GroE dependence of refolding and holoenzyme formation of 6-hydroxy-D-nicotine oxidase.

In Escherichia coli cells expressing 6-hydroxy-D-nicotine oxidase (6-HDNO), a flavoprotein with covalently bound FAD, approximately 40% of the polypeptide is in its apoform. We investigated whether in vivo holoenzyme formation was influenced by the association of the apoenzyme with cellular chaperones. Immunoprecipitation of apoenzyme-containing cell extract with protein-A-Sepharose-bound 6-HDNO- or GroEL-specific antibodies failed to reveal the formation of complexes between these proteins. The limiting factor in holoenzyme formation in vivo appeared to be the intracellular supply of phosphorylated tricarbon compounds (e.g. glycerol-3-P) acting as allosteric effectors in the flavinylation reaction. When holoenzyme formation from purified apo6-HDNO was investigated in vitro, addition of GroEL and GroES to the reaction assays increased the yield of holoenzyme formation. The observed increase in apoenzyme to holoenzyme transition was ATP independent, and the effect of GroE could be simulated by high concentrations of glycerol (40%). Apparently, a nonspecific protein-protein interaction between the GroE proteins and the apo6-HDNO favored holoenzyme formation. The refolding of guanidinium hydrochloride-unfolded holoenzyme, however, was catalyzed by GroEL and GroES in an ATP-dependent reaction. Recovery of the native, enzymatically active, conformation ranged from 30 to 40%. When apo6-HDNO was denatured and refolded, the same dependence on GroE and ATP was observed in the recovery of a conformation able to incorporate FAD and to holoenzyme. [14C] FAD in the refolding assay yielded radioactively labeled 6-HDNO demonstrating the autocatalytical covalent incorporation of FAD into the polypeptide during the folding process.