Binding of FAD to 6-hydroxy-D-nicotine oxidase apoenzyme prevents degradation of the holoenzyme

Abstract

Expression of the 6-hydroxy-D-nicotine oxidase (6-HDNO) gene from Arthrobacter oxidans cloned into Escherichia coli showed a marked temperature-dependence. Transformed E. coli cells grown at 30°C exhibited a several-fold higher 6-HDNO activity than did cells grown at 37°C. This effect did not depend on the promoter used for expression of the cloned gene in E. coli, nor was it an effect of 6-HDNO mRNA instability at 37°C. Studies performed in vivo and in vitro revealed that an increased susceptibility of apo 6-HDNO to proteolytic attack at 37°C was responsible for the observed phenomenon. Extracts from cells grown at 37°C showed on Western blots a decrease in immunologically detectable 6-HDNO polypeptide when compared with extracts from cells grown at 30°C. The 6-HDNO polypeptide is covalently modified by attachment of the cofactor FAD to a histidine residue. It could be shown that covalent flavinylation of the apoenzyme in vitro, i.e. formation of holoenzyme, by incubation of cell extracts with FAD and phosphoenolpyruvate protected the 6-HDNO polypeptide from degradation at 37°C. Of a variety of proteinase inhibitors tested only the cysteine-proteinase inhibitor L-3-trans-carboxyoxiran-3-carbonyl-L-leucylagmatine (E64) prevented degradation, by up to 70%, of the apoenzyme.