2-Haloacrylate reductase, a novel enzyme of the medium chain dehydrogenase/reductase superfamily that catalyzes the reduction of a carbon-carbon double bond of unsaturated organohalogen compounds
2-Haloacrylate reductase, a novel enzyme of the medium chain dehydrogenase/reductase superfamily that catalyzes the reduction of a carbon-carbon double bond of unsaturated organohalogen compounds
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DOI：
10.1074/jbc.m414605200
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发表时间：
2005-05-27
影响因子：
4.8
通讯作者：
Esaki, N
中科院分区：
文献类型：
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作者：
Kurata, A;Kurihara, T;Esaki, N
一种土壤细菌Burkholderia sp. WS生长在2-氯丙烯酸酯作为唯一碳源上。为了鉴定代谢2-氯丙烯酸酯的酶，我们对2-氯丙烯酸酯和乳酸盐生长的细菌细胞的蛋白质进行了比较性的二维凝胶电泳。结果发现，当细胞生长在2-氯丙烯酸酯上时，可诱导合成一种名为CAA 43的蛋白。CAA 43基因被克隆并显示编码333个氨基酸残基（M-r 35，788）的蛋白质，其与来自大肠杆菌的NADPH依赖性醌氧化还原酶（38.2%同一性）具有显著的序列相似性。CAA 43在E. coli中，并纯化至均一。纯化的蛋白质催化NADPH依赖性的2-氯丙烯酸酯的碳-碳双键还原生成（S）-2-氯丙酸酯，其可能进一步被Burkholderia sp. WS的（S）-2-卤酸脱卤酶代谢为（R）-乳酸。尽管与醌氧化还原酶的序列相似，但CAA 43不作用于1，4-苯醌和1，4-萘醌。2-氯代丙烯酸酯类似物，如丙烯酸酯和甲基丙烯酸酯，作为底物也是惰性的。相比之下，2-溴丙烯酸酯作为底物。因此，我们将这种新的酶命名为2-卤代丙烯酸酯还原酶。本研究为不饱和有机卤代物的降解提供了一条新的途径。还值得注意的是，该酶可用于生产（S）-2-氯丙酸酯，该产品用于芳氧苯氧丙酸除草剂的工业生产。
A soil bacterium, Burkholderia sp. WS, grows on 2-chloroacrylate as the sole carbon source. To identify the enzymes metabolizing 2-chloroacrylate, we carried out comparative two-dimensional gel electrophoresis of the proteins from 2-chloroacrylate- and lactate-grown bacterial cells. As a result, we found that a protein named CAA43 was inducibly synthesized when the cells were grown on 2-chloroacrylate. The CAA43 gene was cloned and shown to encode a protein of 333 amino acid residues (M-r 35,788) that shared a significant sequence similarity with NADPH-dependent quinone oxidoreductase from Escherichia coli (38.2% identity). CAA43 was overproduced in E. coli and purified to homogeneity. The purified protein catalyzed the NADPH-dependent reduction of the carbon-carbon double bond of 2-chloroacrylate to produce (S)-2-chloropropionate, which is probably further metabolized to (R)-lactate by (S)-2-haloacid dehalogenase in Burkholderia sp. WS. NADH did not serve as a reductant. Despite the sequence similarity to quinone oxidoreductases, CAA43 did not act on 1,4-benzoquinone and 1,4-naphthoquinone. 2-Chloroacrylate analogs, such as acrylate and methacrylate, were also inert as the substrates. In contrast, 2-bromoacrylate served as the substrate. Thus, we named this novel enzyme 2-haloacrylate reductase. This study revealed a new pathway for the degradation of unsaturated organohalogen compounds. It is also notable that the enzyme is useful for the production of (S)-2-chloropropionate, which is used for the industrial production of aryloxyphenoxypropionic acid herbicides.