Abstract

Phenylobacterium immobile strain E is a soil bacterium with a striking metabolism relying on xenobiotics, such as the herbicide pyrazon, as sole carbon source instead of more bioavailable molecules. Pyrazon is a heterocyclic aromatic compound of environmental concern and its biodegradation pathway has only been reported in P. immobile. The multicomponent pyrazon oxygenase (PPO), a Rieske non-heme iron oxygenase, incorporates molecular oxygen at the 2,3 position of the pyrazon phenyl moiety as first step of degradation, generating a cis-dihydrodiendiol. The aim of this work was to identify the genes encoding for each one of the PPO components and enable their functional assembly in Escherichia coli. P. immobile strain E genome sequencing revealed genes encoding for RO components, such as ferredoxin-, reductase-, α- and β-subunits of an oxygenase. Though, P. immobile E displays three prominent differences with respect to the ROs currently characterized: (1) an operon-like organization for PPO is absent, (2) all the elements are randomly scattered in its DNA, (3) not only one, but 19 different α-subunits are encoded in its genome. Herein, we report the identification of the PPO components involved in pyrazon cis-dihydroxylation in P. immobile, its appropriate assembly, and its functional reconstitution in E. coli. Our results contributes with the essential missing pieces to complete the overall elucidation of the PPO from P. immobile.

Key points

• Phenylobacterium immobile E DSM 1986 harbors the only described pyrazon oxygenase (PPO).

• We elucidated the genes encoding for all PPO components.

• Heterologous expression of PPO enabled pyrazon dihydroxylation in E. coli JW5510.

中文翻译：

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由固定不动的苯基细菌E DSM 1986制成的Rieske非血红素铁加氧酶多组分系统使大肠杆菌中的吡唑酮顺式-二羟基化成为可能

摘要

苯氧不动菌菌株E是一种土壤细菌，具有惊人的新陈代谢，它依赖于诸如除草剂吡嗪等异生物作为唯一的碳源，而不是更多的可生物利用分子。吡唑啉（Pyrazon）是一种涉及环境的杂环芳族化合物，其生物降解途径仅在不动毕赤酵母中有报道。作为降解的第一步，多组分吡唑酮加氧酶（PPO）是一种Rieske非血红素铁加氧酶，在吡唑酮苯基部分的2,3位掺入分子氧，生成顺式-二氢二烯二醇。这项工作的目的是确定编码每个PPO成分的基因，并使它们在大肠杆菌中进行功能装配。不动产菌株E基因组测序揭示了编码RO成分的基因，例如氧化酶的铁氧还蛋白，还原酶，α和β亚基。虽然，固定不动杆菌E与目前表征的RO表现出三个显着差异：（1）缺少PPO的类似操纵子的组织，（2）所有元素随机散布在其DNA中，（3）不仅一个，但是在其基因组中编码了19个不同的α亚基。在这里，我们报告鉴定参与体育不动杆菌吡嗪顺式-二羟基化的PPO成分，其适当的组装和其在大肠杆菌中的功能重构。我们的结果有助于完成一些必要的遗漏，以完成对PPO的整体说明。不可移动。

关键点

•苯丙酮酸不动菌E DSM 1986包含唯一描述的吡唑酮加氧酶（PPO）。

•我们阐明了编码所有PPO成分的基因。

•PPO的异源表达使大肠杆菌JW5510中的吡嗪二羟基化成为可能。