Issue Date: September 17, 2007
Evolved Enzyme Adds Sugars
Glycosyltransferase enzymes are a pretty fussy lot. They catalyze the addition of sugars to other sugars or to natural products, but they'll only do this for a limited range of substrates. A research team has now created a modified glycosyltransferase that is considerably more liberal, catalyzing the addition of a variety of "donor" sugars to a wide range of aryl "acceptor" substrates to yield a range of aryl glycosides.
Sugar-containing compounds tend to be good drug candidates, and the mutant glycosyltransferase will make it easier to synthesize sugar-derivatized compounds that can be tested for bioactivity.
The modified glycosyltransferase was developed by postdocs Gavin J. Williams and Changsheng Zhang and pharmaceutical sciences professor Jon S. Thorson of the University of Wisconsin, Madison. They took a bacterial glycosyltransferase that works with a limited range of donors and acceptors and turned it into one that's much more "promiscuous" (Nat. Chem. Biol., DOI: 10.1038/nchembio.2007.28).
The natural glycosyltransferase they started with accelerates the addition of either of just two sugars, glucose or deoxyglucose, to the natural product oleandomycin in Streptomyces bacteria. Using a technique called directed evolution, the researchers generated random mutations in the gene for the enzyme, introduced the mutated genes into bacteria, and used them to express mutant enzymes. They screened the mutants for activity by using engineered fluorescent acceptor substrates that stopped shining when enzymes attached sugars to them.
This strategy enabled them to identify several mutants with greatly liberalized substrate selectivity. They combined the most promising mutations in one gene to create their promiscuous glycosyltransferase. Whereas the natural enzyme works with just two sugar donors, the mutant version used 15 of 22 the researchers tried. And instead of adding the sugars only to oleandomycin, it adds them to a wide range of aryl organic compounds. Thorson's group is currently studying the enzyme to see whether it works on other substrates as well.
Thorson notes that the only other glycosyltransferase that has been "evolved" before attaches sugars to other sugars. In contrast, the one evolved by Thorson's group adds sugars to aryl compounds, a type of activity likely to be more broadly useful for drug discovery.
"Our understanding of structure and specificity is not yet sufficient for this to be done on a design basis, hence the need for directed-evolution strategies," comments chemistry professor Stephen G. Withers of the University of British Columbia, who evolved the earlier glycosyltransferase. He says the Thorson group's enzyme "will be of particular importance for the generation of antibiotics that contain unusual sugars."
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