Dittmann J, Wenger R M, Kleinkauf H, Lawen A
Institut für Biochemie und Molekulare Biologie, Technische Universität Berlin, Federal Republic of Germany.
J Biol Chem. 1994 Jan 28;269(4):2841-6.
Cyclosporin A is synthesized by cyclosporin synthetase, a multienzyme polypeptide. This enzyme catalyzes at least 40 reaction steps in an assembly belt-like mechanism. It activates all constituent amino acids of cyclosporin A to thioesters via amino acyladenylates and carries out specific N-methylation reactions. During elongation, the activated amino acids are linked by peptide bonds leading to enzyme-bound nascent peptide chains. Some of the linear peptides of the growing cyclosporin A chain were isolated and their N-terminal amino acid was determined. D-Alanine at position 8 of the cyclosporin A molecule was found to be a starting amino acid in the biosynthetic process of cyclosporin A formation. Four intermediate peptides of the growing peptide chain of cyclosporin A could be isolated and identified. All of them represent partial sequences of cyclosporin A starting with D-alanine. That these intermediate peptides were bound by thioester linkage to cyclosporin synthetase could be demonstrated by liberation of the peptides with performic acid. The peptides strongly suggest the stepwise synthesis of a single linear peptide precursor of cyclosporin A.
环孢素A由环孢素合成酶合成，环孢素合成酶是一种多酶多肽。该酶通过类似装配带的机制催化至少40个反应步骤。它通过氨基酰腺苷酸将环孢素A的所有组成氨基酸激活为硫酯，并进行特定的N-甲基化反应。在延伸过程中，活化的氨基酸通过肽键连接，形成与酶结合的新生肽链。分离出了一些正在生长的环孢素A链的线性肽，并确定了它们的N端氨基酸。发现环孢素A分子第8位的D-丙氨酸是环孢素A生物合成过程中的起始氨基酸。可以分离和鉴定环孢素A生长肽链的四个中间肽。它们都代表以D-丙氨酸开头的环孢素A的部分序列。用过甲酸释放肽可以证明这些中间肽通过硫酯键与环孢素合成酶结合。这些肽有力地表明环孢素A的单一线性肽前体是逐步合成的。
