title: Protein structure modeling indicates hexahistidine-tag interference with enzyme activity.
abstract: Unusual kinetic characteristics of tropinone reductase, an enzyme in the family of short chain dehydrogenases, prompted to investigate a possible impact of the hexahistidine affinity tag on catalytic properties. Comparison of enzymes from Solanum dulcamara, Solanaceae, tagged at the N-terminus or at the C-terminus revealed that the C-terminally tagged form was functionally impaired. Protein modeling indicated that the hexahistidine tag attached at the C-terminus but not at the N-terminus of the polypeptide can interfere with the active site by steric or electrostatic interactions. In consequence, protein modeling is suggested before enzyme expression with affinity tags to estimate possible interactions of affinity tags with the active center.
authors.affiliation: Faculty of Science I, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Hoher Weg 8, D-06120 Halle/Saale, Germany.
authors.forename: Anna-Carolin
authors.initials: AC
authors.lastname: Freydank
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Brandt
authors.forename: Birgit
authors.initials: B
authors.lastname: Dräger
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D016415:Sequence Alignment; D032322:Solanum / Q000201:enzymology*; D013379:Substrate Specificity; D014326:Tropanes / Q000737:chemistry / Q000378:metabolism
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D014326:Tropanes; C055033:calystegin; D006639:Histidine; D000429:Alcohol Oxidoreductases; C083753:tropinone reductase