Covalent flavinylation of 6-hydroxy-D-nicotine oxidase analyzed by partial deletions of the gene.
The expression of the enzymatically active 6-hydroxy-D-nicotine oxidase (6-HDNO) from Arthrobacter oxidans requires the covalent attachment of FAD to the polypeptide chain. How this modification takes place and at what time during the synthesis of the polypeptide is not known. We investigated the possibility of cotranslational flavinylation by generating various deletions of the 6-HDNO gene carried on appropriate plasmid vectors. The polypeptides expressed from these plasmids were analyzed for their ability to incorporate [14C]FAD covalently in an Escherichia coli-derived coupled transcription/translation system. The data show that removal of approximately 40% from the carboxy-terminal part of the 6-HDNO polypeptide did not inhibit the covalent flavinylation of the truncated protein. A fusion protein, consisting of the truncated 6-HDNO polypeptide and the beta-lactamase of pBR322, was also covalently flavinylated. The amino acid sequence surrounding the histidine residue, assumed to bind FAD, was shown to be situated approximately 70 amino acid residues from the amino-terminal end of the 6-HDNO polypeptide. Removal of the first 30 amino acids did not abolish covalent flavinylation. Flavinylation could no longer be detected, however, if a short amino acid sequence, consisting of seven residues, replaced the amino acid sequence upstream of the histidine. These findings prove, in our opinion, that cotranslational flavinylation takes place in the synthesis of 6-HDNO.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001173:Arthrobacter / Q000201:enzymology; D001483:Base Sequence; D004262:DNA Restriction Enzymes; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D007120:Immunochemistry; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D014176:Protein Biosynthesis; D014158:Transcription, Genetic
D000596:Amino Acids; D005182:Flavin-Adenine Dinucleotide; D010088:Oxidoreductases; C021413:6-hydroxy-D-nicotine oxidase; D004262:DNA Restriction Enzymes