Studies in vitro on the flavinylation of 6-hydroxy-D-nicotine oxidase.
The gene of 6-hydroxy-D-nicotine oxidase (6-HDNO), a flavoenzyme from Arthrobacter oxidans with covalently bound FAD, was expressed with the aid of an expression vector in a cell-free coupled transcription-translation system derived from Escherichia coli MZ9. Ultraviolet irradiation of the E. coli extract did not affect synthesis of the 6-HDNO polypeptide nor total protein synthesis but enzymatic 6-HDNO activity could not be detected. Addition of FAD to the irradiated cell extract restored the capability of the transcription-translation assays to synthesize enzymatically active 6-HDNO. However, enzymatic activity could not be restored on addition of FAD plus cell-free extract to the ultraviolet-inactivated assays after completion of apo-6-HDNO synthesis (60 min) nor to immunoprecipitates thereof. Under similar conditions, addition of [14C]FAD did not increase the protein-bound radioactivity. These results indicate that under conditions of limited FAD supply in the in vitro system a flavinless apo-6-HDNO-polypeptide was synthesized. It was, however, not possible to bind the cofactor to the completed polypeptide chain. These findings argue for a cotranslational cofactor binding.
D001173:Arthrobacter / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005822:Genetic Vectors; D010088:Oxidoreductases / Q000096:biosynthesis*; D010957:Plasmids; D014176:Protein Biosynthesis / Q000528:radiation effects; D014158:Transcription, Genetic / Q000528:radiation effects; D014466:Ultraviolet Rays
D005182:Flavin-Adenine Dinucleotide; D010088:Oxidoreductases; C021413:6-hydroxy-D-nicotine oxidase