经过基因工程的改造,酶能够用更多种类的糖基来修饰小分子,这是研究人员在9月号在线出版的《自然—化学生物学》(Nature Chemical Biology)期刊上报告的。
自然界的小分子是许多天然药物的基础,而许多天然小分子的活性会因附加其上的糖分子而改变。因此,如何改变糖分子是新药发现的关键所在。
但是,糖分子的改变需要糖基转移酶的参与,而糖基转移酶只与极少数的糖和小分子相互作用,因此,糖分子的改变过程实际上困难重重。
酶的体外定向进化是改造生物催化剂的一种有效的新策略,主要是模拟自然进化进程,在一种酶的特定位置引入随机变异,再经由DNA改组、交错延伸过程、随机引导重组等进行突变基因的体外重组,最终经筛选获得所需活性的酶。利用这种定向进化技术,Jon Thorson和同事对糖基转移酶进行了基因工程改造,让它能够将更多种类的糖基引入多种有重要治疗价值的小分子中。这种“变异”酶可用于新治疗方法的发现。
原始出处:
Nature Chemical Biology
Published online: 9 September 2007 | doi:10.1038/nchembio.2007.28
Expanding the promiscuity of a natural-product glycosyltransferase by directed evolution
Gavin J Williams1, Changsheng Zhang1 and Jon S Thorson1
Natural products, many of which are decorated with essential sugar
residues, continue to serve as a key platform for drug development1.
Adding or changing sugars attached to such natural products can improve
the parent compound's pharmacological properties, specificity at
multiple levels2, and/or even the molecular mechanism of action3. Though
some natural-product glycosyltransferases (GTs) are sufficiently
promiscuous for use in altering these glycosylation patterns, the
stringent specificity of others remains a limiting factor in
natural-product diversification and highlights a need for general GT
engineering and evolution platforms. Herein we report the use of a
simple high-throughput screen based on a fluorescent surrogate acceptor
substrate to expand the promiscuity of a natural-product GT via directed
evolution. Cumulatively, this study presents variant GTs for the
glycorandomization of a range of therapeutically important acceptors,
including aminocoumarins, flavonoids and macrolides, and a potential
template for engineering other natural-product GTs.
As an emerging method to differentially glycosylate natural products,
glycorandomization uses the inherent or engineered substrate promiscuity
of anomeric kinases (Fig. 1a, E1) and nucleotidyltransferases (E2) for
the in vitro synthesis of sugar nucleotide libraries as sugar donors for
natural-product GTs4. Although the successful glycorandomization of
various natural-product scaffolds (including glycopeptides5,
avermectins6 and enediynes7) has been reported, other recent antibiotic
glycorandomization attempts have revealed that aminocoumarin and
macrolide GTs (NovM and EryBV, respectively) accept only 2 alternative
sugar nucleotides out of 25 to 40 potential donors tested8,9. Thus,
though permissive GTs open new opportunities for drug discovery, the
stringent specificity of other GTs remains a limiting factor in
natural-product diversification and highlights a need for general GT
engineering and/or evolution platforms. Despite the wealth of GT
structural and biochemical information10, attempts to alter GT
donor/acceptor specificities via rational engineering have been largely
unsuccessful and primarily limited to sequence-guided single-site
mutagenesis11. Owing to a lack of high-throughput GT screens and
selections, successful reports to alter GT donor/acceptor specificities
via directed evolution are equally sparse. Although an in vivo selection
for the directed evolution of the sialyltransferase CstII (a unique
member of the GT-A superfamily) was recently disclosed12, the directed
evolution of any member of the structurally and functionally distinct
GT-B superfamily has not been achieved.
(a) General overview of enzymatic glycorandomization.
E1 represents a flexible anomeric sugar kinase, E2 represents a flexible
sugar-1-phosphate nucleotidyltransferase, GT represents a flexible
glycosyltransferase and the gray oval represents a complex
natural-product scaffold. (b) The native macrolide glucosyltransferase
reaction catalyzed by OleD (upper reaction), and the
4-methylumbelliferone (4) glucosylation reaction used for OleD directed
evolution (lower reaction).