An NAD-Specific 6-Hydroxy-3-Succinoyl-Semialdehyde-Pyridine Dehydrogenase from Nicotine-Degrading Agrobacterium tumefaciens Strain S33
An NAD-Specific 6-Hydroxy-3-Succinoyl-Semialdehyde-Pyridine Dehydrogenase from Nicotine-Degrading Agrobacterium tumefaciens Strain S33
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DOI：
10.1128/spectrum.00924-21
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发表时间：
2021-09-01
影响因子：
3.7
通讯作者：
Wang，Shuning
中科院分区：
文献类型：
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作者：
Shang，Jinmeng;Wang，Xia;Wang，Shuning
根癌土壤杆菌菌株S33可以通过吡啶和吡咯烷途径的混合物分解代谢尼古丁。除了催化6-羟基-3-琥珀酰基-半醛-吡啶氧化为6-羟基-3-琥珀酰基吡啶的酶外，参与该生化途径的大多数酶已被鉴定和表征。基于先前的基因组和转录组学分析，一个开放的阅读框架（ORF）注释编码乙醛脱氢酶（Ald）在尼古丁降解集群预测负责这一步骤。本研究中，我们异源表达了该酶，并通过生化分析和质谱分析对其功能进行了鉴定。发现Ald催化6-羟基-3-丁二酰基-半醛-吡啶的NAD特异性脱氢生成6-羟基-3-丁二酰基吡啶。以非羟基化类似物3-琥珀酰-半醛-吡啶（SAP）为底物，Ald在pH 9.0时的比活性为10.05 U/mg，对SAP和NAD+的表观Km值分别为58.68 μM和0.41 mM。低温诱导、低盐缓冲液中纯化和保存有助于防止其聚集和沉淀。ald基因的缺失导致菌株S33在烟碱上的生长速率和生物量降低，而在6-羟基-3-琥珀酰吡啶上没有。ALD具有广泛的底物，包括苯甲醛、糠醛和乙醛。携带Ald基因的重组大肠杆菌细胞能够以0.032 mmol min−1g干细胞−1的比速率高效地将糠醛转化为2-糠酸，扩展了Ald在生物基呋喃化合物催化中的应用。这些发现为烟碱降解杂合途径的生化机制以及Ald在工业生物催化中的应用提供了新的见解。重要的是，烟碱是烟草中产生的主要有毒N-杂环芳香生物碱之一。制造烟草和吸烟可能会导致一些环境和公共卫生问题。微生物可以通过多种生化途径降解尼古丁，但尼古丁降解的生化机制尚未完全阐明。在这项研究中，我们确定了醛脱氢酶负责6-羟基-3-琥珀酰-半醛-吡啶氧化为6-羟基-3-琥珀酰吡啶，这是唯一的未表征的酶在杂交的吡啶和吡咯烷途径在根癌农杆菌S33。与已知的醛脱氢酶类似，NAD特异性同二聚体酶在碱性和低盐缓冲液中具有广泛的底物范围和高活性。它不仅能催化烟碱降解产生的醛，还能催化苯甲醛、糠醛和乙醛的降解。结果表明，携带该基因的重组大肠杆菌能够高效地将糠醛转化为有价值的2-糠酸，显示了其在酶催化方面的潜在应用。
Agrobacterium tumefaciens strain S33 can catabolize nicotine via a hybrid of the pyridine and pyrrolidine pathways. Most of the enzymes involved in this biochemical pathway have been identified and characterized, except for the one catalyzing the oxidation of 6-hydroxy-3-succinoyl-semialdehyde-pyridine to 6-hydroxy-3-succinoylpyridine. Based on a previous genomic and transcriptomic analysis, an open reading frame (ORF) annotated to encode aldehyde dehydrogenase (Ald) in the nicotine-degrading cluster was predicted to be responsible for this step. In this study, we heterologously expressed the enzyme and identified its function by biochemical assay and mass spectrum analysis. It was found that Ald catalyzes the NAD-specific dehydrogenation of 6-hydroxy-3-succinoyl-semialdehyde-pyridine to 6-hydroxy-3-succinoylpyridine. With the nonhydroxylated analog 3-succinoyl-semialdehyde-pyridine (SAP) as a substrate, Ald had a specific activity of 10.05 U/mg at pH 9.0 and apparentKmvalues of around 58.68 μM and 0.41 mM for SAP and NAD+, respectively. Induction at low temperature and purification and storage in low-salt buffers were helpful to prevent its aggregation and precipitation. Disruption of thealdgene caused a lower growth rate and biomass of strain S33 on nicotine but not on 6-hydroxy-3-succinoylpyridine. Ald has a broad range of substrates, including benzaldehyde, furfural, and acetaldehyde. Recombinant Escherichia coli cells harboring thealdgene can efficiently convert furfural to 2-furoic acid at a specific rate of 0.032 mmol min−1g dry cells−1, extending the application of Ald in the catalysis of bio-based furan compounds. These findings provide new insights into the biochemical mechanism of the nicotine-degrading hybrid pathway and the possible application of Ald in industrial biocatalysis.IMPORTANCENicotine is one of the major toxicN-heterocyclic aromatic alkaloids produced in tobacco plants. Manufacturing tobacco and smoking may lead to some environmental and public health problems. Microorganisms can degrade nicotine by various biochemical pathways, but the biochemical mechanism for nicotine degradation has not been fully elucidated. In this study, we identified an aldehyde dehydrogenase responsible for the oxidation of 6-hydroxy-3-succinoyl-semialdehyde-pyridine to 6-hydroxy-3-succinoylpyridine; this was the only uncharacterized enzyme in the hybrid of the pyridine and pyrrolidine pathways in Agrobacterium tumefaciens S33. Similar to the known aldehyde dehydrogenase, the NAD-specific homodimeric enzyme presents a broad substrate range with high activity in alkaline and low-salt-containing buffers. It can catalyze not only the aldehyde from nicotine degradation but also those of benzaldehyde, furfural, and acetaldehyde. It was found that recombinant Escherichia coli cells harboring thealdgene could efficiently convert furfural to valuable 2-furoic acid, demonstrating its potential application for enzymatic catalysis.