Glycosynthase activity of Geobacillus stearothermophilus GH52 β-xylosidase:: Efficient synthesis of xylooligosaccharides from α-D-xylopyranosyl fluoride through a conjugated reaction

被引:49
作者
Ben-David, Alon
Bravman, Tsafrir
Balazs, Yael S.
Czjzek, Mirjam
Schomburg, Dietmar
Shoham, Gil
Shoham, Yuval [1 ]
机构
[1] Technion Israel Inst Technol, Dept Food Engn & Biotechnol, IL-32000 Haifa, Israel
[2] Technion Israel Inst Technol, Inst Catalysis Sci & Technol, IL-32000 Haifa, Israel
[3] Technion Israel Inst Technol, Schulich Fac Chem, IL-32000 Haifa, Israel
[4] UPMC, Stn Biol Roscoff, CNRS, UMR Vegetaux Marins & Biomol 7139, F-29682 Roscoff, France
[5] Tech Univ Carolo Wilhelmina Braunschweig, Dept Bioinformat & Biochem, D-38106 Braunschweig, Germany
[6] Hebrew Univ Jerusalem, Dept Inorgan Chem, IL-91904 Jerusalem, Israel
[7] Hebrew Univ Jerusalem, Lab Struct Chem & Biol, IL-91904 Jerusalem, Israel
关键词
enzymes; glycoside hydrolases; glycosynthases; green chemistry; xylo oligosaccharides;
D O I
10.1002/cbic.200700414
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Glycosynthases are mutant glycosidases in which the acidic nucleophile is replaced by a small inert residue. In the presence of glycosyl fluorides of the opposite anomeric configuration (to that of their natural substrates), these enzymes can catalyze glycosidic bond formation with various acceptors. In this study we demonstrate that XynB2E335G, a nucleophile-deficient mutant of a glycoside hydrolase family 52 beta-xylosidase from G. stearothermophilus, can function as an efficient glycosynthase, using alpha-D-xylopyranosyl fluoride as a donor and various aryl sugars as acceptors. The mutant enzyme can also catalyze the self-condensation reaction Of a-D-xylopyranosyl fluoride, providing mainly alpha-D-xylobiosyl fluoride. The self-condensation kinetics exhibited apparent classical Michaelis-Menten behavior, with kinetic constants of 1.3 s(-1) and 2.2 mM for k(cat) and K-M(acceptor),K- respectively, and a k(cat/M(acceptor)) value of. 0.59 s(-1) mM(-1). When the beta-xylosidase E335G mutant was combined with a glycoside hydrolase family 10 glycosynthase, high-molecular-weight xylooligomers were readily obtained from the affordable alpha-D-xylopyranosyl fluoride as the sole substrate.
引用
收藏
页码:2145 / 2151
页数:7
相关论文
共 39 条
[31]   Oligosaccharide synthesis by glycosynthases [J].
Perugino, G ;
Trincone, A ;
Rossi, M ;
Moracci, M .
TRENDS IN BIOTECHNOLOGY, 2004, 22 (01) :31-37
[32]   Toward automated synthesis of oligosaccharides and glycoproteins [J].
Sears, P ;
Wong, CH .
SCIENCE, 2001, 291 (5512) :2344-2350
[33]   Synthesis and medical applications of oligosaccharides [J].
Seeberger, Peter H. ;
Werz, Daniel B. .
NATURE, 2007, 446 (7139) :1046-1051
[34]   A two-component system regulates the expression of an ABC transporter for xylo-oligosaccharides in Geobacillus stearothermophilus [J].
Shulami, Smadar ;
Zaide, Galia ;
Zolotnitsky, Gennady ;
Langut, Yael ;
Feld, Geoff ;
Sonenshein, Abraham L. ;
Shoham, Yuval .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, 73 (03) :874-884
[35]   Characterization of glycosynthase mutants derived from glycoside hydrolase family 10 xylanases [J].
Sugimura, Masahiro ;
Nishimoto, Mamoru ;
Kitaoka, Motomitsu .
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 2006, 70 (05) :1210-1217
[36]   BIOLOGICAL ROLES OF OLIGOSACCHARIDES - ALL OF THE THEORIES ARE CORRECT [J].
VARKI, A .
GLYCOBIOLOGY, 1993, 3 (02) :97-130
[37]   CHANGING ENZYMATIC-REACTION MECHANISMS BY MUTAGENESIS - CONVERSION OF A RETAINING GLUCOSIDASE TO AN INVERTING ENZYME [J].
WANG, QP ;
GRAHAM, RW ;
TRIMBUR, D ;
WARREN, RAJ ;
WITHERS, SG .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1994, 116 (25) :11594-11595
[38]   Glycosynthases: Mutant glycosidases for glycoside synthesis [J].
Williams, SJ ;
Withers, SG .
AUSTRALIAN JOURNAL OF CHEMISTRY, 2002, 55 (1-2) :3-12
[39]   Mapping glycoside hydrolase substrate subsites by isothermal titration calorimetry [J].
Zolotnitsky, G ;
Cogan, U ;
Adir, N ;
Solomon, V ;
Shoham, G ;
Shoham, Y .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2004, 101 (31) :11275-11280