Gene cluster on pAO1 of Arthrobacter nicotinovorans involved in degradation of the plant alkaloid nicotine: Cloning purification. and characterization of 2,6-dihydroxypyridine 3-hydroxylase

A 27,690-bp gene cluster involved in the degradation of the plant alkaloid nicotine was characterized from the plasmid pAO1 of Arthrobacter nicotinovo rans. The genes of the heterotrimeric, molybdopterin cofactor (MoCo)-, flav in adenine dinucleotide (FAD)-, and [Fe-S] cluster-dependent 6-hydroxypseud ooxynicotine (ketone) dehydrogenase (KDH) were identified within this clust er. The gene of the large MoCo subunit of KDH was located 4,266 bp from the FAD and [Fe-S] cluster subunit genes. Deduced functions of proteins encode d by open reading frames (ORFs) of the cluster were correlated to individua l steps in nicotine degradation. The gene for 2,6-dihydroxypyridine 3-hydro xylase was cloned and expressed in Escherichia coli. The purified homodimer ic enzyme of 90 kDa contained 2 mot of tightly bound FAD per mol of dimer. Enzyme activity was strictly NADH-dependent and specific for 2,6-dihydroxyp yridine. 2,3-Dihydroxypyridine and 2,6-dimethoxypyridine acted as irreversi ble inhibitors. Additional ORFs were shown to encode hypothetical proteins presumably required for holoenzyme assembly, interaction with the cell memb rane, and transcriptional regulation, including a MobA homologue predicted to be specific for the synthesis of the molybdopterin cytidine dinucleotide cofactor.