Extended Data Fig. 5: CycN is most closely related to cytochromes from other nicotine degrading species.

CycN (highlighted in red) was used as the template for an NCBI BLAST homology search. The sequences with highest homology were collected, and identical sequences removed. A tree was generated using the NGPhylogeny web server with default settings⁴⁷, then formatted into a figure using the Interactive Tree of Life (iTOL)⁴⁸. Notably, the cytochrome c from Pseudomonas sp. HZN6⁴⁹ (highlighted in red) is not included in the NCBI database, but was added to the sequence set after manual review of that organisms genome. It appears that other known nicotine degrading organisms also contain cytochromes c similar to CycN, suggesting that they use a similar electron transfer pathway^49,50. Sequence analyses of CycN related sequences was complicated by the fact that there is relatively poor annotation of these proteins in nicotine degrading organisms. For example, manual review of the pyrrolidine-pathway nicotine degrading bacteria Pseudomonas sp. HZN6 revealed an unannotated cytochrome c homologous protein just downstream of nicotine oxidoreductase. This is the same genomic architecture as for P. putida S16. This poor annotation led us to manually review other nicotine degrading organism’s genomes, in which we identify a consistent pattern. In organisms that use the pyrrolidine pathway, like P. putida S16, there are nicotine oxidoreductase enzymes similar to NicA2 with neighboring cytochrome-c proteins. In those that metabolize nicotine via the pyridine pathway, there do not appear to be protein-based electron acceptors in the region of their nicotine degrading enzymes. For variant pyrrolidine/pyridine pathway (VPP) organisms, these do not appear to have cytochromes c, but often have pseudoazurin proteins in their nicotine degrading genomic islands. Pseudoazurins are able to participate in a range of electron transport reactions in the periplasm of bacteria⁵¹, though it is unclear if they could serve this role for flavin dependent amine oxidases in these organisms.