A novel R-stereoselective amidase from Pseudomonas sp MCI3434 acting on piperazine-2-tert-butylcarboxamide
A novel R-stereoselective amidase from Pseudomonas sp MCI3434 acting on piperazine-2-tert-butylcarboxamide
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DOI：
10.1111/j.1432-1033.2004.04069.x
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发表时间：
2004-04-01
期刊：
影响因子：
--
通讯作者：
Asano, Y
中科院分区：
文献类型：
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作者：
Komeda, H;Harada, H;Asano, Y
从假单胞菌中分离纯化了一种作用于(R，S)-哌嗪-2-叔丁基甲酰胺的新型酰胺酶。MCI3434，并对其进行了表征。该酶与(R，S)-哌嗪-2-叔丁基甲酰胺发生R-立体选择性作用，生成(R)-哌嗪-2-羧酸，暂定名为R-酰胺酶。该酶的N端氨基酸序列与编码铜绿假单胞菌PAO1假想水解酶的基因PA3598具有较高的同源性。从假单胞菌基因组DNA中克隆了R-酰胺酶基因。MCI3434，并测序。对基因组DNA的1332bp分析表明，存在一个编码R-酰胺酶的开放阅读框架(RAMA)。该酶由274个氨基酸残基组成(相对分子质量为30128 Da)，其推导的氨基酸序列与恶臭假单胞菌KT2440的碳氮水解酶蛋白(PP3846)和铜绿假单胞菌PAO1的PA3598蛋白(同源性分别为72.6%和65.6%)具有较高的同源性，属于碳氮水解酶家族中一个新的亚家族。高效表达RAMA基因的大肠埃希菌无细胞提取液上清液中的R-酰胺酶含量约为假单胞菌的3万倍。MCI3434。该转化子的完整细胞可用于外消旋哌嗪-2-叔丁基甲酰胺的R-立体选择性水解。从高效表达RAMA基因的大肠杆菌转化子的无细胞提取液中，将重组酶纯化为均一的电泳酶。在凝胶过滤层析中，该酶为单体。该酶在45℃、pH 8.0时活性最高，在对氯苯甲酸汞、N-乙基马来酰亚胺、Mn2+、Co2+、Ni2+、Cu2+、Zn2+、Ag+、Cd2+、Hg2+或Pb2+存在下完全失活。RAMA对氨基或亚氨基连接到β-或γ-碳上的羧胺类化合物，如β-丙氨酰胺、(R)-哌嗪-2-羧胺(R)-哌啶-3-甲酰胺、D-谷氨酰胺和(R)-哌嗪-2-叔丁基甲酰胺具有水解活性。然而，尽管该酶的序列与RAMA相似，但它不作用于脂肪酰胺酶的其他酰胺底物。
A novel amidase acting on (R,S)-piperazine-2-tert-butylcarboxamide was purified from Pseudomonas sp. MCI3434 and characterized. The enzyme acted R-stereoselectively on (R,S)-piperazine-2-tert-butylcarboxamide to yield (R)-piperazine-2-carboxylic acid, and was tentatively named R-amidase. The N-terminal amino acid sequence of the enzyme showed high sequence identity with that deduced from a gene named PA3598 encoding a hypothetical hydrolase in Pseudomonas aeruginosa PAO1. The gene encoding R-amidase was cloned from the genomic DNA of Pseudomonas sp. MCI3434 and sequenced. Analysis of 1332 bp of the genomic DNA revealed the presence of one open reading frame (ramA) which encodes the R-amidase. This enzyme, RamA, is composed of 274 amino acid residues (molecular mass, 30 128 Da), and the deduced amino acid sequence exhibits homology to a carbon-nitrogen hydrolase protein (PP3846) from Pseudomonas putida strain KT2440 (72.6% identity) and PA3598 protein from P. aeruginosa strain PAO1 (65.6% identity) and may be classified into a new subfamily in the carbon-nitrogen hydrolase family consisting of aliphatic amidase, beta-ureidopropionase, carbamylase, nitrilase, and so on. The amount of R-amidase in the supernatant of the sonicated cell-free extract of an Escherichia coli transformant overexpressing the ramA gene was about 30 000 times higher than that of Pseudomonas sp. MCI3434. The intact cells of the E. coli transformant could be used for the R-stereoselective hydrolysis of racemic piperazine-2-tert-butylcarboxamide. The recombinant enzyme was purified to electrophoretic homogeneity from cell-free extract of the E. coli transformant overexpressing the ramA gene. On gel-filtration chromatography, the enzyme appeared to be a monomer. It had maximal activity at 45 degreesC and pH 8.0, and was completely inactivated in the presence of p-chloromercuribenzoate, N-ethylmaleimide, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Ag+, Cd2+, Hg2+ or Pb2+. RamA had hydrolyzing activity toward the carboxamide compounds, in which amino or imino group is connected to beta- or gamma-carbon, such as beta-alaninamide, (R)-piperazine-2-carboxamide (R)-piperidine-3-carboxamide, D-glutaminamide and (R)-piperazine-2-tert-butylcarboxamide. The enzyme, however, did not act on the other amide substrates for the aliphatic amidase despite its sequence similarity to RamA.