@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{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}
}
@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}
}