Identification of a novel 4-hydroxyphenylpyruvate dioxygenase from the soil metagenome
Identification of a novel 4-hydroxyphenylpyruvate dioxygenase from the soil metagenome
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DOI：
10.1016/j.bbrc.2008.03.102
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发表时间：
2008-05-30
影响因子：
3.1
通讯作者：
Yoon, Sang-Hong
中科院分区：
文献类型：
--
作者：
Lee, Chang-Muk;Yeo, Yun-Soo;Yoon, Sang-Hong
4-羟基苯基丙酮酸双加氧酶(HPPD)是一种铁(II)依赖的非血红素加氧酶，能将4-羟基苯基丙酮酸转化为同质酸。植物中的主要辅因子，如叶绿体二酚和生育酚，是通过依赖于HPPD的合成代谢途径产生的。为了用不依赖培养的方法分离一个新的HPPD，我们从韩国的土壤中构建了一个粘粒亚基因组文库。通过对大肠杆菌元基因组文库的筛选，获得了一株在添加L酪氨酸的Luria-Bertani培养基中产生深褐色色素的阳性克隆YS103B。YS103B的体外转座子诱变表明，1.3kb的插入片段足以产生溶血棕色素。YS103B的序列分析揭示了一个开放阅读框架，编码一个41.4 kDa的蛋白，具有HPPD家族酶家族中保守的原核加氧酶基序。HPPD特异性的β-三酮除草剂舒可曲酮抑制YS103B的色素沉着。在大肠杆菌中表达的重组蛋白产生了均一酸。因此，我们提出了从一个未培养的土壤细菌中成功地异源表达以前未描述的HPPD基因。(C)2008 Elsevier Inc.保留所有权利。
4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a Fe(II)-dependent, non-heme oxygenase that converts 4-hydroxyphenylpyruvate to homogentisate. Essential cofactors, such as plastoquinone and tocopherol, are produced by HPPD-dependent anabolic pathways in plants. To isolate a novel hppd using culture-independent method, a cosmid metagenomic library was constructed from soil in Korea. Screening of Escherichia coli metagenomic libraries led to the identification of a positive clone, YS103B, producing dark brown pigment in Luria-Bertani medium supplemented with L-tyrosine. In vitro transposon mutagenesis of YS103B showed that the 1.3 kb insert was sufficient to produce the hemolytic brown pigment. Sequence analysis of YS103B disclosed one open reading frame encoding a 41.4 kDa protein with the well-conserved prokaryotic oxygenase motif of the HPPD family of enzymes. The HPPD-specific beta-triketone herbicide, sulcotrione, inhibited YS103B pigmentation. The recombinant protein expressed in E. coli generated homogentisic acid. Thus, we present the successful heterologous expression of a previously uncharacterized hppd gene from an uncultured soil bacterium. (C) 2008 Elsevier Inc. All rights reserved.