Cysteine to serine replacements in 6-hydroxy-D-nicotine oxidase. Consequences for enzyme activity, cofactor incorporation, and formation of high molecular weight protein complexes with molecular chaperones (GroEL).

6-Hydroxy-D-nicotine oxidase, an enzyme with FAD covalently attached to the protein, contains 6 cysteine residues in positions 45 (Cys1), 59 (Cys2), 136 (Cys3), 173 (Cys4), 260 (Cys5) and 433 (Cys6). Cys2, 3, 5, and 6 were replaced with serine by site-directed mutagenesis. The effects of these exchanges on enzyme activity, the autocatalytic incorporation of the cofactor, and the interaction of the mutant proteins with molecular chaperones were analyzed. The flavinylation of 6-hydroxy-D-nicotine oxidase is dependent on the presence of allosteric effectors, e.g. glycerol 3-phosphate or other phosphorylated tricarbon compounds. Replacement of Cys2 or Cys5 abolished this dependence. Covalent incorporation of FAD was reduced to an undetectable level in the Cys3 and Cys5 mutants. Replacement of Cys6 by Ser had no significant effect on enzyme activity and cofactor attachment. Deletion of two amino acids, Phe and Arg, situated 12 and 11 amino acid residues, respectively, from the carboxyl terminus of the protein, resulted in an inactive enzyme with no covalently bound FAD. This result indicates that almost the entire protein chain has to be synthesized before the cofactor can be incorporated, making a cotranslational flavinylation step rather unlikely. The distribution of the 6-hydroxy-D-nicotine oxidase polypeptide between the high molecular weight complexes and the free soluble form was analyzed by gel filtration on Sephacryl S-200. The wild-type holoenzyme as well as the wild-type apoenzyme were recovered in the eluent fraction of the column while the mutant proteins were retained in high molecular weight complexes, predominantly in those associated with GroEL, as revealed by immunoprecipitation. The extent of complex formation with this molecular chaperone depended on the position of the mutated Cys residue within the protein. Complex formation was highest with protein from the mutants Cys2 and Cys3, less with the Cys5, and absent with the Cys6 mutant protein. Thus, alterations in the amino-terminal part of the 6-hydroxy-D-nicotine oxidase appear more important for the interaction with molecular chaperones than alterations situated in the carboxyl-terminal part of the protein.