Functional Identification of Two Novel Genes from Pseudomonas sp Strain HZN6 Involved in the Catabolism of Nicotine
Functional Identification of Two Novel Genes from Pseudomonas sp Strain HZN6 Involved in the Catabolism of Nicotine
复制标题
假单胞菌HZN6菌株中两个参与尼古丁分解代谢的新基因的功能鉴定
DOI：
10.1128/aem.07025-11
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发表时间：
2012-04-01
影响因子：
4.4
通讯作者：
Liu, Weiping
中科院分区：
文献类型：
--
作者：
Qiu, Jiguo;Ma, Yun;Liu, Weiping
尼古丁是烟草产生的一种天然生物碱，其被微生物降解的机制多种多样。在本研究中，我们报告了两个新的基因参与尼古丁降解新分离的假单胞菌菌株HZN 6的突变，克隆和鉴定。转座子诱变鉴定了HZN 6突变体，其中尼古丁降解途径被阻断在pseudooxycotinic。通过自身形成的衔接子PCR获得了位于转座子插入位点侧翼的3，874-bp的DNA片段。两个开放阅读框架（命名为pao和sap）进行分析，推导的氨基酸序列与节杆菌nicotinovorans的6-羟基-L-尼古丁氧化酶的同源性为29%，与汉赛巴尔通体（Bartonella henselae）的醛脱氢酶的同源性为49%。pao和sap均在重组大肠杆菌BL 21中得到克隆和功能性表达。pao基因编码一种新的假氧烟碱胺氧化酶，其与黄素腺嘌呤二核苷酸（FAD）非共价结合，并表现出底物特异性，通过形成3-琥珀酰半胱氨酸-吡啶和过氧化氢从假氧烟碱中去除甲胺。sap基因编码NADP（+）依赖的3-琥珀酰半胱氨酸-吡啶脱氢酶，其催化3-琥珀酰半胱氨酸-吡啶脱氢为3-琥珀酰-吡啶。遗传分析表明，pao基因在菌株HZN 6的烟碱或假氧化烟碱矿化中起重要作用，而sap基因则不起作用。本研究为假单胞菌在基因水平上的尼古丁降解机制提供了新的见解。
Nicotine is a natural alkaloid produced by tobacco plants, and the mechanisms of its catabolism by microorganisms are diverse. In the present study, we reported the mutation, cloning, and identification of two novel genes involved in nicotine degradation from the newly isolated Pseudomonas sp. strain HZN6. Transposon mutagenesis identified a HZN6 mutant in which the nicotine-degrading pathway was blocked at pseudooxynicotine. A 3,874-bp DNA fragment flanking the transposon insertion site was obtained through self-formed adaptor PCR. Two open reading frames (designated pao and sap) were analyzed, and the deduced amino acid sequences shared 29% identity with 6-hydroxy-L-nicotine oxidase from Arthrobacter nicotinovorans and 49% identity with an aldehyde dehydrogenase from Bartonella henselae. Both pao and sap were cloned and functionally expressed in recombinant Escherichia coli BL21. The pao gene encoded a novel pseudooxynicotine amine oxidase with noncovalently bound flavin adenine dinucleotide (FAD) and exhibited substrate specificity removing the methylamine from pseudooxynicotine with the formation of 3-succinoylsemialdehyde-pyridine and hydrogen dioxide. The sap gene encoded a NADP(+)-dependent 3-succinoylsemialdehyde-pyridine dehydrogenase that catalyzed the dehydrogenation of 3-succinoylsemialdehyde-pyridine to 3-succinoyl-pyridine. Genetic analyses indicated that the pao gene played an essential role in nicotine or pseudooxynicotine mineralization in strain HZN6, whereas the sap gene did not. This study provides novel insight into the nicotine-degrading mechanism at the genetic level in Pseudomonas spp.