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 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.

ETFDH Mutations and Flavin Adenine Dinucleotide Homeostasis Disturbance Are Essential for Developing Riboflavin‐Responsive Multiple Acyl–Coenzyme A Dehydrogenation Deficiency

“…This phenomenon indicated that ETF:QO protein synthesis was not affected and should not be the cause of ETF:QO protein reduction. This, in vivo, indirectly confirmed the hypothesis of previous research, that the decrease in ETF:QO protein might possibly be due to a faster degradation of mutant ETF:QO proteins. The defective FAD binding to ETF:QO in RR‐MADD patients should be explained on the basis of a subtly altered protein structure that partially, but not entirely, hampers the affinity of FAD to flavoproteins.…”

Emerging concepts in the flavinylation of succinate dehydrogenase

“…The steady-state levels of the mature SDH complex is lower when the covalent bond is disrupted either by mutation of the His 90 residue or by deletion of Sdh5. This is probably not caused by an impaired import or processing of Sdh1 [8]; rather, the possibilities include an increase in protease sensitivity [33] or an inherently less stable holo-complex in vivo that is prone to disassembly [32]. …”

What’s in a covalent bond?

“…The second step in the bacterial degradation of nicotine is catalyzed by 6‐HDNO, which was one of the first discovered covalent flavoproteins and has been extensively studied [41–43]. By incubating the apo form of 6‐HDNO with [ 14 C] FAD, it was shown that in vitro covalent flavinylation is a self‐catalytic process [44].…”