ywfE in Bacillus subtilis codes for a novel enzyme, L-amino acid ligase
ywfE in Bacillus subtilis codes for a novel enzyme, L-amino acid ligase
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DOI：
10.1128/jb.187.15.5195-5202.2005
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发表时间：
2005-08-01
影响因子：
3.2
通讯作者：
Hashimoto, S
中科院分区：
文献类型：
--
作者：
Tabata, K;Ikeda, H;Hashimoto, S
已知ATP依赖性羧酸-胺/硫醇连接酶超家族含有催化各种类型的肽（如D-丙氨酰-D-丙氨酸、聚谷氨酸和γ-肽）形成的酶，但奇怪的是，没有已知合成L-氨基酸的α-二肽的酶。我们试图找到这样一种酶。通过基于超家族的共有序列的计算机筛选，随后用纯化的酶进行体外测定以避免合成的肽的降解，发现枯草芽孢杆菌的ywfE编码由L-丙氨酸和L-谷氨酰胺形成L-丙氨酰-L-谷氨酰胺的活性，其中ATP水解为ADP。没有形成AMP，支持这种酶属于超家族的观点。令人惊讶的是，这种酶能接受多种L-氨基酸。在231种L-氨基酸组合中，有111种组合得到了反应产物，经高效液相色谱分析确认了44种α-二肽，而没有检测到三肽或更长的肽，D-氨基酸是惰性的。从这些结果，我们建议，ywjE编码的超家族，L-氨基酸连接酶的新成员。
The ATP-dependent carboxylate-amine/thiol ligase superfamily is known to contain enzymes catalyzing the formation of various types of peptide, such as D-alanyl-D-alanine, polyglutamate, and gamma-peptide, but, curiously, no enzyme synthesizing alpha-dipeptides of L-amino acids is known. We attempted to find such an enzyme. By in silico screening based on the consensus sequence of the superfamily followed by an in vitro assay with purified enzyme to avoid the degradation of the peptide(s) synthesized, ywfE of Bacillus subtilis was found to code for the activity forming L-alanyl-L-glutamine from L-alanine and L-glutamine with hydrolysis of ATP to ADP. No AMP was formed, supporting the idea that the enzyme belongs to the superfamily. Surprisingly, the enzyme accepted a wide variety Of L-amino acids. Among 231 combinations of L-amino acids tested, reaction products were obtained for 111 combinations and 44 kinds of alpha-dipeptides were confirmed by high-performance liquid chromatography analyses, while no tripeptide or longer peptide was detected and the D-amino acids were inert. From these results, we propose that ywjE encodes a new member of the superfamily, L-amino acid ligase.