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

J Bacteriol. 2001 Sep;183(18):5262-7. doi: 10.1128/JB.183.18.5262-5267.2001.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Arthrobacter / enzymology*
  • Arthrobacter / genetics
  • Biodegradation, Environmental
  • Cloning, Molecular
  • Genes, Bacterial*
  • Mixed Function Oxygenases / genetics*
  • Mixed Function Oxygenases / isolation & purification
  • Mixed Function Oxygenases / metabolism
  • Molecular Sequence Data
  • Multigene Family
  • Nicotine / metabolism*
  • Open Reading Frames
  • Plasmids / genetics*
  • Pyridines / metabolism

Substances

  • Pyridines
  • hydroxypyridines
  • Nicotine
  • Mixed Function Oxygenases
  • 2,6-dihydroxypyridine 3-hydroxylase

Associated data

  • GENBANK/AF373840