A Novel ( S )-6-Hydroxynicotine Oxidase Gene from Shinella sp. Strain HZN7

Nicotine is an important environmental toxicant in tobacco waste.Shinella sp. strain HZN7 can metabolize nicotine into nontoxic compounds via variations of the pyridine and pyrrolidine pathways. However, the catabolic mechanism of this variant pathway at the gene or enzyme level is still unknown. In this study, two 6-hydroxynicotine degradation-deficient mutants, N7-M9 and N7-W3, were generated by transposon mutagenesis. The corresponding mutant genes, designatednctB andtnp2 , were cloned and analyzed. ThenctB gene encodes a novel flavin adenine dinucleotide-containing (S )-6-hydroxynicotine oxidase that converts (S )-6-hydroxynicotine into 6-hydroxy-N -methylmyosmine and then spontaneously hydrolyzes into 6-hydroxypseudooxynicotine. The deletion and complementation of thenctB gene showed that this enzyme is essential for nicotine or (S )-6-hydroxynicotine degradation. Purified NctB could also convert (S )-nicotine intoN -methylmyosmine, which spontaneously hydrolyzed into pseudooxynicotine. The kinetic constants of NctB toward (S )-6-hydroxynicotine (Km = 0.019 mM,k cat = 7.3 s−1 ) and nicotine (Km = 2.03 mM,k cat = 0.396 s−1 ) indicated that (S )-6-hydroxynicotine is the preferred substratein vivo . NctB showed no activities toward theR enantiomer of nicotine or 6-hydroxynicotine. Strain HZN7 could degrade (R )-nicotine into (R )-6-hydroxynicotine without any further degradation. Thetnp2 gene from mutant N7-W3 encodes a putative transposase, and its deletion did not abolish the nicotine degradation activity. This study advances the understanding of the microbial diversity of nicotine biodegradation.