Anatomy of glycosynthesis: Structure and kinetics of the Humicola insolens Cel7B E197A and E197S glycosynthase mutants

V M A  Ducros; C A  Tarling; D L  Zechel; A M  Brzozowski; T P  Frandsen; I  von Ossowski; M  Schulein; S G  Withers; G J  Davies

Anatomy of glycosynthesis: Structure and kinetics of the Humicola insolens Cel7B E197A and E197S glycosynthase mutants

V M A Ducros, C A Tarling, D L Zechel, A M Brzozowski, T P Frandsen, I von Ossowski, M Schulein, S G Withers, G J Davies

Chemistry

Research output: Contribution to journal › Article › peer-review

Abstract

The formation of glycoconjugates and oligosaccharides remains one of the most challenging chemical syntheses. Chemo-enzymatic routes using retaining glycosidases have been successfully harnessed but require tight kinetic or thermodynamic control. "Glycosynthases," specifically engineered glycosidases that catalyze the formation of glycosidic bonds from glycosyl donor and acceptor alcohol, are an emerging range of synthetic tools in which catalytic nucleophile mutants are harnessed together with glycosyl fluoride donors to generate powerful and versatile catalysts. Here we present the structural and kinetic dissection of the Humicola insolens Cel7B glycosynthases in which the nucleophile of the wild-type enzyme is mutated to alanine and serine (E197A and E197S). 3-D structures reveal the acceptor and donor subsites and the basis for substrate inhibition. Kinetic analysis shows that the E197S mutant is considerably more active than the corresponding alanine mutant due to a 40-fold increase in k(cat).

Original language	English
Pages (from-to)	619-628
Number of pages	10
Journal	Chemistry & Biology
Volume	10
Issue number	7
Publication status	Published - Jul 2003

Keywords

CATALYTIC NUCLEOPHILE
OLIGOSACCHARIDE SYNTHESIS
BETA-GALACTOSIDASE
MASS-SPECTROMETRY
ENDOGLUCANASE-I
ENZYME INTERMEDIATE
FUSARIUM-OXYSPORUM
CELLULOMONAS-FIMI
ESCHERICHIA-COLI
ACTIVE-SITE

Cite this

@article{e7012f0472c342df9a3b5955ef22e438,

title = "Anatomy of glycosynthesis: Structure and kinetics of the Humicola insolens Cel7B E197A and E197S glycosynthase mutants",

abstract = "The formation of glycoconjugates and oligosaccharides remains one of the most challenging chemical syntheses. Chemo-enzymatic routes using retaining glycosidases have been successfully harnessed but require tight kinetic or thermodynamic control. {"}Glycosynthases,{"} specifically engineered glycosidases that catalyze the formation of glycosidic bonds from glycosyl donor and acceptor alcohol, are an emerging range of synthetic tools in which catalytic nucleophile mutants are harnessed together with glycosyl fluoride donors to generate powerful and versatile catalysts. Here we present the structural and kinetic dissection of the Humicola insolens Cel7B glycosynthases in which the nucleophile of the wild-type enzyme is mutated to alanine and serine (E197A and E197S). 3-D structures reveal the acceptor and donor subsites and the basis for substrate inhibition. Kinetic analysis shows that the E197S mutant is considerably more active than the corresponding alanine mutant due to a 40-fold increase in k(cat).",

keywords = "CATALYTIC NUCLEOPHILE, OLIGOSACCHARIDE SYNTHESIS, BETA-GALACTOSIDASE, MASS-SPECTROMETRY, ENDOGLUCANASE-I, ENZYME INTERMEDIATE, FUSARIUM-OXYSPORUM, CELLULOMONAS-FIMI, ESCHERICHIA-COLI, ACTIVE-SITE",

author = "Ducros, {V M A} and Tarling, {C A} and Zechel, {D L} and Brzozowski, {A M} and Frandsen, {T P} and {von Ossowski}, I and M Schulein and Withers, {S G} and Davies, {G J}",

year = "2003",

month = jul,

language = "English",

volume = "10",

pages = "619--628",

journal = "Chemistry & Biology",

issn = "1074-5521",

publisher = "Elsevier",

number = "7",

}

TY - JOUR

T1 - Anatomy of glycosynthesis: Structure and kinetics of the Humicola insolens Cel7B E197A and E197S glycosynthase mutants

AU - Ducros, V M A

AU - Tarling, C A

AU - Zechel, D L

AU - Brzozowski, A M

AU - Frandsen, T P

AU - von Ossowski, I

AU - Schulein, M

AU - Withers, S G

AU - Davies, G J

PY - 2003/7

Y1 - 2003/7

N2 - The formation of glycoconjugates and oligosaccharides remains one of the most challenging chemical syntheses. Chemo-enzymatic routes using retaining glycosidases have been successfully harnessed but require tight kinetic or thermodynamic control. "Glycosynthases," specifically engineered glycosidases that catalyze the formation of glycosidic bonds from glycosyl donor and acceptor alcohol, are an emerging range of synthetic tools in which catalytic nucleophile mutants are harnessed together with glycosyl fluoride donors to generate powerful and versatile catalysts. Here we present the structural and kinetic dissection of the Humicola insolens Cel7B glycosynthases in which the nucleophile of the wild-type enzyme is mutated to alanine and serine (E197A and E197S). 3-D structures reveal the acceptor and donor subsites and the basis for substrate inhibition. Kinetic analysis shows that the E197S mutant is considerably more active than the corresponding alanine mutant due to a 40-fold increase in k(cat).

AB - The formation of glycoconjugates and oligosaccharides remains one of the most challenging chemical syntheses. Chemo-enzymatic routes using retaining glycosidases have been successfully harnessed but require tight kinetic or thermodynamic control. "Glycosynthases," specifically engineered glycosidases that catalyze the formation of glycosidic bonds from glycosyl donor and acceptor alcohol, are an emerging range of synthetic tools in which catalytic nucleophile mutants are harnessed together with glycosyl fluoride donors to generate powerful and versatile catalysts. Here we present the structural and kinetic dissection of the Humicola insolens Cel7B glycosynthases in which the nucleophile of the wild-type enzyme is mutated to alanine and serine (E197A and E197S). 3-D structures reveal the acceptor and donor subsites and the basis for substrate inhibition. Kinetic analysis shows that the E197S mutant is considerably more active than the corresponding alanine mutant due to a 40-fold increase in k(cat).

KW - CATALYTIC NUCLEOPHILE

KW - OLIGOSACCHARIDE SYNTHESIS

KW - BETA-GALACTOSIDASE

KW - MASS-SPECTROMETRY

KW - ENDOGLUCANASE-I

KW - ENZYME INTERMEDIATE

KW - FUSARIUM-OXYSPORUM

KW - CELLULOMONAS-FIMI

KW - ESCHERICHIA-COLI

KW - ACTIVE-SITE

M3 - Article

SN - 1074-5521

VL - 10

SP - 619

EP - 628

JO - Chemistry & Biology

JF - Chemistry & Biology

IS - 7

ER -