Zum Mechanismus der Induktion nicotinabbauender Enzyme in Artbrobacter oxydans

Abstract

Die Induktion der Enzyme Nicotin-oxidase, L-6-Hydroxynicotin-oxidase, D-6-Hydroxynicotinoxidase und Keton-oxidase in Arthrobacter oxydans durch die optischen Antipoden und das Racemat des Nicotins und 6-Hydroxynicotins wurde in Gegenwart einer leicht metabolisierbaren C- und N-Quelle untersucht. Die Induktion der beiden Schlüsselenzyme des Nicotinabbaus, L-6-Hydroxynicotin-oxidase und D-6-Hydroxynicotin-oxidase, erfolgte nicht absolut stereospezifisch durch die optischen Antipoden des Nicotins oder 6-Hydroxynicotins, jedoch stimulierten die D-Antipoden stark die Induktion von D-6-Hydroxynicotin-oxidase. Eine Nicotin- oder eine 6-Hydroxynicotin-racemase ließ sich nicht nachweisen. D,L-6-Amino-nicotin erwies sich als ein schwacher Inducer für die nicotinabbauenden Enzyme in Arthrobacter oxydans.Während sich aus Wachstumsversuchen eine Koordination für alle vier Enzyme ergab, führte die kinetische Analyse der Induktion zu einer Aufteilung in die Gruppen Nicotin-oxidase, L-6-Hydroxynicotin-oxidase, Keton-oxidase und D-6-Hydroxynicotin-oxidase. Die Induktion wurde durch D-Chloramphenicol und durch Actinomycin C gehemmt.

Sequence of the 165-Kilobase Catabolic Plasmid pAO1 fromArthrobacter nicotinovoransand Identification of a pAO1-Dependent Nicotine Uptake System

“…An A. nicotinovorans strain not carrying pAO1 was transformed with pKGT452␤, and Cm r colonies appeared on selection plates with frequencies of 10 to 100 colonies per g of pKGT452␤ DNA. Bacteria not carrying pAO1 are unable to degrade nicotine and do not turn dark, in contrast to pAO1-carrying bacteria, when grown on citrate plates with nicotine (14,21 14 C]nicotine and the time-dependent nicotine uptake was measured, only those bacteria harboring the pAO1 plasmid showed L-nicotine accumulation (Fig. 2).…”

“…Since the tobacco plant produces only L-nicotine, the presence of a 6-hydroxy-D-nicotine-specific oxidase may be surprising (21). The need for an enzyme with D-nicotine specificity could be warranted if a D-nicotine derivative would arise as a natural product of nicotine metabolism.…”

“…A. nicotinovorans harboring the catabolic plasmid pAO1 (8) and a cured derivative not harboring pAO1 were grown on citrate medium supplemented with mineral salts and vitamins (15,21). pAO1 DNA was extracted as described previously (32).…”

Cell‐free synthesis of 6‐hydroxy‐D‐nicotine oxidase containing covalently bound FAD

“…Polysomes were isolated both from induced and uninduced A. oxidans cells. The activity of 6-HDNO begins to appear after A. oxidans has reached the stationary phase of growth [2] , and therefore it would be expected that only a limited number of proteins are synthesized during that period.…”

“…6-Hydroxy-D-nicotine oxidase (6-HDNO) (EC 1.5.3.6) is an inducible flavoprotein involved in the degradation of D-nicotine by Arthrobucter oxidans [ 1,2] . The enzyme contains FAD bound covalently via a linkage between the &-methyl group of the flavin nucleus and N(3) of a histidyl residue of the apoprotein [3], In recent years, several enzymes were reported to contain FAD covalently bound to the apoprotein (reviewed [4]).…”