title: Covalent flavinylation of 6-hydroxy-D-nicotine oxidase involves an energy-requiring process.
abstract: E. coli cells harbouring the recombinant plasmid pDB222 with the 6-HDNO gene under the control of the tac-promotor were induced with IPTG to synthesize a high amount of 6-HDNO protein. Part of this protein was present as 6-HDNO apoenzyme. The proportion of 6-HDNO apoenzyme formed could be increased when the induction of 6-HDNO synthesis by IPTG was performed in the presence of the inhibitor diphenyleneiodonium. The 6-HDNO apoenzyme thus formed could be transformed into enzymatically active holoenzyme in the presence of FAD by a process requiring an energy-generating system consisting of ATP, phosphoenolpyruvate and pyruvate kinase. This finding suggests that an enzymatic step(s) is (are) involved in the covalent flavinylation of 6-HDNO.
authors.affiliation: Biochemisches Institut der Universität, Freiburg, FRG.
authors.forename: R
authors.initials: R
authors.lastname: Brandsch
authors.forename: V
authors.initials: V
authors.lastname: Bichler
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001051:Apoenzymes / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D010728:Phosphoenolpyruvate / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011770:Pyruvate Kinase / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D001051:Apoenzymes; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D005182:Flavin-Adenine Dinucleotide; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; C021413:6-hydroxy-D-nicotine oxidase; D011770:Pyruvate Kinase