Crystal Structure of Truncated Fibrobacter
succinogenes 1,3-1,4-b-
D
-Glucanase in Complex with
b-1,3-1,4-Cellotriose
Li-Chu Tsai
1
*
, Lie-Fen Shyur
2
, Yi-Sheng Cheng
3
and Shu-Hua Lee
2
1
Department of Molecular
Science and Engineering
National Taipei University of
Technology, Taipei 10608,
Taiwan
2
Institute of BioAgricultural
Sciences, Academia Sinica
Taipei 11529, Taiwan
3
Bioinformatics-Biology Service
Core, Institute of Molecular
Biology, Academia Sinica
Taipei 11529, Taiwan
Fibrobacter succinogenes 1,3-1,4-b-
D
-glucanase (Fsb-glucanase) catalyzes the
specific hydrolysis of b-1,4 glycosidic bonds adjacent to b-1,3 linkages in
b-
D
-glucans or lichenan. This is the first report to elucidate the crystal
structure of a truncated Fsb-glucanase (TFsb-glucanase) in complex with
b-1,3-1,4-cellotriose, a major product of the enzyme reaction. The crystal
structures, at a resolution of 2.3 A
˚
, reveal that the overall fold of TFsb-
glucanase remains virtually unchanged upon sugar binding. The enzyme
accommodates five glucose residues, forming a concave active cleft. The
b-1,3-1,4-cellotriose with subsites K3toK1 bound to the active cleft of
TFsb-glucanase with its reducing end subsite K1 close to the key catalytic
residues Glu56 and Glu60. All three subsites of the b-1,3-1,4-cellotriose
adopted a relaxed
4
C
1
conformation, with a b-1,3 glycosidic linkage
between subsites K2 and K1, and a b-1,4 glycosidic linkage between
subsites K3 and K2. On the basis of the enzyme–product complex
structure observed in this study, a catalytic mechanism and substrate
binding conformation of the active site of TFsb-glucanase is proposed.
q 2005 Elsevier Ltd. All rights reserved.
Keywords: glucanase; 1,3-1,4-b-
D
-glucanase; b-1,3-1,4-cellotriose (CLTR);
active cleft
*Corresponding author
Introduction
Enzyme–carbohydrate interactions and selective
hydrolysis of glycosidic bonds are crucial for
cellular processes, energy uptake, and degra-
dation.
1
Glycosyl hydrolases have the ability to
recognize and bind particular carbohydrates and
then cleave the specific glycosidic bond between
either two or more carbohydrates or between a
carbohydrate and a non-carbohydrate moiety. To
date, approximately 100 families of glycosyl hydro-
lases containing thousands of protein members
have been classified†. Enzymatic hydrolysis of
glycosidic bonds has been classified into two
major mechanisms, retaining and inverting,
2
that
take place via general acid/base catalysis and that
require two critical residues, a general acid/base
and a nucleophile/base, giving rise to either an
overall retention or an inversion cleavage mechan-
ism.
Bacterial b-glucanases (1,3-1,4-b-
D
-glucan 4-glu-
canohydrolases, EC 3.2.1.73), belonging to glycosyl
hydrolases family 16 (GHF16), hydrolyze and
cleave b-1,4-glycosidic bonds precisely when b-1,
3-glycosidic linkages are located prior to b-1,4-
glycosidic linkages in lichenan or b-
D
-glucans.
Lichenan from Cetraria islandica,mossstarch
polyglucan, is a linear polysaccharide structure
composed of mixed-linked b-1,3- and b-1,4-glyco-
sidic bonds, with a proportion of 25–30% b-1,3-
bonds,
3
similar to b-
D
-glucans from grain endo-
sperm cell walls.
4
Hydrolysis of lichenan yields 82%
cellotriose and 9.5% cellopentaose as the major
products, while hydrolysis of barley b-glucans
yields 63.5% cellotriose and 29.5% cellotetraose as
the major products.
3
However, 1,3-1,4-b-
D
-gluca-
nase cannot hydrolyze b-1,3-laminarin or b-1,4-
cellulose, which contain only b-1,3- or b-1,4-
glycosidic linkages, respectively.
Crystal structures of bacterial b-glucanases have
been reported from many Bacillus species.
5–7
The
overall topology of bacterial b-glucanases consists
mainly of two eight-stranded anti-parallel b-sheets
0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
Abbreviations used: GHF, glycosyl hydrolases family;
TFsb-glucanase, 1,3-1,4-b-
D
-glucanase; CLTR, b-1,3-1,4-
cellotriose; ECLTR, extended b-1,3-1,4-cellotriose.
E-mail address of the corresponding author:
lichu@ntut.edu.tw
† http://afmb.cnrs-mrs.fr/CAZY/
doi:10.1016/j.jmb.2005.09.041 J. Mol. Biol. (2005) 354, 642–651