Enzymatic steps in nicotine degradation by A. nicotinovorans. The enzymes are NDH, 6HLNO, and KDH. The intermediates are HPON [γ-N-methylaminopropyl-(6-hydroxypyridyl)-ketone], DHPON [γ-N-methylaminopropyl-(2,6-dihydroxypyridyl)-ketone], DHMM, and DHP. Shown in brackets are two possible tautomeric forms of 2,6-dihydroxypseudooxynicotine that may be cleaved, at the position marked by the bar, by a 1,3-β-ketolase, or a 1,5-β-ketolase activity, as indicated by the numbering. See the text for a discussion.

Identification of 2,6-dihydroxypyridine and γ-N-methylaminobutyrate as products of the reaction catalyzed by 2,6-dihydroxypseudooxynicotine hydrolase. (A, a) HPLC analysis of 5 μl of a 10 mM 2,6-dihydroxypyridine standard. (A, b) HPLC analysis of 10 μl of coupled assay. (B, a) Ten microliters of 1 mM phenylisothiocyanate-derivatized γ-N-methylaminobutyrate standard, which eluted directly after phenylthiohydantoin-glutamate. (P, b) Ten microliters of phenylisothiocyanate-derivatized coupled assay analyzed in a protein sequencer.

pAO1 ORFs addressed in this study and their nicotine-dependent transcription. (A) Transcriptional orientation of kdhL, ORF367, and ORF116. The fragment number and fragment size amplified by PCR from each ORF are shown. Primers are indicated schematically by arrows, and nucleotide positions of ORFs on pAO1 DNA are given. (B) RT-PCR was performed as described in reference 19 with RNA extracted from nicotine-induced (lanes 1 to 12) and uninduced (lanes 13 to 21) A. nicotinovorans pAO1. pAO1 DNA as positive control, RNA as negative control, and RNA-derived cDNA were employed as templates in PCR performed with primer pairs derived from each of the ORFs. Nicotine-induced: I, kdhL transcripts (lane 1, pAO1 DNA; lane 2, RNA; lane 3, cDNA); II, ORF367 transcripts (lane 4, pAO1; lane 5, RNA; lane 6, cDNA); III, ORF116 transcripts (lane 7, pAO1 DNA; lane 8, RNA; lane 9, cDNA); II + III, transcripts obtained with a forward primer from ORF367 and a reverse primer from ORF116 (lane 10, pAO1 DNA; lane 11, RNA; lane 12, cDNA). Nicotine uninduced: I (lanes 13 to 15), II (lanes 16 to 18), III (lanes 19 to 21) (same order as for induced). M, molecular size markers.