Designer enzymes for glycosphingolipid synthesis by directed evolution
Designer enzymes for glycosphingolipid synthesis by directed evolution
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DOI：
10.1038/nchembio.191
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发表时间：
2009-07-01
影响因子：
14.8
通讯作者：
Withers, Stephen G.
中科院分区：
文献类型：
--
作者：
Hancock, Susan M.;Rich, Jamie R.;Withers, Stephen G.
尽管鞘糖脂作为癌症、HIV、神经退行性疾病和自身免疫性疾病的治疗剂具有巨大的潜力，但其生物学作用的广泛研究和作为药物的开发都受到其合成困难的限制，特别是在大规模合成方面。在这里，我们解决了这个限制，通过合理的诱变和定向进化与ELISA为基础的筛选策略相结合，扩大了鞘糖脂合成酶的合成范围。我们的目标是低水平的混杂底物活性和催化剂的整体催化效率，我们确定了几个增强活性的突变体。这些新的催化剂，这是能够产生广泛的均匀的样品，代表了一个显着的进步，朝着简便，大规模的合成鞘糖脂，并证明了这种方法的通用性，对设计师的鞘糖脂合成酶的创建。
Though glycosphingolipids have great potential as therapeutics for cancer, HIV, neurodegenerative diseases and auto-immune diseases, both extensive study of their biological roles and development as pharmaceuticals are limited by difficulties in their synthesis, especially on large scales. Here we addressed this restriction by expanding the synthetic scope of a glycosphingolipid-synthesizing enzyme through a combination of rational mutagenesis and directed evolution with an ELISA-based screening strategy. We targeted both a low-level promiscuous substrate activity and the overall catalytic efficiency of the catalyst, and we identified several mutants with enhanced activities. These new catalysts, which are capable of producing a broad range of homogeneous samples, represent a significant advance toward the facile, large-scale synthesis of glycosphingolipids and demonstrate the general utility of this approach toward the creation of designer glycosphingolipid-synthesizing enzymes.