A new type of aminoacyltransferase from Saccharothrix sp. AS-2 favorable for the synthesis of D-amino acid-containing peptides.
A new type of aminoacyltransferase from Saccharothrix sp. AS-2 favorable for the synthesis of D-amino acid-containing peptides.
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来自糖丝菌属的新型氨酰基转移酶。
DOI：
10.1093/oxfordjournals.jbchem.a003095
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发表时间：
2002
影响因子：
2.7
通讯作者：
Y. Tominaga
中科院分区：
文献类型：
--
作者：
A. Sugihara;Y. Shimada;S. Sugihara;T. Nakai;T. Kakuno;T. Nagao;Yomi Watanabe;Y. Tominaga
从嗜铬菌AS-2的发酵液中分离纯化了一种具有某些性质的独特的D-氨基酸多肽合成酶。纯化步骤包括硫酸铵分级分离、CM-Toyopolymer 650 M和ProtEx Butyl上的色谱以及蔗糖密度梯度等电聚焦。该酶由四个56 kDa的亚基组成，在pH 8.2和35 ℃左右显示出最大的转移活性，等电点为5.8。该酶从D-Asp（OMe）、D-Met、D-Phe、D-Trp、D-Tyr和L-Glu（OMe）的甲酯产生同源寡聚物，但对所测试的任何D-或L-氨基酸甲酯均不显示水解活性。同源寡聚体不是由相应的游离氨基酸形成的。Ac-D-Phe-OMe与DL-Ala-NH（2）、DL-Leu-NH（2）、DL-Phe-NH（2）或DL-Trp-NH（2）的反应被该酶有效地催化，产生预期的N-乙酰二肽的DD-和DL-立体异构体。所得二肽即使在48小时孵育后仍保持未水解。此外，它对酪蛋白、diAla、diMet和diPhe的非对映体、D-/L-氨基酸酰胺或D-/L-氨基酸对硝基苯胺没有可检测的水解活性，表明该酶没有导致生长肽的二次水解的肽酶活性。该酶的活性被苯甲磺酰氟强烈抑制，但不是由青霉素G或氨苄青霉素，这表明该蛋白质是一种丝氨酸酶缺乏青霉素结合能力。这些观察结果使我们得出结论，该酶的特征在于在本研究中从藻属物种AS-2是一种新型的氨酰基转移酶的氨基酸酯作为酰基供体，并具有潜在的效用作为催化剂的合成D-氨基酸的肽。
A unique enzyme with some properties favorable for the synthesis of D-amino acid-containing peptides has been purified from the culture broth of Saccharothrix sp. AS-2. The purification steps included ammonium sulfate fractionation, chromatographies on CM-Toyopearl 650M and ProtEx Butyl, and sucrose density-gradient isoelectric focusing. The enzyme, consisting of four subunits of 56 kDa, showed its maximum transfer activity at around pH 8.2 and 35 degrees C, and had an isoelectric point of 5.8. The enzyme yielded homooligomers from methyl esters of D-Asp(OMe), D-Met, D-Phe, D-Trp, D-Tyr, and L-Glu(OMe), but showed no hydrolytic activity toward any of the D- or L-amino acid methyl esters tested. The homooligomers were not formed from the corresponding free amino acids. The reaction of Ac-D-Phe-OMe with DL-Ala-NH(2), DL-Leu-NH(2), DL-Phe-NH(2), or DL-Trp-NH(2) was effectively catalyzed by the enzyme, both the DD- and DL-stereoisomers of the expected N-acetyldipeptide being yielded. The resulting dipeptides remained unhydrolyzed even after 48 h incubation. Also, it showed no detectable hydrolytic activity toward casein, diastereomers of diAla, diMet, and diPhe, D-/L-amino acid amides, or D-/L-amino acid p-nitroanilides, indicating that the enzyme had no peptidase activity leading to secondary hydrolysis of the growing peptide. The enzyme activity was strongly depressed by phenylmethanesulfonyl fluoride, but not by penicillin G or ampicillin, suggesting that the protein is a serine enzyme lacking penicillin-binding ability. These observations lead us to the conclusion that the enzyme from Saccharothrix sp. AS-2 characterized in this study is a new type of aminoacyltransferase with an amino acid ester as the acyl donor, and has potential utility as a catalyst for the synthesis of D-amino acid-containing peptides.