Cloning of a Novel Nicotine Oxidase Gene from Pseudomonas sp Strain HZN6 Whose Product Nonenantioselectively Degrades Nicotine to Pseudooxynicotine
Cloning of a Novel Nicotine Oxidase Gene from Pseudomonas sp Strain HZN6 Whose Product Nonenantioselectively Degrades Nicotine to Pseudooxynicotine
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从假单胞菌 HZN6 菌株中克隆新型尼古丁氧化酶基因，其产物非对映选择性地将尼古丁降解为假氧尼古丁
DOI：
10.1128/aem.03824-12
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发表时间：
2013-04-01
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
假单胞菌菌株HZN 6利用尼古丁作为其碳、氮和能量的唯一来源。然而，其分解代谢机制尚未阐明。在这项研究中，自我形成的衔接子PCR进行扩增的假羟烟碱胺氧化酶基因的上游序列。发现一个1，437-bp的开放阅读框架（指定为nox）编码尼古丁氧化酶（NOX），其显示与来自节杆菌nicotinovorans的6-羟基-L-尼古丁氧化酶30%的氨基酸序列同一性。将nox基因克隆到广宿主范围克隆载体中，并转移到非尼古丁降解细菌大肠杆菌DH 5 α（DH-nox）和恶臭假单胞菌KT 2440（KT-nox）中。转化子KT-nox具有降解烟碱的能力，产生等摩尔量的假氧化烟碱，而DH-nox则不能。逆转录-PCR显示，nox基因在DH 5 α和KT 2440中均表达，这表明尼古丁降解所需的额外因子存在于假单胞菌菌株中，但不存在于大肠杆菌中。杆菌突变株HZN 6在氮氧化物破坏后失去了降解尼古丁的能力，但假氧化尼古丁没有被降解。这些结果表明，nox基因负责尼古丁降解的第一步。（RS）-尼古丁降解结果表明，两种对映体以大致相同的速率降解，表明NOX不显示手性选择性。定点突变表明，保守的黄素腺嘌呤二核苷酸（FAD）结合GXGXXG基序和His 456是尼古丁降解活性所必需的。
Pseudomonas sp. strain HZN6 utilizes nicotine as its sole source of carbon, nitrogen, and energy. However, its catabolic mechanism has not been elucidated. In this study, self-formed adaptor PCR was performed to amplify the upstream sequence of the pseudooxynicotine amine oxidase gene. A 1,437-bp open reading frame (designated nox) was found to encode a nicotine oxidase (NOX) that shows 30% amino acid sequence identity with 6-hydroxy-L-nicotine oxidase from Arthrobacter nicotinovorans. The nox gene was cloned into a broad-host-range cloning vector and transferred into the non-nicotine-degrading bacteria Escherichia coli DH5 alpha (DH-nox) and Pseudomonas putida KT2440 (KT-nox). The transconjugant KT-nox obtained nicotine degradation ability and yielded an equimolar amount of pseudooxynicotine, while DH-nox did not. Reverse transcription-PCR showed that the nox gene is expressed in both DH5 alpha and KT2440, suggesting that additional factors required for nicotine degradation are present in a Pseudomonas strain(s), but not in E. coli. The mutant of strain HZN6 with nox disrupted lost the ability to degrade nicotine, but not pseudooxynicotine. These results suggested that the nox gene is responsible for the first step of nicotine degradation. The (RS)-nicotine degradation results showed that the two enantiomers were degraded at approximately the same rate, indicating that NOX does not show chiral selectivity. Site-directed mutagenesis revealed that both the conserved flavin adenine dinucleotide (FAD)-binding GXGXXG motif and His456 are essential for nicotine degradation activity.