Structure, mechanism and engineering of a nucleotidylyltransferase as a first step toward glycorandomization
Structure, mechanism and engineering of a nucleotidylyltransferase as a first step toward glycorandomization
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DOI：
10.1038/88618
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发表时间：
2001-06-01
期刊：
影响因子：
--
通讯作者：
Nikolov, DB
中科院分区：
文献类型：
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作者：
Barton, WA;Lesniak, J;Nikolov, DB
代谢物糖基化受三类酶的影响：核苷酸基转移酶，它激活糖作为核苷酸二磷酸衍生物，中间糖修饰酶和糖基转移酶。将最终衍生化的活性糖转移到糖醛酸底物。据报道，在2.0埃和2.1埃的分辨率下，沙门氏菌α - d -葡萄糖氨基酰基磷酸腺苷基转移酶（E-p）与产物（UDP-Glc）和底物（dTTP）的复合物，是负责该级联第一步的酶的第一个晶体结构之一。这些结构，结合E-p的动力学表征，阐明了这类重要酶的催化机理。E-p的结构工程产生了能够利用野生型E-p不接受的“非天然”磷酸糖的修饰酶。通过设计改变核苷酸基转移酶特异性的能力是为生产各种糖随机化文库而开发的体外糖基化系统的一个组成部分。
Metabolite glycosylation is affected by three classes of enzymes: nucleotidylyltransferases, which activate sugars as nucleotide diphospho-derivatives, intermediate sugar-modifying enzymes and glycosyltransferases. which transfer the final derivatized activated sugars to aglycon substrates. One of the first crystal structures of an enzyme responsible for the first step in this cascade, alpha -D-glucopyranosyl phosphate thymidylyltransferase (E-p) from Salmonella, in complex with product (UDP-Glc) and substrate (dTTP) is reported at 2.0 Angstrom and 2.1 Angstrom resolution, respectively. These structures, in conjunction with the kinetic characterization of E-p, clarify the catalytic mechanism of this important enzyme class. Structure-based engineering of E-p produced modified enzymes capable of utilizing 'unnatural' sugar phosphates not accepted by wild type E-p. The demonstrated ability to alter nucleotidylyltransferase specificity by design is an integral component of in vitro glycosylation systems developed for the production of diverse glycorandomized libraries.