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.