Horizontal gene transfer involved in the convergent evolution of the plasmid-encoded enantioselective 6-hydroxynicotine oxidases

The D- and L-specific nicotine oxidases are flavoproteins involved in the o xidative degradation of nicotine by the Gram-positive soil bacterium Arthro bacter nicotinovorans. Their structural genes are located on a 160-kbp plas mid together with those of other nicotine-degrading enzymes. They are struc turally unrelated at the DNA as well as at the protein level. Each of these oxidases possesses a high degree of substrate specificity; their catalytic stereoselectivity is absolute, although they are able to bind both enantio meric substrates with a similar affinity. It appears that the existence of these enzymes is the result of convergent evolution. The amino acid sequenc e of 6-hydroxy-L-nicotine oxidase (EC 1.5.3.6) as derived from the respecti ve structural gene shows considerable structural similarity with eukaryotic monoamine oxidases (EC 1.4.3.4) but not with monoamine oxidases from proka ryotic bacteria including those of the genus Arthrobacter. These similariti es are not confined to the nucleotide-binding sites. A 100-amino acid stret ch at the N-terminal regions of 6-hydroxy-L-nicotine oxidase and human mono amine oxidases A possess a 35% homology. Overall, 27.0, 26.4, and 25.8% of the amino acid positions of the monoamine oxidases of Aspergillus niger (N) , humans (A), and rainbow trout (Salmo gairdneri) are identical to those of 6-hydroxy-L-nicotine oxidase (Smith-Waterman algorithm). In addition, the G+C content of the latter enzyme is in the range of that of eukaryotic mono amine oxidases and definitely lower than that of the A. nicotinovorans DNA and even that of the pAO1 DNA. The primary structure of 6-hydroxy-D-nicotin e oxidase (EC 1.5.3.5) does not reveal its evolutionary history as easily. Significant similarities are found with a mitomycin radical oxidase from St reptomyces lavendulae (23.3%) and a "hypothetical protein" from Mycobacteri um tuberculosis (26.0%). It is proposed that the plasmid-encoded gene of 6- hydroxy-L-nicotine oxidase evolved after horizontal transfer from an eukary otic source.