6-hydroxy-3-succinoylpyridine hydroxylase catalyzes a central step of nicotine degradation in Agrobacterium tumefaciens S33.
6-hydroxy-3-succinoylpyridine hydroxylase catalyzes a central step of nicotine degradation in Agrobacterium tumefaciens S33.
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DOI：
10.1371/journal.pone.0103324
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Wang S
中科院分区：
文献类型：
--
作者：
Li H;Xie K;Huang H;Wang S
尼古丁是烟草中的主要生物碱，也是烟草废弃物中的主要有毒物质。它可以通过吡啶途径或吡咯烷途径被细菌降解。此前，有人提出根癌农杆菌S33降解尼古丁的途径是吡啶途径和吡咯烷途径的融合，其中6-羟基-3-琥珀酰吡啶（HSP）是连接这两条途径的关键中间体。本文报道了一种依赖于NADH的热休克蛋白羟化酶的纯化和性质。根瘤菌S33。90 kDa的同源二聚体黄素蛋白在pH 8.0的条件下，在NADH和FAD的存在下，以约18.8±1.85 µmol min−1 mg protein−1的特定速率催化HSP氧化脱羧为2，5-二羟基吡啶（2，5-DHP）。通过对纯化蛋白的N-末端氨基酸残基的比对，对该菌的基因组草图进行了鉴定。它编码由391个氨基酸组成的蛋白质，与来自恶臭假单胞菌S16的HSP羟化酶（HspB）具有62%的同一性，其通过吡咯烷途径降解尼古丁。考虑到2，5-DHP在农业和医药上的应用潜力，我们开发了一条利用重组HSP羟化酶和NADH再生系统（甲酸、NAD+和甲酸脱氢酶）将HSP转化为2，5-DHP的路线，通过该路线，从0.76±0.01 mM HSP转化为约0.53±0.03 mM 2，5-DHP，摩尔转化率为69.7%。本研究对吡啶和吡咯烷融合烟碱降解途径中的关键酶HSP羟化酶的生化性质进行了研究，并提出了一条新的绿色生化合成功能化2，5-DHP的路线。
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.
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