Schulman M D, Acton S L, Valentino D L, Arison B H
Department of Fermentation Microbiology, Merck Sharpe & Dohme Research Laboratories, Rahway, New Jersey 07065.
J Biol Chem. 1990 Oct 5;265(28):16965-70.
The nucleotide sugar precursor of the oleandrose units of the avermectins has been purified from a mutant of Streptomyces avermitilis, which does not synthesize any avermectins but which converts avermectin aglycones to their respective disaccharides. This precursor has been identified as dTDP-oleandrose. The purification was achieved by anion exchange and reverse phase high performance liquid chromatography. The purified nucleotide sugar had an absorption spectra characteristic of thymidine, released dTMP when treated with phosphodiesterase, and possessed an NMR spectrum in which three resonances characteristic of oleandrose were seen in addition to the thymidine signals. The enzyme, avermectin aglycone dTDP-oleandrose glycosyltransferase, which catalyzes the stepwise addition of oleandrose to the avermectin aglycones, has been demonstrated in cell-free extracts and (NH4)2SO4 fractions of cell-free extracts of S. avermitilis. The enzyme is specific for dTDP-oleandrose as the glycosyl donor but utilizes all avermectin aglycones as glycosyl acceptors. The stoichiometry between dTDP-oleandrose consumed in the reaction and oleandrose units transferred to the avermectin mono- and disaccharide was found to be 1:1.
阿维菌素中夹竹桃糖单元的核苷酸糖前体已从阿维链霉菌的一个突变体中纯化出来，该突变体不合成任何阿维菌素，但能将阿维菌素苷元转化为各自的二糖。这种前体已被鉴定为dTDP - 夹竹桃糖。纯化是通过阴离子交换和反相高效液相色谱法实现的。纯化后的核苷酸糖具有胸腺嘧啶核苷的吸收光谱特征，用磷酸二酯酶处理时会释放出dTMP，并且其核磁共振谱除了胸腺嘧啶核苷信号外，还出现了夹竹桃糖的三个特征共振峰。催化夹竹桃糖逐步添加到阿维菌素苷元上的酶——阿维菌素苷元dTDP - 夹竹桃糖糖基转移酶，已在阿维链霉菌的无细胞提取物和无细胞提取物的硫酸铵分级分离物中得到证实。该酶对作为糖基供体的dTDP - 夹竹桃糖具有特异性，但利用所有阿维菌素苷元作为糖基受体。发现反应中消耗的dTDP - 夹竹桃糖与转移到阿维菌素单糖和二糖上的夹竹桃糖单元之间的化学计量比为1:1。
