally inactive hAOX1 allelic variants as well as variants encoding enzymes with different catalytic activities. Our results indicate that the presence of these allelic variants should be considered for the design of future drugs.}, language = {en} } @article{BoehmerHartmannLeimkuehler2014, author = {Boehmer, Nadine and Hartmann, Tobias and Leimk{\"u}hler, Silke}, title = {The chaperone FdsC for Rhodobacter capsulatus formate dehydrogenase binds the bis-molybdopterin guanine dinucleotide cofactor}, series = {FEBS letters : the journal for rapid publication of short reports in molecular biosciences}, volume = {588}, journal = {FEBS letters : the journal for rapid publication of short reports in molecular biosciences}, number = {4}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0014-5793}, doi = {10.1016/j.febslet.2013.12.033}, pages = {531 -- 537}, year = {2014}, abstract = {Molybdoenzymes are complex enzymes in which the molybdenum cofactor (Moco) is deeply buried in the enzyme. Most molybdoenzymes contain a specific chaperone for the insertion of Moco. For the formate dehydrogenase FdsGBA from Rhodobacter capsulatus the two chaperones FdsC and FdsD were identified to be essential for enzyme activity, but are not a subunit of the mature enzyme. Here, we purified and characterized the FdsC protein after heterologous expression in Escherichia coli. We were able to copurify FdsC with the bound Moco derivate bis-molybdopterin guanine dinucleotide. This cofactor successfully was used as a source to reconstitute the activity of molybdoenzymes. Structured summary of protein interactions: FdsC and FdsC bind by molecular sieving (View interaction) FdsD binds to RcMobA by surface plasmon resonance (View interaction) FdsC binds to RcMobA by surface plasmon resonance (View interaction) FdsC binds to FdsA by surface plasmon resonance (View interaction)}, language = {en} } @misc{HartmannSchwanholdLeimkuehler2015, author = {Hartmann, Tobias and Schwanhold, Nadine and Leimk{\"u}hler, Silke}, title = {Assembly and catalysis of molybdenum or tungsten-containing formate dehydrogenases from bacteria}, series = {Biochimica et biophysica acta : Proteins and proteomics}, volume = {1854}, journal = {Biochimica et biophysica acta : Proteins and proteomics}, number = {9}, publisher = {Elsevier}, address = {Amsterdam}, issn = {1570-9639}, doi = {10.1016/j.bbapap.2014.12.006}, pages = {1090 -- 1100}, year = {2015}, abstract = {The global carbon cycle depends on the biological transformations of C-1 compounds, which include the reductive incorporation of CO2 into organic molecules (e.g. in photosynthesis and other autotrophic pathways), in addition to the production of CO2 from formate, a reaction that is catalyzed by formate dehydrogenases (FDHs). FDHs catalyze, in general, the oxidation of formate to CO2 and H+. However, selected enzymes were identified to act as CO2 reductases, which are able to reduce CO2 to formate under physiological conditions. This reaction is of interest for the generation of formate as a convenient storage form of H-2 for future applications. Cofactor-containing FDHs are found in anaerobic bacteria and archaea, in addition to facultative anaerobic or aerobic bacteria. These enzymes are highly diverse and employ different cofactors such as the molybdenum cofactor (Moco), FeS clusters and flavins, or cytochromes. Some enzymes include tungsten (W) in place of molybdenum (Mo) at the active site. For catalytic activity, a selenocysteine (SeCys) or cysteine (Cys) ligand at the Mo atom in the active site is essential for the reaction. This review will focus on the characterization of Mo- and W-containing FDHs from bacteria, their active site structure, subunit compositions and its proposed catalytic mechanism. We will give an overview on the different mechanisms of substrate conversion available so far, in addition to providing an outlook on bio-applications of FDHs. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications. (C) 2014 Elsevier B.V. All rights reserved.}, language = {en} } @inproceedings{LeimkuehlerHartmannGarattinietal.2011, author = {Leimk{\"u}hler, Silke and Hartmann, Tobias and Garattini, Enrico and Jones, Jeffrey P.}, title = {Structure-function studies on human aldehyde oxidase and the impact of polymorphisms on enzyme activity}, series = {Drug metabolism reviews : biotransformation and disposition of xenobiotics ; official journal of the International Society for the Study of Xenobiotics}, volume = {43}, booktitle = {Drug metabolism reviews : biotransformation and disposition of xenobiotics ; official journal of the International Society for the Study of Xenobiotics}, number = {6}, publisher = {Taylor \& Francis Group}, address = {London}, issn = {0360-2532}, pages = {13 -- 13}, year = {2011}, language = {en} } @article{HartmannSchrapersUteschetal.2016, author = {Hartmann, Tobias and Schrapers, Peer and Utesch, Tillmann and Nimtz, Manfred and Rippers, Yvonne and Dau, Holger and Mroginski, Maria Andrea and Haumann, Michael and Leimk{\"u}hler, Silke}, title = {The Molybdenum Active Site of Formate Dehydrogenase Is Capable of Catalyzing C-H Bond Cleavage and Oxygen Atom Transfer Reactions}, series = {Biochemistry}, volume = {55}, journal = {Biochemistry}, publisher = {American Chemical Society}, address = {Washington}, issn = {0006-2960}, doi = {10.1021/acs.biochem.6b00002}, pages = {2381 -- 2389}, year = {2016}, abstract = {Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pK(a) of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase.}, language = {en} } @article{FotiHartmannCoelhoetal.2016, author = {Foti, Alessandro and Hartmann, Tobias and Coelho, Catarina and Santos-Silva, Teresa and Romao, Maria Joao and Leimk{\"u}hler, Silke}, title = {Optimization of the Expression of Human Aldehyde Oxidase for Investigations of Single-Nucleotide Polymorphisms}, series = {Drug metabolism and disposition : the biological fate of chemicals}, volume = {44}, 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.115.068395}, pages = {1277 -- 1285}, year = {2016}, abstract = {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.}, language = {en} } @article{CoelhoFotiHartmannetal.2015, author = {Coelho, Catarina and Foti, Alessandro and Hartmann, Tobias and Santos-Silva, Teresa and Leimk{\"u}hler, Silke and Romao, Maria Joao}, title = {Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase}, series = {Nature chemical biology}, volume = {11}, journal = {Nature chemical biology}, number = {10}, publisher = {Nature Publ. Group}, address = {New York}, issn = {1552-4450}, doi = {10.1038/NCHEMBIO.1895}, pages = {779 -- +}, year = {2015}, abstract = {Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-angstrom resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-angstrom resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.}, language = {en} } @article{CazellesLalaouiHartmannetal.2016, author = {Cazelles, R. and Lalaoui, N. and Hartmann, Tobias and Leimk{\"u}hler, Silke and Wollenberger, Ursula and Antonietti, Markus and Cosnier, S.}, title = {Ready to use bioinformatics analysis as a tool to predict immobilisation strategies for protein direct electron transfer (DET)}, series = {Polymer : the international journal for the science and technology of polymers}, volume = {85}, journal = {Polymer : the international journal for the science and technology of polymers}, publisher = {Elsevier}, address = {Oxford}, issn = {0956-5663}, doi = {10.1016/j.bios.2016.04.078}, pages = {90 -- 95}, year = {2016}, language = {en} }