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

Abstract

6‐Hydroxy‐d‐nicotine oxidase is an inducible flavoprotein of Arthrobacter oxiduns in which one mole of FAD is bound covalently to the polypeptide chain. During cell‐free translation of poly‐ somes 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 8α‐methyl group to the N3 atom of a histidyl residue. From the translation products an aminoacyl‐riboflavin was isolated and its identity with synthetic 8α‐(N3‐histidyl)‐riboflavin was shown.

Bacteria belonging to the extremely versatile genus Arthrobacter as novel source of natural pigments with extended hue range

“…isolated from soil e.g. Arthrobacter globiformis excreted a yellow pigment during exponential growth which was identified as riboflavin, C 17 H 20 N 4 O 6, 7,8-dimethyl-10-(1′-D-ribityl) isoalloxazine (Hamm & Decker, 1980;Veldkamp, Venema, Harder, & Konings, 1966;Yamane, Nakamra, Okamoto, Oshima, & Kato, 1995) . Yamane et al (1995) investigated the riboflavin production by an Arthrobacter sp.…”

Covalent flavinylation of 6‐hydroxy‐D‐nicotine oxidase involves an energy‐requiring process

“…We were able to conCorrespondence address: R. Brandsch, Biochemisches Institut der Universit~it, Hermann-Herder-Str. 7, D-7800 Freiburg, FRG Abbreviations: 6-HDNO, 6-hydroxy-D-nicotine oxidase; IPTG, isopropyl-~'-D-thiogalactopyranoside; DPI, diphenyleneiodonium; HPLC, high-performance liquid chromatography; CCCP, carbonylcyanide chlorophenylhydrazone; PEP, phosphoenolpyruvate; PK, pyruvate kinase; Cm, chloramphenicol firm previous reports [4] showing that covalent flavinylation of 6-HDNO can take place cotranslationally [5]. Here we show that under certain conditions in a genetically engineered, 6-HDNO overproducing E. coli strain 6-HDNO apoenzyme was synthesized.…”