Identification and functional analysis of two aromatic-ring-hydroxylating dioxygenases from a Sphingomonas strain that degrades various polycyclic aromatic hydrocarbons
Identification and functional analysis of two aromatic-ring-hydroxylating dioxygenases from a Sphingomonas strain that degrades various polycyclic aromatic hydrocarbons
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DOI：
10.1128/aem.70.11.6714-6725.2004
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发表时间：
2004-11-01
影响因子：
4.4
通讯作者：
Jouanneau, Y
中科院分区：
文献类型：
--
作者：
Demanèche, S;Meyer, C;Jouanneau, Y
在本研究中，黄原氨酸降解菌Sphingomonas sp.中参与多环芳烃(PAH)降解的酶。对菌株CRY-1进行了研究。[C-14]大黄素矿化实验表明，多环芳烃生长的细菌产生了高水平的大黄素分解代谢活性。其中一个多环芳烃诱导的蛋白质与环羟化双加氧酶β亚基相似，第二个多环芳烃诱导的蛋白质与外源双加氧酶相似。对编码这些蛋白的基因进行了克隆，序列分析显示有两个不同的基因位点包含聚集性分解代谢基因，与在新环果藻F199中发现的相应基因具有很强的相似性。在第一个座位上，两个可能编码末端双加氧酶成分的基因被命名为PhnI，紧随其后的是编码芳香醇脱氢酶(PhnB)的基因。第二个基因包含5个基因，分别编码外源双加氧酶(PhnC)、铁氧还蛋白(PhnA3)、加氧酶组分(PhnII)和异构酶(PhnD)。研究发现，PhnI能够将几种多环芳烃，包括大黄素，转化为相应的二氢二醇。编码铁氧还蛋白(PhnA3)和还原酶(PhnA4)的基因共表达后，PhnI的活性大大增强。编码PhnIα亚单位的phnA1(A)基因的破坏导致了一种突变菌株，该突变菌株已经失去了在多环芳烃上生长的能力。在大肠杆菌中高效表达的重组PhnII酶具有水杨酸1-羟基酶的功能。PhnII还使用了水杨酸甲酯和邻氨基苯甲酸酯作为底物。我们的结果表明，在菌株CRY-1中，一个单一的酶(PhnI)负责包括大黄素在内的一系列多环芳烃的最初攻击。此外，水杨酸盐转化为邻苯二酚的催化作用是由一个与已知的水杨酸羟基酶无关的三组分加氧酶催化的。
In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysene-degrading organism Sphingomonas sp. strain CRY-1 were investigated. [C-14]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in Novosphingobium aromaticivorans F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (phnB). The second locus contained five genes encoding an extradiol dioxygenase (phnC), a ferredoxin (phnA3), another oxygenase component (PhnII), and an isomerase (phnD). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (phnA3) and a reductase (phnA4). Disruption of the phnA1(a) gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in Escherichia coli functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CRY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known sallicylate hydroxylases.