title: Mechanism of the 6-hydroxy-3-succinoyl-pyridine 3-monooxygenase flavoprotein from Pseudomonas putida S16.
abstract: 6-Hydroxy-3-succinoyl-pyridine (HSP) 3-monooxygenase (HspB), a flavoprotein essential to the pyrrolidine pathway of nicotine degradation, catalyzes pyridine-ring β-hydroxylation, resulting in carbon-carbon cleavage and production of 2,5-dihydroxypyridine. Here, we generated His6-tagged HspB in Escherichia coli, characterized the properties of the recombinant enzyme, and investigated its mechanism of catalysis. In contrast to conclusions reported previously, the second product of the HspB reaction was shown to be succinate, with isotope labeling experiments providing direct evidence that the newly introduced oxygen atom of succinate is derived from H2O. Phylogenetic analysis reveals that HspB is the most closely related to two p-nitrophenol 4-monooxygenases, and the experimental results exhibit that p-nitrophenol is a substrate of HspB. The reduction of HspB (with maxima at 375 and 460 nm, and a shoulder at 485 nm) by NADH was followed by stopped-flow spectroscopy, and the rate constant for reduction was shown to be stimulated by HSP. Reduced HspB reacts with oxygen to form a C(4a)-(hydro)peroxyflavin intermediate with an absorbance maximum at ∼400 nm within the first few milliseconds before converting to the oxidized flavoenzyme species. The formed C(4a)-hydroperoxyflavin intermediate reacts with HSP to form an intermediate that hydrolyzes to the products 2,5-dihydroxypyridine and succinate. The investigation on the catalytic mechanism of a flavoprotein pyridine-ring β-position hydroxylase provides useful information for the biosynthesis of pyridine derivatives.
authors.affiliation: From the State Key Laboratory of Microbial Metabolism, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China and.
authors.forename: Hao
authors.initials: H
authors.lastname: Yu
authors.affiliation: the Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Hausinger
authors.affiliation: From the State Key Laboratory of Microbial Metabolism, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China and.
authors.forename: Hong-Zhi
authors.initials: HZ
authors.lastname: Tang
authors.affiliation: From the State Key Laboratory of Microbial Metabolism, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China and pingxu@sjtu.edu.cn.
authors.forename: Ping
authors.initials: P
authors.lastname: Xu
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002384:Catalysis; D005420:Flavoproteins / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D009243:NAD / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D010802:Phylogeny; D016958:Pseudomonas putida / Q000201:enzymology*; D011725:Pyridines / Q000737:chemistry; D013053:Spectrophotometry; D013379:Substrate Specificity; D013386:Succinates / Q000737:chemistry; D014867:Water / Q000737:chemistry
chemical_list: C507268:6-hydroxy-3-succinoyl-pyridine; D001426:Bacterial Proteins; D005420:Flavoproteins; D011725:Pyridines; D013386:Succinates; D014867:Water; D009243:NAD; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; C023666:pyridine; D010100:Oxygen
keywords: Bacteria; Enzyme Mechanism; Flavoprotein; Metabolism; Monooxygenase; Protein Purification; Pyridine Derivative