Catalytic properties of a mutant beta-galactosidase from Xanthomonas manihotis engineered to synthesize galactosyl-thio-beta-1,3 and -beta-1,4-glycosides.
The identity of the acid/base catalyst of the Family 35 beta-galactosidases from Xanthomonas manihotis (BgaX) has been confirmed as Glu184 by kinetic analysis of mutants 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 glucosides and galactosides in high (>80%) yields. Kinetic analysis of the thioglycoligase reveals glycosyl donor K(m) values of 1.5-21 microM and glycosyl acceptor K(m) values from 180 to 500 microM. This mutant should be a valuable catalyst for the synthesis of metabolically stable analogues of this important glycosidic linkage.
Kim
Young-Wan
YW
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1.
Chen
Hongming
H
Kim
Jin Hyo
JH
Withers
Stephen G
SG
D019943:Amino Acid Substitution; D001386:Azides / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D006031:Glycosylation; D007700:Kinetics; D008025:Ligases / Q000378:metabolism; D009154:Mutation; D013865:Thioglycosides / Q000096:biosynthesis* / Q000737:chemistry; D014974:Xanthomonas / Q000201:enzymology*; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D001386:Azides; D001426:Bacterial Proteins; D013865:Thioglycosides; D018698:Glutamic Acid; D001616:beta-Galactosidase; D008025:Ligases