Protein structure modeling indicates hexahistidine-tag interference with enzyme activity.
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.
Freydank
Anna-Carolin
AC
Faculty of Science I, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Hoher Weg 8, D-06120 Halle/Saale, Germany.
Brandt
Wolfgang
W
Dräger
Birgit
B
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
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D014326:Tropanes; C055033:calystegin; D006639:Histidine; D000429:Alcohol Oxidoreductases; C083753:tropinone reductase