Cloning of a Novel Nicotine Oxidase Gene from Pseudomonas sp. Strain HZN6 Whose Product Nonenantioselectively Degrades Nicotine to Pseudooxynicotine

Pseudomonas sp. strain HZN6 utilizes nicotine as its sole source of carbon, nitrogen, and energy. However, its catabolic mechanism has not been elucidated. In this study, self-formed adaptor PCR was performed to amplify the upstream sequence of the pseudooxynicotine amine oxidase gene. A 1,437-bp open reading frame (designatednox ) was found to encode a nicotine oxidase (NOX) that shows 30% amino acid sequence identity with 6-hydroxy-l-nicotine oxidase fromArthrobacter nicotinovorans . Thenox gene was cloned into a broad-host-range cloning vector and transferred into the non-nicotine-degrading bacteriaEscherichia coli DH5α (DH-nox) andPseudomonas putida KT2440 (KT-nox). The transconjugant KT-nox obtained nicotine degradation ability and yielded an equimolar amount of pseudooxynicotine, while DH-nox did not. Reverse transcription-PCR showed that thenox gene is expressed in both DH5α and KT2440, suggesting that additional factors required for nicotine degradation are present in aPseudomonas strain(s), but not inE. coli . The mutant of strain HZN6 withnox disrupted lost the ability to degrade nicotine, but not pseudooxynicotine. These results suggested that thenox gene is responsible for the first step of nicotine degradation. The (RS )-nicotine degradation results showed that the two enantiomers were degraded at approximately the same rate, indicating that NOX does not show chiral selectivity. Site-directed mutagenesis revealed that both the conserved flavin adenine dinucleotide (FAD)-binding GXGXXG motif and His456 are essential for nicotine degradation activity.