title: A novel (S)-6-hydroxynicotine oxidase gene from Shinella sp. strain HZN7.
abstract: 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, designated nctB and tnp2, were cloned and analyzed. The nctB 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 the nctB gene showed that this enzyme is essential for nicotine or (S)-6-hydroxynicotine degradation. Purified NctB could also convert (S)-nicotine into N-methylmyosmine, which spontaneously hydrolyzed into pseudooxynicotine. The kinetic constants of NctB toward (S)-6-hydroxynicotine (Km = 0.019 mM, kcat = 7.3 s(-1)) and nicotine (Km = 2.03 mM, kcat = 0.396 s(-1)) indicated that (S)-6-hydroxynicotine is the preferred substrate in vivo. NctB showed no activities toward the R enantiomer of nicotine or 6-hydroxynicotine. Strain HZN7 could degrade (R)-nicotine into (R)-6-hydroxynicotine without any further degradation. The tnp2 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.
authors.affiliation: Key Laboratory of Environmental Remediation and Ecosystem Health, Ministry of Education, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, China.
authors.forename: Jiguo
authors.initials: J
authors.lastname: Qiu
authors.affiliation: Research Center of Environmental Science, College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, China.
authors.forename: Yin
authors.initials: Y
authors.lastname: Wei
authors.affiliation: Research Center of Environmental Science, College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Research Center of Environmental Science, College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, China.
authors.forename: Rongti
authors.initials: R
authors.lastname: Wen
authors.affiliation: Key Laboratory of Environmental Remediation and Ecosystem Health, Ministry of Education, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, China.
authors.forename: Yuezhong
authors.initials: Y
authors.lastname: Wen
authors.affiliation: Key Laboratory of Environmental Remediation and Ecosystem Health, Ministry of Education, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, China wliu@zju.edu.cn.
authors.forename: Weiping
authors.initials: W
authors.lastname: Liu
mesh_terms: D004251:DNA Transposable Elements; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D007700:Kinetics; D008562:Membrane Glycoproteins; D016254:Mutagenesis, Insertional; D009538:Nicotine / Q000031:analogs & derivatives* / Q000378:metabolism*; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012230:Rhizobiaceae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: C502317:6-hydroxynicotine; D004251:DNA Transposable Elements; D008562:Membrane Glycoproteins; C055764:S-layer proteins; D009538:Nicotine; D010088:Oxidoreductases