title: A novel NADH-dependent and FAD-containing hydroxylase is crucial for nicotine degradation by Pseudomonas putida.
abstract: Nicotine, the main alkaloid produced by Nicotiana tabacum and other Solanaceae, is very toxic and may be a leading toxicant causing preventable disease and death, with the rise in global tobacco consumption. Several different microbial pathways of nicotine metabolism have been reported: Arthrobacter uses the pyridine pathway, and Pseudomonas, like mammals, uses the pyrrolidine pathway. We identified and characterized a novel 6-hydroxy-3-succinoyl-pyridine (HSP) hydroxylase (HspB) using enzyme purification, peptide sequencing, and sequencing of the Pseudomonas putida S16 genome. The HSP hydroxylase has no known orthologs and converts HSP to 2,5-dihydroxy-pyridine and succinic semialdehyde, using NADH. (18)O(2) labeling experiments provided direct evidence for the incorporation of oxygen from O(2) into 2,5-dihydroxy-pyridine. The hspB gene deletion showed that this enzyme is essential for nicotine degradation, and site-directed mutagenesis identified an FAD-binding domain. This study demonstrates the importance of the newly discovered enzyme HspB, which is crucial for nicotine degradation by the Pseudomonas strain.
authors.affiliation: State Key Laboratory of Microbial Metabolism & School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Hongzhi
authors.initials: H
authors.lastname: Tang
authors.forename: Yuxiang
authors.initials: Y
authors.lastname: Yao
authors.forename: Dake
authors.initials: D
authors.lastname: Zhang
authors.forename: Xiangzhou
authors.initials: X
authors.lastname: Meng
authors.forename: Lijuan
authors.initials: L
authors.lastname: Wang
authors.forename: Hao
authors.initials: H
authors.lastname: Yu
authors.forename: Lanying
authors.initials: L
authors.lastname: Ma
authors.forename: Ping
authors.initials: P
authors.lastname: Xu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D016680:Genome, Bacterial / Q000502:physiology; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009538:Nicotine / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011725:Pyridines / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000031:analogs & derivatives
chemical_list: D001426:Bacterial Proteins; D011725:Pyridines; D005680:gamma-Aminobutyric Acid; D009538:Nicotine; D006899:Mixed Function Oxygenases; C009338:succinic semialdehyde; C065452:2,5-dihydroxypyridine