In vivo and in vitro expression of the 6-hydroxy-D-nicotine oxidase gene of Arthrobacter oxidans, cloned into Escherichia coli, as an enzymatically active, covalently flavinylated polypeptide.
The 6-hydroxy-D-nicotine oxidase gene of Arthrobacter oxidans was cloned into E.coli with the aid of the expression vector pKK223-3. This enzyme, as well as the E.coli enzymes succinate dehydrogenase and fumarate reductase, bears the cofactor FAD covalently attached to the polypeptide through a His-N3-8 alpha-linkage. The amino acid sequence surrounding the histidine residue involved in FAD binding in 6-hydroxy-D-nicotine oxidase and the two E.coli enzymes, however, show no homology. Nevertheless, 6-hydroxy-D-nicotine oxidase is expressed in E.coli in vivo and in an E.coli-derived coupled transcription-translation system as a covalently flavinylated, enzymatically active polypeptide.
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005420:Flavoproteins / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D007700:Kinetics; D008970:Molecular Weight; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D012256:Riboflavin / Q000378:metabolism*
D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide; D010088:Oxidoreductases; C021413:6-hydroxy-D-nicotine oxidase; D012256:Riboflavin