6-hydroxy-3-succinoyl-pyridine and pyridine

Topics: Agrobacterium tumefaciens; Bacterial Proteins; Butanones; Electron Transport; Electron Transport Chain Complex Proteins; Electron-Transferring Flavoproteins; Electrons; Gene Expression Regulation, Bacterial; Metabolic Networks and Pathways; Nicotine; Oxidation-Reduction; Oxidoreductases; Oxygen; Pyridines; Recombinant Proteins; Succinates; Transcriptome

Treatment of solid and liquid tobacco wastes with high nicotine content remains a longstanding challenge. Here, we explored an environmentally friendly approach to replace tobacco waste disposal with resource recovery by genetically engineering Pseudomonas putida. The biosynthesis of 3-succinoyl-pyridine (SP), a precursor in the production of hypotensive agents, from the tobacco waste was developed using whole cells of the engineered Pseudomonas strain, S16dspm. Under optimal conditions in fed-batch biotransformation, the final concentrations of product SP reached 9.8 g/L and 8.9 g/L from aqueous nicotine solution and crude suspension of the tobacco waste, respectively. In addition, the crystal compound SP produced from aqueous nicotine of the tobacco waste in batch biotransformation was of high purity and its isolation yield on nicotine was 54.2%. This study shows a promising route for processing environmental wastes as raw materials in order to produce valuable compounds.

Topics: Bacterial Proteins; Batch Cell Culture Techniques; Biocatalysis; Biodegradation, Environmental; Genetic Engineering; Industrial Waste; Magnetic Resonance Spectroscopy; Nicotiana; Nicotine; Plant Leaves; Pseudomonas putida; Pyridines; Refuse Disposal; Spectrometry, Mass, Electrospray Ionization; Succinates

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.

Topics: Bacterial Proteins; Catalysis; Flavoproteins; Hydrogen Peroxide; Kinetics; Mass Spectrometry; Mixed Function Oxygenases; NAD; Oxygen; Phylogeny; Pseudomonas putida; Pyridines; Spectrophotometry; Substrate Specificity; Succinates; Water