Construction of modified ribosomes for incorporation of D-amino acids into proteins
Construction of modified ribosomes for incorporation of D-amino acids into proteins
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DOI：
10.1021/bi060986a
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发表时间：
2006-12-26
期刊：
影响因子：
2.9
通讯作者：
Hecht, Sidney M.
中科院分区：
文献类型：
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作者：
Dedkova, Larisa M.;Fahmi, Nour Eddine;Hecht, Sidney M.
虽然许多生物活性肽含有 D-氨基酸，但此类物质的精制并非通过核糖体合成进行。事实上，细菌核糖体强烈反对 D-氨酰基-tRNA 中 D-氨基酸的掺入。为了允许使用体外蛋白质合成系统将 D-氨基酸掺入蛋白质中，已经开发了一种策略来制备修饰的核糖体，该核糖体在肽基转移酶中心和 23S rRNA 的螺旋 89 内含有改变。发现源自含有具有改变的 23S rRNA 的核糖体的集落的 S-30 制剂对 D-氨基酸具有增强的耐受性，并允许在预定位点加工含有 D-氨基酸的蛋白质。将大肠杆菌二氢叶酸还原酶和萤火虫荧光素酶中的5个特定氨基酸替换为D-苯丙氨酸和D-蛋氨酸，并测定所得酶的比活性。
While numerous biologically active peptides contain D-amino acids, the elaboration of such species is not carried out by ribosomal synthesis. In fact, the bacterial ribosome discriminates strongly against the incorporation of D-amino acids from D-aminoacyl-tRNAs. To permit the incorporation of D-amino acids into proteins using in vitro protein-synthesizing systems, a strategy has been developed to prepare modified ribosomes containing alterations within the peptidyltransferase center and helix 89 of 23S rRNA. S-30 preparations derived from colonies shown to contain ribosomes with altered 23S rRNAs were found to exhibit enhanced tolerance for D-amino acids and to permit the elaboration of proteins containing D-amino acids at predetermined sites. Five specific amino acids in Escherichia coli dihydrofolate reductase and Photinus pyralis luciferase were replaced with D-phenylalanine and D-methionine, and the specific activities of the resulting enzymes were determined.