Binding of FAD to 6-hydroxy-D-nicotine oxidase apoenzyme prevents degradation of the holoenzyme.
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 degrees C exhibited a several-fold higher 6-HDNO activity than did cells grown at 37 degrees 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 degrees C. Studies performed in vivo and in vitro revealed that an increased susceptibility of apo-6-HDNO to proteolytic attack at 37 degrees C was responsible for the observed phenomenon. Extracts from cells grown at 37 degrees C showed on Western blots a decrease in immunologically detectable 6-HDNO polypeptide when compared with extracts from cells grown at 30 degrees 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 degrees C. Of a variety of proteinase inhibitors tested only the cysteine-proteinase inhibitor L-3-trans-carboxyoxiran-2-carbonyl-L-leucylagmatine (E64) prevented degradation, by up to 70%, of the apoenzyme.
Brandsch
R
R
Biochemisches Institut, Universität Freiburg, Federal Republic of Germany.
Bichler
V
V
Krauss
B
B
D001051:Apoenzymes / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D010728:Phosphoenolpyruvate / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism; D013696:Temperature
D001051:Apoenzymes; D001059:Apoproteins; D011480:Protease Inhibitors; D005182:Flavin-Adenine Dinucleotide; D010728:Phosphoenolpyruvate; D010088:Oxidoreductases; C021413:6-hydroxy-D-nicotine oxidase