FAD is covalently attached to peptidyl-tRNA during cell-free synthesis of 6-hydroxy-D-nicotine oxidase.
The process, by which FAD is attached covalently to the 6-hydroxy-D-nicotine oxidase apoprotein in D-nicotine-induced cells of Arthrobacter oxidans was studied in vitro. [3H]Adenine-labelled FAD prepared biosynthetically in Clostridium kluyveri was incorporated into the 6-hydroxy-D-nicotine oxidase molecule during cell-free translation. FAD rather than FMN or riboflavin was thus shown to be the flavin derivative transferred to the polypeptide chain. After short-term protein synthesis on ribosomes from induced A. oxidans cells in the presence of an Escherichia coli MRE 600 supernatant fraction and [adenine-2-3H]FAD, THE PEPTIDYL-TRNA fraction was separated from completed polypeptides. Labelled FAD was found to be covalently attached to the tRNA-bound polypeptides. Cleavage of the tRNA-peptide bond released labelled polypeptides the largest of which migrated as authentic 6-hydroxy-D-nicotine oxidase during dodecylsulfate/polyacrylamide gel electrophoresis. These results strongly suggest that FAD is incorporated into the nascent polypeptide chains of 6-hydroxy-D-nicotine oxidase during ribosomal translation.
D000225:Adenine / Q000378:metabolism; D001173:Arthrobacter / Q000378:metabolism; D002474:Cell-Free System; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D008970:Molecular Weight; D009538:Nicotine / Q000031:analogs & derivatives; D010088:Oxidoreductases / Q000096:biosynthesis*; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism*; D011132:Polyribosomes / Q000378:metabolism; D012343:RNA, Transfer / Q000302:isolation & purification / Q000378:metabolism*; D012256:Riboflavin / Q000378:metabolism
D010455:Peptides; D005182:Flavin-Adenine Dinucleotide; D009538:Nicotine; D012343:RNA, Transfer; D010088:Oxidoreductases; C021413:6-hydroxy-D-nicotine oxidase; D000225:Adenine; D012256:Riboflavin