FAD Is Covalently Attached to Peptidyl‐tRNA during Cell‐free Synthesis of 6‐Hydroxy‐d‐nicotine Oxidase

Abstract

The process, by which FAD is attached covalently to the 6-hydroxy-~-nicotine oxidase apoprotein in D-nicotine-induced cells of Arthrobucter oxiduns was studied in vitro.[3H]Adenine-labelled FAD prepared biosynthetically in Clostridium kluyveri was incorporated into the 6-hydroxy-~-nicotine oxidase molecule during cell-free translation. FAD rather than FMN or riboflavin was thus shown to be the flavin derivative transferred to the polypeptide chain. After short-term protein synthesis on ribosomes from induced A . oxiduns cells in the presence of an Escherichiu coli MRE 600 supernatant fraction and [~denine-2-~H]FAD, the peptidyl-tRNA fraction was separated from completed polypeptides. Labelled FAD was found to be covalently attached to the tRNA-bound polypeptides. Cleavage of the tRNA-peptide bond released labelled polypeptides the largest of which migrated as authentic 6-hydroxy-~-nicotine oxidase during dodecylsulfate/polyacrylamide gel electrophoresis. These results strongly suggest that FAD is incorporated into the nascent polypeptide chains of 6-hydroxy-~-nicotine oxidase during ribosomal translation.In the last few years an increasing number of flavoproteins have been reported to contain covalently bound coenzymes (for review see [l]). However, the flavin-binding process during the biosynthesis of these proteins is not yet understood. In Arthrobucter oxidans, 6-hydroxy-~-nicotine oxidase is induced in the presence of D-nicotine [2]; the coenzyme, FAD, was shown [3] to be covalently attached through its 8cc-methyl group to N-3 of a histidyl residue of the apoprotein. This enzyme is synthesized during the early stationary phase of growth [4] where only a limited number of proteins is assumed to be produced. Studies with a riboflavin-requiring mutant on the flavin-binding process in vivo indicated that the function of intact ribosomes is necessary for the formation of enzymatically active holoenzyme, even during flavin-limited 6-hydroxy-~-nicotine oxidase synthesis With isolated polysomes and the translation system of the ribonuclease-free strain (MRE 600) of Esche-151.

Succinate Dhydrogenase Mutants of Bacillus Suybtilis Lacking Covalently Bound Flavin in the Flavoprotien Subunit

“…The flavin of A . oxiduns 6-hydroxy-~-nicotine oxidase has been shown to be attached in the form of FAD to the apoprotein cotranslationally [28). Binding of the FAD molecule seems to occur when about 40% or more of the nascent polypeptide has been translated.…”

Stalled flavodoxin binds its cofactor while fully exposed outside the ribosome

“…Heme has been suggested to bind to nascent globin [7], which may imply that the protein folds co-translationally. Flavin adenine dinucleotide (FAD) was proposed to covalently attach to nascent 6-hydroxy-D-nicotine oxidase [22], but its co-translational flavinylation is now judged unlikely [23].…”