Purification, cloning, and sequencing of a 3,5-dichlorophenol reductive dehalogenase from Desulfitobacterium frappieri PCP-1
Purification, cloning, and sequencing of a 3,5-dichlorophenol reductive dehalogenase from Desulfitobacterium frappieri PCP-1
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DOI：
10.1128/aem.70.8.4532-4537.2004
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发表时间：
2004-08-01
影响因子：
4.4
通讯作者：
Beaudet, R
中科院分区：
文献类型：
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作者：
Thibodeau, J;Gauthier, A;Beaudet, R
从 Desulfitobacter frappieri PCP-1 中分离出膜相关的 3,5-二氯苯酚还原脱卤酶。在 22°C 培养的生物量中观察到最高的脱卤酶活性，而在 30°C 和 37°C 培养的细胞悬浮液的活性分别低 2.2 倍和 9.6 倍。还原性脱卤酶纯化 12.7 倍，达到明显的同质性。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示出一条表观分子量为 57 kDa 的单条带。其脱氯活性不受硫酸盐和硝酸盐抑制，但被2.5 mM亚硫酸盐和10 mM KCN完全抑制。碘丙烷和柠檬酸钛的混合物引起脱氯活性的光可逆抑制，表明类咕啉辅助因子的参与。几种多氯酚在间位和对位脱氯。在甲基紫精浓度为 2 mM 时，3,5-二氯苯酚的表观 K-m 为 49.3 +/- 3.1 muM。通过质谱法对六种内部胰蛋白酶肽进行测序。在含有这些肽序列的哈夫尼脱硫杆菌基因组中发现了一个开放阅读框（ORF）。该ORF对应于编码CprA型还原脱卤酶的基因。克隆并测序了 D. frappieri PCP-1 中相应的 ORF（命名为 cprA5）。 cprA5 基因编码 548 个氨基酸的蛋白质，其中包含双精氨酸型分泌信号。该基因产物具有一个钴胺素结合位点基序和两个铁硫结合基序，并与一些四氯乙烯还原脱卤酶表现出 66% 的同一性（76% 至 77% 相似性）。这是第一个可以在间位和对位脱氯氯酚的 CprA 型还原脱卤酶。
A membrane-associated 3,5-dichlorophenol reductive dehalogenase was isolated from Desulfitobacterium frappieri PCP-1. The highest dehalogenase activity was observed with the biomass cultured at 22degreesC, compared to 30 and 37degreesC, where the cell suspensions were 2.2 and 9.6 times less active, respectively. The reductive dehalogenase was purified 12.7-fold to apparent homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 57 kDa. Its dechlorinating activity was not inhibited by sulfate and nitrate but was completely inhibited by 2.5 mM sulfite and 10 mM KCN. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activities, suggesting the involvement of a corrinoid cofactor. Several polychlorophenols were dechlorinated at the meta and para positions. The apparent K-m for 3,5-dicholorophenol was 49.3 +/- 3.1 muM at a methyl viologen concentration of 2 mM. Six internal tryptic peptides were sequenced by mass spectrometry. One open reading frame (ORF) was found in the Desulfitobacterium hafniense genome containing these peptide sequences. This ORF corresponds to a gene coding for a CprA-type reductive dehalogenase. The corresponding ORF (named cprA5) in D. frappieri PCP-1 was cloned and sequenced. The cprA5 gene codes for a 548-amino-acid protein that contains a twin-arginine-type signal for secretion. The gene product has a cobalamin binding site motif and two iron-sulfur binding motifs and shows 66% identity (76 to 77% similarity) with some tetrachloroethene reductive dehalogenases. This is the first CprA-type reductive dehalogenase that can dechlorinate chlorophenols at the meta and para positions.