Cell-Free Synthesis of a Flavoprotein Containing the 8alpha-(N3- Histidyl)-Riboflavin Linkage

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Abstract
6-Hydroxy-D-nicotine oxidase is an inducible flavoprotein of Arthrobacter oxydans in which 1 mol of FAD is bound covalently to the polypeptide chain. During cell-free translation of polysomes from nicotine-induced A. oxydans cells in the presence of an Escherichia coli (MRE 600) supernatant fraction, labeled FAD, leucine and histidine were incorporated into 6-hydroxy-D-nicotine oxidase in the same ratio found in the enzyme isolated from whole cells. This indicates that 1 mol FAD is covalently attached per mol of 6-hydroxy-D-nicotine oxidase synthesized in vitro. In the native enzyme the coenzyme molecule is bound via its 8.alpha.-methyl group to the N-3 atom of a histidyl residue. From the translation products an aminoacyl-riboflavine was isolated and identified with synthetic 8.alpha.-(N3-histidyl)-riboflavine.