<jats:title>ABSTRACT</jats:title><jats:p>A newly isolated strain, SJY1, identified as<jats:named-content content-type="genus-species">Ochrobactrum</jats:named-content>sp., utilizes nicotine as a sole source of carbon, nitrogen, and energy. Strain SJY1 could efficiently degrade nicotine via a variant of the pyridine and pyrrolidine pathways (the VPP pathway), which highlights bacterial metabolic diversity in relation to nicotine degradation. A 97-kbp DNA fragment containing six nicotine degradation-related genes was obtained by gap closing from the genome sequence of strain SJY1. Three genes, designated<jats:italic>vppB</jats:italic>,<jats:italic>vppD</jats:italic>, and<jats:italic>vppE</jats:italic>, in the VPP pathway were cloned and heterologously expressed, and the related proteins were characterized. The<jats:italic>vppB</jats:italic>gene encodes a flavin-containing amine oxidase converting 6-hydroxynicotine to 6-hydroxy-<jats:italic>N</jats:italic>-methylmyosmine. Although VppB specifically catalyzes the dehydrogenation of 6-hydroxynicotine rather than nicotine, it shares higher amino acid sequence identity with nicotine oxidase (38%) from the pyrrolidine pathway than with its isoenzyme (6-hydroxy-<jats:sc>l</jats:sc>-nicotine oxidase, 24%) from the pyridine pathway. The<jats:italic>vppD</jats:italic>gene encodes an NADH-dependent flavin-containing monooxygenase, which catalyzes the hydroxylation of 6-hydroxy-3-succinoylpyridine to 2,5-dihydroxypyridine. VppD shows 62% amino acid sequence identity with the hydroxylase (HspB) from<jats:named-content content-type="genus-species">Pseudomonas putida</jats:named-content>strain S16, whereas the specific activity of VppD is ∼10-fold higher than that of HspB. VppE is responsible for the transformation of 2,5-dihydroxypyridine. Sequence alignment and phylogenetic analysis suggested that the VPP pathway, which evolved independently from nicotinic acid degradation, might have a closer relationship with the pyrrolidine pathway. The proteins and functional pathway identified here provide a sound basis for future studies aimed at a better understanding of molecular principles of nicotine degradation.