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

Abstract

A 27,690-bp gene cluster involved in the degradation of the plant alkaloid nicotine was characterized from the plasmid pAO1 of Arthrobacter nicotinovorans. The genes of the heterotrimeric, molybdopterin cofactor (MoCo)-, flavin adenine dinucleotide (FAD)-, and [Fe-S] cluster-dependent 6-hydroxypseudooxynicotine (ketone) dehydrogenase (KDH) were identified within this cluster. 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 encoded by open reading frames (ORFs) of the cluster were correlated to individual steps in nicotine degradation. The gene for 2,6-dihydroxypyridine 3-hydroxylase was cloned and expressed in Escherichia coli. The purified homodimeric enzyme of 90 kDa contained 2 mol of tightly bound FAD per mol of dimer. Enzyme activity was strictly NADH-dependent and specific for 2,6-dihydroxypyridine. 2,3-Dihydroxypyridine and 2,6-dimethoxypyridine acted as irreversible inhibitors. Additional ORFs were shown to encode hypothetical proteins presumably required for holoenzyme assembly, interaction with the cell membrane, and transcriptional regulation, including a MobA homologue predicted to be specific for the synthesis of the molybdopterin cytidine dinucleotide cofactor.

FIG. 1

Overview of the steps in nicotine degradation to blue pigment by A. nicotinovorans pAO1. The reaction catalyzed by 2,6-DHPH, cloned and purified in this work, is framed. NDH, nicotine dehydrogenase; 6HLNO, 6-hydroxy-l-nicotine oxidase; KDH, ketone dehydrogenase.

FIG. 2

Characterization of the 27,690-bp region of A. nicotinovorans pAO1 involved in nicotine degradation, showing the physical and genetic map of the nic gene cluster. Genes encoding known enzymes of nicotine degradation are shaded dark gray and are indicated by the corresponding abbreviation. ORFs proposed to encode proteins involved in nicotine degradation are shaded light gray, ORFs encoding hypothetical transcription factors are indicated in black, and ORFs with no similarity to known ORFs in data banks are unshaded. Gaps between gene subclusters are indicated in base pairs (bp) above the schematically drawn ORFs. Tn, transposase.