2-Chloro-4-nitrophenol (2C4NP) is the most common chlorinated nitrophenol pollutant, and its environmental fate is of great concern. Cupriavidus sp. CNP-8, a Gram-negative bacterium, has been reported to degrade 2C4NP via the 1,2,4-benzenetriol (BT) pathway, significantly different from the (chloro)hydroquinone pathways reported in all other Gram-negative 2C4NP-utilizers. Herein, the BT pathway of the catabolism of 2C4NP in this strain was characterized at the molecular, biochemical, and genetic levels. The hnp gene cluster was suspected to be involved in the catabolism of 2C4NP because the hnp genes are significantly upregulated in the 2C4NP-induced strain CNP-8 compared to the uninduced strain. HnpAB, a two-component FAD-dependent monooxygenase, catalyzes the conversion of 2C4NP to BT via chloro-1,4-benzoquinone, with a Km of 2.7 ± 1.1 μΜ and a kcat/Km of 0.17 ± 0.03 μΜ-1 min-1. hnpA is necessary for strain CNP-8 to utilize 2C4NP in vivo. HnpC, a BT 1,2-dioxygenase, was proved to catalyze BT ring-cleavage with formation of maleylacetate by HPLC-MS analysis. Phylogenetic analysis indicated that HnpA likely has different evolutionary origin compared to other functionally identified 2C4NP monooxygenases. To our knowledge, this is the first report revealing the catabolic mechanism of 2C4NP via the BT pathway in a Gram-negative bacterium, increasing our knowledge of the catabolic diversity for microbial 2C4NP degradation at the molecular and biochemical level.

中文翻译：

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在革兰氏阴性细菌中通过1,2,4-苯三酚途径降解2-氯-4-硝基苯酚的分子和生化特征。

2-氯-4-硝基苯酚（2C4NP）是最常见的氯化硝基苯酚污染物，其环境命运备受关注。Cupriavidus sp。据报道，革兰氏阴性细菌CNP-8可通过1,2,4-苯三醇（BT）途径降解2C4NP，这与所有其他革兰氏阴性2C4NP利用者中报道的（氯）对苯二酚途径显着不同。在此，在分子，生化和遗传水平上表征了该菌株中2C4NP分解代谢的BT途径。hnp基因簇被怀疑与2C4NP的分解代谢有关，因为与未诱导的菌株相比，在2C4NP诱导的菌株CNP-8中hnp基因显着上调。HnpAB是一种二组分FAD依赖性单加氧酶，可催化2C4NP通过氯-1,4-苯醌转化为BT，Km为2.7±1。1μM和kcat / Km为0.17±0.03μM-1min-1。hnpA是菌株CNP-8在体内利用2C4NP所必需的。HnpC是一种BT 1,2-双加氧酶，通过HPLC-MS分析证明其可催化BT环裂解并生成马来酸乙酸酯。系统发育分析表明，与其他功能识别的2C4NP单加氧酶相比，HnpA可能具有不同的进化起源。据我们所知，这是第一份报道通过革兰氏阴性细菌中的BT途径揭示2C4NP的分解代谢机制，从而增加了我们对微生物2C4NP在分子和生化水平降解的分解代谢多样性的认识。系统发育分析表明，与其他功能识别的2C4NP单加氧酶相比，HnpA可能具有不同的进化起源。据我们所知，这是第一份报道通过革兰氏阴性细菌中的BT途径揭示2C4NP的分解代谢机理，从而增加了我们对微生物2C4NP在分子和生化水平降解的分解代谢多样性的认识。系统发育分析表明，与其他功能识别的2C4NP单加氧酶相比，HnpA可能具有不同的进化起源。据我们所知，这是第一份报道通过革兰氏阴性细菌中的BT途径揭示2C4NP的分解代谢机理，从而增加了我们对微生物2C4NP在分子和生化水平降解的分解代谢多样性的认识。