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.