earticle

영어

Thioglycoligases are mutant enzymes derived from retaining glycosidases in which the acid/base carboxylic acid residue has been replaced by an amino acid that has no negative charge. The engineered glycosidase mutants catalyze the formation of a thio-glycosidic linkage with substrates bearing a good leaving group, such as dinitrophenol or fluoride, and sugar acceptors bearing a suitably-positioned thiol. The glycosidase-resistant thioglycoside analogues of the original oligosaccharides are attractive candidates as potential therapeutics. Here, a collection of galactosyl-thio-β -glycosides was prepared by a thioglycoligase derived from a β-galactosidase from Xanthomonas manihotis (BgaX), and a potent inhibitor against human lysosomal β- galactosidase. The identity of the acid/base catalyst of BgaX has been confirmed as Glu184 by kinetic analysis of mutant modified at that position. The Glu184Ala mutant of BgaX is shown to function as an efficient thioglycoligase, which synthesises thiogalactosides with linkages to the 3 and 4 positions of glucoside and galactoside in high yields. Amongst five galactosyl-thio-β-glycosides synthesized by BgaX-E184A, pNP-β-galactosyl-β- 1,3-N-acetyl-glucosamine was expected to be the best inhibitor against human lysosomal β-galactosidase based on its substrate specificity. However, pNP-β- galactosyl-β-1,3-glucoside was the best inhibitor with a reasonable potency (Ki = 8 μM) against the human enzyme. Interestingly, the inhibitor did not inhibit bacterial β- galactosidases which not only belongs to the same glycosidase family but also possess similar substrate specificity to that of the human enzyme. Test of the compound as an inhibitor toward β-galactosidases belonging to other two families of such enzyme revealed that β-galactosidase from E. coli (LacZ) showed a similar order of affinity, but β-galactosidase from B. subtilis was not inhibited. In conclusion, our result opens the interesting possibility of finding novel, and unpredicted inhibitors of enzymes of interest through this relatively simple strategy of library generation in which aglycone-diverse thioglycosides are created by thioglycoligases. From a modest set of thiosugar acceptors it is reasonable to envisage the assembly of a substantial library of thio-disaccharides for testing as inhibitors of glycosidases or carbohydrate binding proteins of interest.