N-Hydroxyarylamine O-Acetyltransferases Catalyze Acetylation of 3-Amino-4-Hydroxyphenylarsonic Acid in the 4-Hydroxy-3-Nitrobenzenearsonic Acid Transformation Pathway of Enterobacter sp. Strain CZ-1
N-Hydroxyarylamine O-Acetyltransferases Catalyze Acetylation of 3-Amino-4-Hydroxyphenylarsonic Acid in the 4-Hydroxy-3-Nitrobenzenearsonic Acid Transformation Pathway of Enterobacter sp. Strain CZ-1
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N-羟基芳胺 O-乙酰基转移酶催化肠杆菌属 4-羟基-3-硝基苯胂酸转化途径中 3-氨基-4-羟基苯胂酸的乙酰化。
DOI：
10.1128/aem.02050-19
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发表时间：
2019-11
影响因子：
4.4
通讯作者：
Fang-jie Zhao
中科院分区：
文献类型：
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作者：
Ke Huang;Fan Gao;X. Chris Le;Fang-jie Zhao
Roxarsone（ROX）是一种有机砷饲料添加剂，70多年来已广泛用于家禽业，用于促进生长，控制球虫病和改善肉色。大多数ROX被排泄在垃圾中并分散到环境中，在那里它被微生物转化为不同的含砷化合物。ROX转化的主要产物是N-乙酰基-4-羟基-间阿散酸（N-AHPAA），其也用作治疗难治性细菌性阴道病的临床药物。本文报道了肠杆菌CZ-1菌株中编码N-羟基芳胺O-乙酰基转移酶的两个基因NhoA 1和NhoA 2的克隆和功能特性，这两个基因催化由ROX还原成N-AHPAA形成的3-氨基-4-羟基苯胂酸（3-AHPAA）的乙酰化。本研究为N-羟基芳胺O-乙酰基转移酶在重要有机砷化合物转化中的作用提供了新的认识。摘要有机砷饲料添加剂4-羟基-3-硝基苯胂酸（roxarsone [ROX]）被广泛使用并释放到环境中。我们先前展示了肠杆菌属菌株CZ-1转化ROX的两步途径，包括将ROX还原为3-氨基-4-羟基苯胂酸（3-AHPAA）和将3-AHPAA乙酰化为N-乙酰基-4-羟基-间阿散酸（N-AHPAA）（K. Huang，H. Peng，F.高角，加-地Liu等人，Environ Pollut 247：482-487，2019，https：//doi.org/10.1016/j.envpol.2019.01.076）。在这项研究中，我们确定了两个nhoA基因（nhoA 1和nhoA 2），编码N-羟基芳胺O-乙酰转移酶，负责3-AHPAA乙酰化肠杆菌属菌株CZ-1。遗传破坏和互补的结果表明，这两个nhoA基因参与ROX的生物转化和nhoA 1是主要的3-AHPAA乙酰转移酶基因。定量逆转录-PCR分析表明，nhoA 1的相对表达水平比nhoA 2高3倍。每种重组NhoAs在大肠杆菌BL 21中过表达，并通过亲和层析作为二聚体均匀纯化。两种纯化的NhoAs都催化乙酰辅酶A依赖的3-AHPAA乙酰化。3-AHPAA对NhoA 1和NhoA 2的Km值分别为151.5和428.3 μM。定点突变实验表明，每个NhoA的两个保守的精氨酸和半胱氨酸残基是必要的酶活性。Roxarsone（ROX）是一种有机砷饲料添加剂，70多年来一直广泛用于家禽业，用于促进生长、控制球虫病和改善肉色。大多数ROX被排泄在垃圾中并分散到环境中，在那里它被微生物转化为不同的含砷化合物。ROX转化的主要产物是N-乙酰基-4-羟基-间阿散酸（N-AHPAA），其也用作治疗难治性细菌性阴道病的临床药物。本文报道了肠杆菌CZ-1菌株中编码N-羟基芳胺O-乙酰基转移酶的两个基因NhoA 1和NhoA 2的克隆和功能特性，这两个基因催化由ROX还原成N-AHPAA形成的3-氨基-4-羟基苯胂酸（3-AHPAA）的乙酰化。本研究为N-羟基芳胺O-乙酰基转移酶在重要有机砷化合物转化中的作用提供了新的认识。
Roxarsone (ROX) is an organoarsenic feed additive that has been widely used in poultry industries for growth promotion, coccidiosis control, and meat pigmentation improvement for more than 70 years. Most ROX is excreted in the litter and dispersed into the environment, where it is transformed by microbes into different arsenic-containing compounds. A major product of ROX transformation is N-acetyl-4-hydroxy-m-arsanilic acid (N-AHPAA), which is also used as a clinical drug for treating refractory bacterial vaginosis. Here, we report the cloning and functional characterization of two genes encoding N-hydroxyarylamine O-acetyltransferases, NhoA1 and NhoA2, in Enterobacter sp. strain CZ-1, which catalyze the acetylation of 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA) formed by the reduction of ROX to N-AHPAA. This study provides new insights into the function of N-hydroxyarylamine O-acetyltransferase in the transformation of an important organoarsenic compound. ABSTRACT The organoarsenical feed additive 4-hydroxy-3-nitrobenzenearsonic acid (roxarsone [ROX]) is widely used and released