3- and 4-alkylphenol degradation pathway in Pseudomonas sp strain KL28:: genetic organization of the lap gene cluster and substrate specificities of phenol hydroxylase and catechol 2,3-dioxygenase
3- and 4-alkylphenol degradation pathway in Pseudomonas sp strain KL28:: genetic organization of the lap gene cluster and substrate specificities of phenol hydroxylase and catechol 2,3-dioxygenase
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DOI：
10.1099/mic.0.26628-0
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发表时间：
2003-11-01
期刊：
影响因子：
2.8
通讯作者：
Lee, K
中科院分区：
文献类型：
--
作者：
Jeong, JJ;Kim, JH;Lee, K
在需氧细菌中，负责高级烷基酚分解代谢的酶和基因还没有被表征。假单胞菌属菌株KL28可以利用广泛的烷基酚，包括4-正烷基酚(C-1-C-5)。从染色体DNA中克隆了编码分解代谢途径酶的基因，命名为LAP(长链烷基/苯酚)，并进行了测序。LAP基因位于一个13(.)2kb的区域，有14个ORF，序列为loRBKLMNOPCEHIFG，具有相同的转录方向。LapR基因是独立转录的，编码XylR/DmpR正向转录调控因子的一个成员。LapB是LAP操纵子的第一个基因，编码儿茶酚2，3-双加氧酶(C230)。LapKLMNOP和LapCEHIFG基因分别编码多组份苯酚羟化酶(MPH)和将2-羟基粘果半醛(HMS)的衍生物降解为TCA循环中间体的酶。P-LapB启动子在-24(GG)和-12(GC)位置含有基序，这些基序通常存在于依赖西格玛(54)的启动子中。用P-LapB：：GFP转录融合载体进行的启动子分析表明，LapB启动子的活性是可诱导的，并且它对广泛的烷基苯酚有反应。这种分解代谢所需的酶的编码结构基因与来自典型苯酚降解菌假单胞菌的分解代谢pVI150质粒上编码的基因相似(42-69%)。CF600。然而，LAP基因座不包括编码HMS水解酶和铁氧还蛋白的基因。已知后者在功能上与使用4-烷基儿茶酚作为底物的C230有关。LAP分解代谢基因的排列在其他代谢操纵子中是不常见的。底物专一性研究表明，MPH优先氧化3-和4-烷基酚为4-烷基儿茶酚。C230优先通过近端(2，3)裂解氧化4-烷基儿茶酚。这表明这两个关键酶具有独特的底物偏好，并导致在菌株KL28中建立LAP途径的初始步骤。
The enzymes and genes responsible for the catabolism of higher alkylphenols have not been characterized in aerobic bacteria. Pseudomonas sp. strain KL28 can utilize a wide range of alkylphenols, which include the 4-n-alkylphenols (C-1-C-5). The genes, designated as lap (for long-chain alky/phenols), encoding enzymes for the catabolic pathway were cloned from chromosomal DNA and sequenced. The lap genes are located in a 13(.)2 kb region with 14 ORFs in the order lapRBKLMNOPCEHIFG and with the same transcriptional orientation. The lapR gene is transcribed independently and encodes a member of the XylR/DmpR positive transcriptional regulators. lapB, the first gene in the lap operon, encodes catechol 2,3-dioxygenase (C230). The lapKLMNOP and lapCEHIFG genes encode a multicomponent phenol hydroxylase (mPH) and enzymes that degrade derivatives of 2-hydroxymuconic semialdehyde (HMS) to TCA cycle intermediates, respectively. The P-lapB promoter contains motifs at positions -24(GG) and -12(GC) which are typically found in sigma(54)-dependent promoters. A promoter assay using a P-lapB: :gfp transcriptional fusion plasmid showed that lapB promoter activity is inducible and that it responds to a wide range of (alkyl)phenols. The structural genes encoding enzymes required for this catabolism are similar (42-69%) to those encoded on a catabolic pVI150 plasmid from an archetypal phenol degrader, Pseudomonas sp. CF600. However, the lap locus does not include genes encoding HMS hydrolase and ferredoxin. The latter is known to be functionally associated with C230 for use of 4-alkylcatechols as substrates. The arrangement of the lap catabolic genes is not commonly found in other meta-cleavage operons. Substrate specificity studies show that mPH preferentially oxidizes 3- and 4-alkylphenols to 4-alkylcatechols. C230 preferentially oxidizes 4-alkylcatechols via proximal (2,3) cleavage. This indicates that these two key enzymes have unique substrate preferences and lead to the establishment of the initial steps of the lap pathway in strain KL28.