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Journal of Bacteriology, September 2001, p. 5262-5267, Vol. 183, No. 18
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.18.5262-5267.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Daniel Baitsch,1 Cristinel Sandu,1 Roderich Brandsch,1,* and Gabor L. Igloi2

Institute of Biochemistry and Molecular Biology,1 and Institute of Biology III,2 University of Freiburg, Freiburg, Germany

Received 3 April 2001/Accepted 29 June 2001

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.

* Corresponding author. Mailing address: Institute of Biochemistry and Molecular Biology, Hermann-Herder-Str. 7, D-79104 Freiburg, Germany. Phone: 49-761-203-5231. Fax: 49-761-203-5253. E-mail: brandsch{at}ruf.uni-freiburg.de.

Journal of Bacteriology, September 2001, p. 5262-5267, Vol. 183, No. 18
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.18.5262-5267.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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