into the environment. We previously showed a two-step pathway of ROX transformation by Enterobacter sp. strain CZ-1 involving the reduction of ROX to 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA) and the acetylation of 3-AHPAA to N-acetyl-4-hydroxy-m-arsanilic acid (N-AHPAA) (K. Huang, H. Peng, F. Gao, Q. Liu, et al., Environ Pollut 247:482–487, 2019, https://doi.org/10.1016/j.envpol.2019.01.076). In this study, we identified two nhoA genes (nhoA1 and nhoA2), encoding N-hydroxyarylamine O-acetyltransferases, as responsible for 3-AHPAA acetylation in Enterobacter sp. strain CZ-1. The results of genetic disruption and complementation showed that both nhoA genes are involved in ROX biotransformation and that nhoA1 is the major 3-AHPAA acetyltransferase gene. Quantitative reverse transcription-PCR analysis showed that the relative expression level of nhoA1 was 3-fold higher than that of nhoA2. Each of the recombinant NhoAs was overexpressed in Escherichia coli BL21 and homogenously purified as a dimer by affinity chromatography. Both purified NhoAs catalyzed acetyl coenzyme A-dependent 3-AHPAA acetylation. The Km values of 3-AHPAA for NhoA1 and NhoA2 were 151.5 and 428.3 μM, respectively. Site-directed mutagenesis experiments indicated that two conserved arginine and cysteine residues of each NhoA were necessary for their enzyme activities. IMPORTANCE Roxarsone (ROX) is an organoarsenic feed additive that has been widely used in poultry industries for growth promotion, coccidiosis control, and meat pigmentation improvement for more than 70 years. Most ROX is excreted in the litter and dispersed into the environment, where it is transformed by microbes into different arsenic-containing compounds. A major product of ROX transformation is N-acetyl-4-hydroxy-m-arsanilic acid (N-AHPAA), which is also used as a clinical drug for treating refractory bacterial vaginosis. Here, we report the cloning and functional characterization of two genes encoding N-hydroxyarylamine O-acetyltransferases, NhoA1 and NhoA2, in Enterobacter sp. strain CZ-1, which catalyze the acetylation of 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA) formed by the reduction of ROX to N-AHPAA. This study provides new insights into the function of N-hydroxyarylamine O-acetyltransferase in the transformation of an important organoarsenic compound.
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DOI：
10.1002/anie.201700736
发表时间：
2017-06-06
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
--
作者：
Peng H;Hu B;Liu Q;Li J;Li XF;Zhang H;Le XC
通讯作者：
Le XC
DOI：
10.1196/annals.1419.006
发表时间：
2008-01-01
期刊：
INCREDIBLE ANAEROBES: FROM PHYSIOLOGY TO GENOMICS TO FUELS
影响因子：
--
作者：
Fisher, Edward;Dawson, Asia M.;Stolz, John F.
通讯作者：
Stolz, John F.
影响因子：
--
作者：
W. Weber;C. King
通讯作者：
W. Weber;C. King
影响因子：
--
作者：
W. Weber;C. King
通讯作者：
W. Weber;C. King
DOI：
10.1038/76783
发表时间：
2000-07
期刊：
Nature Structural Biology
影响因子：
--
作者：
J. Sinclair;J. Sandy;R. Delgoda;E. Sim;M. Noble
通讯作者：
J. Sinclair;J. Sandy;R. Delgoda;E. Sim;M. Noble