Maillard Julien, Schumacher Wolfram, Vazquez Francisco, Regeard Christophe, Hagen Wilfred R, Holliger Christof
ENAC-Laboratory for Environmental Biotechnology, Swiss Federal Institute of Technology (EPFL) CH-1015 Lausanne, The Netherlands.
Appl Environ Microbiol. 2003 Aug;69(8):4628-38. doi: 10.1128/AEM.69.8.4628-4638.2003.
The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of Dehalobacter restrictus, was purified 25-fold to apparent electrophoretic homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 60 +/- 1 kDa, whereas the native molecular mass was 71 +/- 8 kDa according to size exclusion chromatography in the presence of the detergent octyl-beta-D-glucopyranoside. The monomeric enzyme contained (per mol of the 60-kDa subunit) 1.0 +/- 0.1 mol of cobalamin, 0.6 +/- 0.02 mol of cobalt, 7.1 +/- 0.6 mol of iron, and 5.8 +/- 0.5 mol of acid-labile sulfur. Purified PceA catalyzed the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with a specific activity of 250 +/- 12 nkat/mg of protein. In addition, several chloroethanes and tetrachloromethane caused methyl viologen oxidation in the presence of PceA. The K(m) values for tetrachloroethene, trichloroethene, and methyl viologen were 20.4 +/- 3.2, 23.7 +/- 5.2, and 47 +/- 10 micro M, respectively. The PceA exhibited the highest activity at pH 8.1 and was oxygen sensitive, with a half-life of activity of 280 min upon exposure to air. Based on the almost identical N-terminal amino acid sequences of PceA of Dehalobacter restrictus, Desulfitobacterium hafniense strain TCE1 (formerly Desulfitobacterium frappieri strain TCE1), and Desulfitobacterium hafniense strain PCE-S (formerly Desulfitobacterium frappieri strain PCE-S), the pceA genes of the first two organisms were cloned and sequenced. Together with the pceA genes of Desulfitobacterium hafniense strains PCE-S and Y51, the pceA genes of Desulfitobacterium hafniense strain TCE1 and Dehalobacter restrictus form a coherent group of reductive dehalogenases with almost 100% sequence identity. Also, the pceB genes, which may code for a membrane anchor protein of PceA, and the intergenic regions of Dehalobacter restrictus and the three desulfitobacteria had identical sequences. Whereas the cprB (chlorophenol reductive dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always located upstream of cprA, all pceB genes known so far are located downstream of pceA. The possible consequences of this feature for the annotation of putative reductive dehalogenase genes are discussed, as are the sequence around the iron-sulfur cluster binding motifs and the type of iron-sulfur clusters of the reductive dehalogenases of Dehalobacter restrictus and Desulfitobacterium dehalogenans identified by electron paramagnetic resonance spectroscopy.
膜结合四氯乙烯还原脱卤酶（PCE-RDase）（PceA；EC 1.97.1.8）是限制脱卤杆菌呼吸链的末端组分，经纯化后达到表观电泳均一性，纯化倍数为25倍。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示有一条单一的条带，表观分子量为60±1 kDa，而在去污剂辛基-β-D-吡喃葡萄糖苷存在下，根据尺寸排阻色谱法测定其天然分子量为71±8 kDa。单体酶（每摩尔60 kDa亚基）含有1.0±0.1摩尔钴胺素、0.6±0.02摩尔钴、7.1±0.6摩尔铁和5.8±0.5摩尔酸不稳定硫。纯化的PceA催化四氯乙烯和三氯乙烯还原脱氯生成顺式-1,2-二氯乙烯，比活性为250±12 nkat/mg蛋白质。此外，几种氯乙烷和四氯化碳在PceA存在下会引起甲基紫精氧化。四氯乙烯、三氯乙烯和甲基紫精的K(m)值分别为20.4±3.2、23.7±5.2和47±10 μM。PceA在pH 8.1时表现出最高活性，对氧气敏感，暴露于空气中时活性半衰期为280分钟。基于限制脱卤杆菌、哈夫尼脱硫肠杆菌菌株TCE1（以前的弗氏脱硫肠杆菌菌株TCE1）和哈夫尼脱硫肠杆菌菌株PCE-S（以前的弗氏脱硫肠杆菌菌株PCE-S）的PceA几乎相同的N端氨基酸序列，克隆并测序了前两种生物的pceA基因。与哈夫尼脱硫肠杆菌菌株PCE-S和Y51的pceA基因一起，哈夫尼脱硫肠杆菌菌株TCE1和限制脱卤杆菌的pceA基因形成了一组具有几乎100%序列同一性的还原脱卤酶。此外，可能编码PceA膜锚定蛋白的pceB基因以及限制脱卤杆菌和三种脱硫肠杆菌的基因间区域具有相同的序列。虽然氯酚脱卤呼吸细菌的cprB（氯酚还原脱卤酶）基因总是位于cprA的上游，但迄今为止已知的所有pceB基因都位于pceA的下游。讨论了这一特征对推定还原脱卤酶基因注释的可能影响，以及通过电子顺磁共振光谱鉴定的限制脱卤杆菌和脱卤脱硫肠杆菌还原脱卤酶的铁硫簇结合基序周围的序列和铁硫簇类型。
