Phosphoenolpyruvate-dependent flavinylation of 6-hydroxy-D-nicotine oxidase.
The reaction leading to the flavinylation of apo-6-hydroxy-D-nicotine oxidase was investigated in cell-free extracts of Eschericia coli carrying the 6-hydroxy-D-nicotine oxidase (6-HDNO) gene on the expression plasmid pDB222. It was demonstrated that the reaction required phosphoenolpyruvate (P-pyruvate) in addition to FAD. When [32P]P-pyruvate or [14C]P-pyruvate were used in the reaction with apo-6-HDNO, no phosphorylated or pyruvylated apo-protein could be detected, however. In order to drive the reaction to completion, FAD and P-pyruvate had to be present simultaneously in the reaction mixture. When apo-6-HDNO, highly purified by affinity chromatography, was used in the reaction with P-pyruvate and FAD, no additional protein fraction was required. A possible reaction scheme for the formation of holoenzyme from 6-HDNO is discussed.
Nagursky
H
H
Biochemisches Institut, Universität Freiburg, Federal Republic of Germany.
Bichler
V
V
Brandsch
R
R
D000244:Adenosine Diphosphate / Q000494:pharmacology; D001051:Apoenzymes / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004734:Energy Metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D007700:Kinetics; D009861:Onium Compounds / Q000494:pharmacology; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010728:Phosphoenolpyruvate / Q000494:pharmacology*; D010766:Phosphorylation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011770:Pyruvate Kinase / Q000494:pharmacology; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid
D001051:Apoenzymes; D009861:Onium Compounds; D011773:Pyruvates; D005182:Flavin-Adenine Dinucleotide; D000244:Adenosine Diphosphate; C007517:diphenyleneiodonium; D010728:Phosphoenolpyruvate; D019289:Pyruvic Acid; D010088:Oxidoreductases; C021413:6-hydroxy-D-nicotine oxidase; D011770:Pyruvate Kinase