通过“热点”饱和诱变优化糖基转移酶特异性为新生霉素糖基随机化提供了一种催化剂。
Optimizing glycosyltransferase specificity via "hot spot" saturation mutagenesis presents a catalyst for novobiocin glycorandomization.
作者信息
Williams Gavin J, Goff Randal D, Zhang Changsheng, Thorson Jon S
机构信息
Laboratory for Biosynthetic Chemistry, Pharmaceutical Sciences Division, School of Pharmacy, National Cooperative Drug Discovery Program, University of Wisconsin-Madison, 777 Highland Avenue, Madison, WI 53705, USA.
出版信息
Chem Biol. 2008 Apr;15(4):393-401. doi: 10.1016/j.chembiol.2008.02.017.
Abstract
A comprehensive two-phase "hot spot" saturation mutagenesis strategy for the rapid evolution of glycosyltransferase (GT) specificity for nonnatural acceptors is described. Specifically, the application of a high-throughput screen (based on the fluorescent acceptor umbelliferone) was used to identify key amino acid hot spots that contribute to GT proficiency and/or promiscuity. Saturation mutagenesis of the corresponding hot spots facilitated the utilization of a lower-throughput screen to provide OleD prodigy capable of efficiently glycosylating the nonnatural acceptor novobiocic acid with an array of unique sugars. Incredibly, even in the absence of a high-throughput screen for novobiocic acid glycosylation, this approach rapidly led to improvements in the desired catalytic activity of several hundred-fold.
摘要
描述了一种全面的两阶段“热点”饱和诱变策略，用于糖基转移酶（GT）对非天然受体特异性的快速进化。具体而言，应用高通量筛选（基于荧光受体伞形酮）来鉴定有助于GT活性和/或通用性的关键氨基酸热点。对相应热点进行饱和诱变有助于利用较低通量的筛选，以提供能够用一系列独特糖类有效糖基化非天然受体新生霉素酸的OleD变体。令人惊讶的是，即使在没有针对新生霉素酸糖基化的高通量筛选的情况下，这种方法也能迅速使所需的催化活性提高数百倍。
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