Interest in the metabolic fate of the alkaloid nicotine is of long standing in my laboratory. Arthrobacter nicotinovorans (formerly known as Arthrobacter oxidans), an aerobic bacterium isolated from the soil around tobacco plants, was found to utilize D‑ or L‑nicotine as the sole source of carbon and nitrogen for growth. The enzymes responsible for the degradation of the alkaloid are encoded on a 160 kb plasmid, pAO1, present in this organism.
The first enzyme of the nicotine pathway , nicotine dehydrogenase, introduces a hydroxyl group into the 6‑position of the pyridine ring of both L‑ and D‑nicotine. The oxidation of the 6-hydroxynicotines by the “enantiozymes” 6‑hydroxy‑L‑nicotine oxidase (6‑HLNO) and 6‑hydroxy‑D‑nicotine oxidase (6‑HDNO), respectively, involves the asymmetric C’2 of the pyrrolidine ring and leads to the optically inactive 6‑hydroxy‑N‑methylmyosmine that hydrolyzes spontaneously to 3‑(6‑hydroxypyridyl‑)‑(g‑N‑methylaminopropyl‑)ketone. Although both enzymes are strictly stereospecific, the respective inactive enantiomeric 6-hydroxynicotine is an inhibitor with high affinity to the binding site.
6‑HLNO consists of two identical subunits of Mr = 46’985, each bearing one non‑covalently bound FAD molecule; in contrast, the monomeric 6‑HDNO (Mr = 48’981) carries the cofactor FAD covalently bound to a histidine residue.
Since D-nicotine is not produced naturally (traces of D-nornicotine, however, have been found in Nicotiana plants), the synthesis of an absolutely D-specific enzyme is surprising. The more so, as it turned out that the primary structures of these enantiozymes are not homologous and may have a different evolutionary history. It appears that the genetic information resulting in the 6‑hlno gene was acquired by Arthrobacter nicotinovorans from an eukaryotic source by horizontal gene transfer.
The aim of the ongoing investigations, carried out in collaboration with Dr. Bartuniks group (MPG-ASMB) at DESY (Hamburg), centers on the 3D-structure of 6-HLNO.