Catalytic properties of a mutant beta-galactosidase from Xanthomonas manihotis engineered to synthesize galactosyl-thio-beta-1,3 and -beta-1,4-glycosides |
| |
Authors: | Kim Young-Wan Chen Hongming Kim Jin Hyo Withers Stephen G |
| |
Affiliation: | Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1. |
| |
Abstract: | 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. |
| |
Keywords: | DNP, 2,4-dinitrophenyl PNP, 4-nitrophenyl BgaX, β-galactosidase from Xanthomonas manihotis Abg, β-glucosidase from Agrobacterium sp. GH, glycoside hydrolase TLC, thin layer chromatography NMR, nuclear magnetic resonance ESI, electrospray ionization DNPGal, 3,4-dinitrophenyl β- smallcaps" >d-galactopyranoside PNPGal, 4-nitrophenyl β- smallcaps" >d-galactopyranoside PNP4SGlc, 4-nitrophenyl 4-deoxy-4-thio-β- smallcaps" >d-glucopyranoside PNP3SGlc, 4-nitrophenyl 3-deoxy-3-thio-β- smallcaps" >d-glucopyranoside PNP6SGlc, 4-nitrophenyl 6-deoxy-6-thio-β- smallcaps" >d-glucopyranoside PNP4SGal, 4-nitrophenyl 4-deoxy-4-thio-β- smallcaps" >d-galactopyranoside PNP3SGal, 4-nitrophenyl 3-deoxy-3-thio-β- smallcaps" >d-galactopyranoside |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|