Enzymatic thioglycosylation: current knowledge and challenges - Carbohydrate Chemistry: Chemical and Biological Approaches - page 182

one highlighted the arising of S-GT activity from bioengineering. A triple
mutant of OleD, from Streptomyces antibioticus (GT1 family), was
screened for GT activity with more than 130 drug-like acceptors. 36 Among
these acceptors, 4 thiol-containing products proved to be recognized as
acceptors by the OleD wild-type and triple mutant protein, indicating that
these two enzymes were able to catalyze S-glycosylation.
In 2012, yeast was bioengineered and used as an expression platform
for the synthesis of glucosinolates. 38 The whole biosynthesis gene path-
way from A. thaliana was cloned in yeast, including the S-glycosyl-
transferase S-UGT74B1 (see 2.2 and Fig. 3). This is the first example of
using S-GT to artificially synthesize S-glycosylated compounds, although
the enzyme was not used alone, but among a whole metabolic pathway.
2 Glycoside Hydrolases to thioglycoligases: A
mechanism-based evolution of natural enzymes
2.1 Glycoside hydrolases: an overview
Glycoside hydrolases (GHs) are widespread carbohydrate-specific
enzymes involved in many processes in biological systems. In mammals,
GHs are mostly extracellular, such as in saliva or digestive tract, and act
to the degradation of glycans for nutritive or anti-bacterial needs. How-
ever, some of GHs are located in the Endoplasmic Reticulum or Golgi
apparatus participating to post-translational processes. 39,40
This group of enzyme (EC 3.2.1-xx) catalyzes the hydrolysis of glycosidic
bonds in carbohydrate-carbohydrate or carbohydrate-aglycone (non-
carbohydrate moiety) entities. They can be classified in two distinctive
groups as they are able to cleave internal or external osidic linkages
(Fig. 4):
- endo-glycosidases cleave the middle intersugar linkage of oligo-
saccharidic chains,
- exo-glycosidases hydrolyze the external osidic bond at the non-
reducing end of oligosaccharides.
Carbohydrates constitute one the of most complex structures occurring
in nature (with nucleic acids and proteins) due to: i) the monosaccharide
diversity, ii) the type of linkage and iii) the nature of carbohydrate-linked
molecules. 10 As a result, GHs present a wide range of activities, which
have necessitated the creation of a specific classification more explicit
than the IUB Enzyme Nomenclature. In 1998, the Carbohydrate-Active
Enzymes (CAZy) database was created, gathering glycosidases in families
based on amino-acid sequence and protein structures similarities. 7,41
Fig. 4 Endo/Exo-Glycoside Hydrolases