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Identification and Functional Analysis of Two Aromatic-Ring-Hydroxylating Dioxygenases from a Sphingomonas Strain That Degrades Various Polycyclic Aromatic Hydrocarbons

机译:鞘氨醇单胞菌菌株降解两种多环芳烃的两种芳香环加氧双加氧酶的鉴定和功能分析

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摘要

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysene-degrading organism Sphingomonas sp. strain CHY-1 were investigated. [14C]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 phnA1a 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 CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases.
机译:在这项研究中,参与降解降解生物的鞘氨醇单胞菌属物种鞘氨醇单胞菌(Sphingomonas sp。)中的多环芳烃(PAH)的酶。研究了菌株CHY-1。 [14C]铬矿化实验表明,多环芳烃(PAH)生长的细菌产生了高水平的铬分解代谢活性。一种PAH诱导的蛋白显示与环羟基化双加氧酶β亚基的相似性,另一种PAH诱导的蛋白显示与二醇外双加氧酶的相似性。克隆了编码这些蛋白质的基因,并进行序列分析,发现了两个不同的基因座,它们包含成簇的分解代谢基因,与在新孢子虫芳香族F199中发现的相应基因具有高度相似性。在第一个基因座中,两个可能编码末端双加氧酶成分的基因称为PhnI,然后是编码芳基醇脱氢酶(phnB)的基因。第二个基因座包含五个编码外二醇双加氧酶(phnC),铁氧还蛋白(phnA3),另一种加氧酶成分(PhnII)和异构酶(phnD)的基因。发现PhnI能够将几种PAH(包括,)转化为相应的二氢二醇。当编码铁氧还蛋白(phnA3)和还原酶(phnA4)的基因共表达时,PnI的活性大大增强。编码PhnIα亚基的phnA1a基因的破坏导致突变株失去了在PAHs上生长的能力。在大肠杆菌中过量产生的重组PhnII酶起水杨酸酯1-羟化酶的作用。 PhnII还使用了甲基水杨酸酯和邻氨基苯甲酸酯作为底物。我们的结果表明,一种单一的酶(PhnI)引起了CHY-1菌株中一系列PAH(包括initial)的初始攻击。此外,水杨酸酯向儿茶酚的转化是通过与已知水杨酸酯羟化酶无关的三组分加氧酶催化的。

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