Cell-free synthesis of a flavoprotein containing the 8 alpha-(N3-histidyl)-riboflavin linkage

Abstract

6-Hydroxy-D-nicotine oxidase is an inducible flavorprotein of Arthrobacter oxidans in which one mole of FAD is bound covalently to the polypeptide chain. During cell-free translation of polysomes from nicotine-induced A. oxidans cells in the presence of an Escherichia coli (MRE 600) supernatant fraction, labelled 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 one mole 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-riboflavin was isolated and its identity with synthetic 8 alpha-(N3-histidyl)-riboflavin was shown.