6-hydroxy-3-succinoylpyridine hydroxylase catalyzes a central step of nicotine degradation in Agrobacterium tumefaciens S33.

Huili Li,Haiyan Huang,Shuning Wang,Kebo Xie,Andrea Motta

doi:10.1371/journal.pone.0103324

Huili Li, Haiyan Huang + Show 3 more

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https://doi.org/10.1371/journal.pone.0103324

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Journal: PloS one	Publication Date: Jul 23, 2014
Citations: 42	License type: CC BY 4.0

Affiliation: Shandong University, Shandong First Medical University

Abstract

Nicotine is a main alkaloid in tobacco and is also the primary toxic compound in tobacco wastes. It can be degraded by bacteria via either pyridine pathway or pyrrolidine pathway. Previously, a fused pathway of the pyridine pathway and the pyrrolidine pathway was proposed for nicotine degradation by Agrobacterium tumefaciens S33, in which 6-hydroxy-3-succinoylpyridine (HSP) is a key intermediate connecting the two pathways. We report here the purification and properties of an NADH-dependent HSP hydroxylase from A. tumefaciens S33. The 90-kDa homodimeric flavoprotein catalyzed the oxidative decarboxylation of HSP to 2,5-dihydroxypyridine (2,5-DHP) in the presence of NADH and FAD at pH 8.0 at a specific rate of about 18.8±1.85 µmol min−1 mg protein−1. Its gene was identified by searching the N-terminal amino acid residues of the purified protein against the genome draft of the bacterium. It encodes a protein composed of 391 amino acids with 62% identity to HSP hydroxylase (HspB) from Pseudomonas putida S16, which degrades nicotine via the pyrrolidine pathway. Considering the application potential of 2,5-DHP in agriculture and medicine, we developed a route to transform HSP into 2,5-DHP with recombinant HSP hydroxylase and an NADH-regenerating system (formate, NAD+ and formate dehydrogenase), via which around 0.53±0.03 mM 2,5-DHP was produced from 0.76±0.01 mM HSP with a molar conversion as 69.7%. This study presents the biochemical properties of the key enzyme HSP hydroxylase which is involved in the fused nicotine degradation pathway of the pyridine and pyrrolidine pathways and a new green route to biochemically synthesize functionalized 2,5-DHP.

Highlights

Microbial degradation of nicotine has drawn an increasing interest recently since it has various biochemical and physiological mechanisms and represents a promising biological method to treat the tobacco leaves and wastes [1,2,3,4,5,6]
Because 2,5DHP, the product of the reaction catalyzed by HSP hydroxylase, is a valuable precursor for the chemical synthesis of 5-aminolevulinic acid, which is applied as a plant growth hormone, a herbicide and in cancer therapy, an efficient process to transform HSP into 2,5DHP was developed here with heterologously expressed HSP hydroxylase and NADH-regenerating system
The reaction was performed in a 10-ml tube containing 5 ml of mixture composed of 50 mM sodium phosphate buffer, 0.75 mM HSP, NADH-regenerating system (1.67 mM NAD+, 83.5 mM sodium formate, 5 units of formate dehydrogenase), and 8 units of recombinant HSP hydroxylase

Summary

Introduction

Microbial degradation of nicotine has drawn an increasing interest recently since it has various biochemical and physiological mechanisms and represents a promising biological method to treat the tobacco leaves and wastes [1,2,3,4,5,6]. Nicotine can be modified into important functionalized pyridines by biocatalytic processes that are difficult to synthesize via chemical methods [15] It can be transformed by Arthrobacter sp. A novel pathway different from the reported pathways mentioned above in Agrobacterium tumefaciens S33 was characterized and proposed, that is, by way of the identification of its intermediates and measurement of key enzymes activities in cell extracts and partially enriched enzymes [20,24] In this novel pathway (Figure 1), nicotine is firstly transformed into 6-hydroxy-pseudooxynicotine via the pyridine pathway through 6-hydroxy-L-nicotine and 6-hydroxy-N-methylmyosmine, and it turns to the pyrrolidine pathway with the formation of HSP and 2,5-DHP. Because 2,5DHP, the product of the reaction catalyzed by HSP hydroxylase, is a valuable precursor for the chemical synthesis of 5-aminolevulinic acid, which is applied as a plant growth hormone, a herbicide and in cancer therapy, an efficient process to transform HSP into 2,5DHP was developed here with heterologously expressed HSP hydroxylase and NADH-regenerating system (formate, NAD+ and formate dehydrogenase from Candida boidinii)

Materials and Methods

Results and Discussion

Conclusion