Regulation of flavoprotein synthesis studied in vivo in a riboflavin-requiring mutant of Arthrobacter oxidans

Abstract

The biosynthesis of two flavoproteins, 6-hydroxy-D-nicotine oxidase with covalently bound FAD and 6-hydroxy-L-nicotine oxidase containing non-covalently bound FAD, was studied in wild-type cells and in a riboflavin-requiring mutant of Arthrobacter oxidans. In the mutant cells, the rate of synthesis and the maximal activity level of both enzymes after induction by nicotine depended on the amount of added riboflavin. The low rate of synthesis in the presence of 2 micron riboflavin could be enhanced during the induction phase by further addition of riboflavin (33 micron). Inhibitors of translation (chloramphenicol or streptomycin) completely blocked the synthesis of both flavoproteins. Inhibitors of transcription (rifamycin S or actinomycin D) stopped the synthesis of both enantiozymes in wild-type cells and in the mutant grown in the presence of a saturating supply of riboflavin (15 micron). Under conditions of restricted flavoprotein synthesis (2 micron riboflavin in the medium), however, the mutant cells continued to synthesize the enzyme for 2--3 h after the addition of the transcription inhibitors. It appears, that in these cells a rather stable m-RNA accumulated during riboflavin-limited flavoprotein synthesis. The dependence of the effect of transcription inhibitors on the extracellular supply of riboflavin suggests that the regulation of the synthesis of both flavoproteins occurs not only by control of gene expression (induction by nicotine), but also at the level of translation through the availability of FAD.

Structural analysis and molybdenum‐dependent expression of the pAO1‐encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans

“…Bacterial strains, culture media and growth conditions A. nicotinovorans was grown with vigorous shaking at 30 C in citrate medium supplemented with 0.05% yeast extract and trace elements (Hamm and Decker, 1978). Induction of the degradative enzymes of A. nicotinovorans by nicotine was performed in media of defined mineral salt and vitamin composition (Freudenberg et ai, 1988).…”

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

“…The detection of [3H]FAD bound covalently to peptidyl-tRNA is taken as evidence that the flavin-binding process during 6-hydroxy-D-nicotine oxidase biosynthesis is a cotranslational event. The requirement of an intact protein synthesis for coenzyme attachment was indicated already by previous experiments in vivo [5] using a riboflavin-requiring mutant and inhibitors of translation. It is, to our knowledge, the first instance of a covalent cofactor attachment to a nascent polypeptide chain.…”

“…A riboflavin-requiring mutant of Arthrobacter oxidans was grown in the presence of 15 pM riboflavin and 0.1 % m-nicotine [5], until the maximal rate of 6-hydroxy-~-nicotine oxidase synthesis was reached. After rapid cooling to 4 "C, the cells were centrifuged and washed with ice-cold standard buffer (10 mM Tris .…”

Regulation of Gene Expression by Cofactors Derived from B Vitamins.

“…Thus, expression of the put operon is regulated by changes in the redox state of the PutA protein. A riboflavin-dependentJ expression of genes not involved in the biosynthesis of the cofactor was described for the nicotine-degrading enzymes of Arthrobacter oxidans (27). Expression in this soil bacterium of the nicotine-degrading flavoenzymes is inducible by nicotine (28).…”