This article expounds on the topics of directed evolution and development of selection and screening methods. Here, Jakeman et al. employ a strategy for the in vivo directed evolution of glycosynthetases. Glycosynthetases are glycosidases that aid in the synthesis if glycosidic linkages and are designed by mutation of the catalytic nucleophile in a glycosidase. This strategy hinges on the ability to identify active mutant enzyme catalysts. They were able to develop the first glycosynthetase capable of glycosylating amino sugar acceptor substrates with a β-(1,2) glycosidic linkage. This should allow for the solubility enhancement and selective targeting of pharmaceuticals.
"This should allow for the solubility enhancement and selective targeting of pharmaceuticals."
ReplyDeleteMaybe. Probably not. The reaction performed here is naturally catalyzed by multiple glycosyltransferases.
Glycosynthases have a very specific niche. They're wonderful at attaching sugars to other sugars. IIRC, only one example of a glycosynthase accepting a non-sugar acceptor substrate has been reported.
Glycosynthases don't exist naturally, they are engineered by removing the hydrolytic function of glycosidases. So if you have a molecule R-O-Sugar and know a glycosidase that cleaves that sugar to form R-OH, you can sometimes engineer it to do the reverse reaction instead. After doing that, the goal is to engineer it to accept other sugars, so that you can make analogues of the original molecule (R-O-Sugar*).
The major limitation to glycosynthases compared to glycosyltransferases is that glycosidase structure (and thus glycosynthase structure) is extremely diverse, while glycosyltransferase structure is highly conserved. Extensive study of glycosyltransferase structure/function is predicted to yield knowledge that will assist in modifying the properties of any glycosyltransferase. With glycosynthases, you have to start from scratch every time.
The major advantage of glycosynthases is that they don't need activated sugar donor substrates (NDP-sugars), which have traditionally been a huge pain to make. The glycosyl fluorides that glycosynthases accept are relatively simple to make. However, the Thorson group has recently developed a simple strategy to produce NDP-sugars (http://www.nature.com/nchembio/journal/v7/n10/full/nchembio.638.html). With the Thorson system, there's no good reason to use the glycosynthase described in this paper instead of a glycosyltransferase.
tl;dr version: glycosyltransferase > glycosynthase
I think you make important points. One being that glycosyltransferases conserved structure allowing for modification of properties of any glycosyltransferase without having to start from scratch. I think that the Thorson approach would be more useful.
ReplyDelete