The	O
Mechanism	O
by	O
Which	O
Arabinoxylanases	B-protein_type
Can	O
Recognize	O
Highly	B-protein_state
Decorated	I-protein_state
Xylans	B-chemical
*	O

The	O
enzymatic	O
degradation	O
of	O
plant	B-taxonomy_domain
cell	O
walls	O
is	O
an	O
important	O
biological	O
process	O
of	O
increasing	O
environmental	O
and	O
industrial	O
significance	O
.	O

Xylan	B-chemical
,	O
a	O
major	O
component	O
of	O
the	O
plant	B-taxonomy_domain
cell	O
wall	O
,	O
consists	O
of	O
a	O
backbone	O
of	O
β	B-chemical
-	I-chemical
1	I-chemical
,	I-chemical
4	I-chemical
-	I-chemical
xylose	I-chemical
(	O
Xylp	B-chemical
)	O
units	O
that	O
are	O
often	O
decorated	O
with	O
arabinofuranose	B-chemical
(	O
Araf	B-chemical
)	O
side	O
chains	O
.	O

A	O
large	O
penta	B-protein_type
-	I-protein_type
modular	I-protein_type
enzyme	I-protein_type
,	O
CtXyl5A	B-protein
,	O
was	O
shown	O
previously	O
to	O
specifically	O
target	O
arabinoxylans	B-chemical
.	O

Here	O
we	O
report	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
arabinoxylanase	B-protein_type
and	O
the	O
enzyme	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
ligands	B-chemical
.	O

The	O
data	O
showed	O
that	O
four	O
of	O
the	O
protein	O
modules	O
adopt	O
a	O
rigid	O
structure	O
,	O
which	O
stabilizes	O
the	O
catalytic	B-structure_element
domain	I-structure_element
.	O

The	O
C	O
-	O
terminal	O
non	B-structure_element
-	I-structure_element
catalytic	I-structure_element
carbohydrate	I-structure_element
binding	I-structure_element
module	I-structure_element
could	O
not	O
be	O
observed	O
in	O
the	O
crystal	B-evidence
structure	I-evidence
,	O
suggesting	O
positional	O
flexibility	O
.	O

The	O
structure	B-evidence
of	O
the	O
enzyme	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
Xylp	B-chemical
-	I-chemical
β	I-chemical
-	I-chemical
1	I-chemical
,	I-chemical
4	I-chemical
-	I-chemical
Xylp	I-chemical
-	I-chemical
β	I-chemical
-	I-chemical
1	I-chemical
,	I-chemical
4	I-chemical
-	I-chemical
Xylp	I-chemical
-[	I-chemical
α	I-chemical
-	I-chemical
1	I-chemical
,	I-chemical
3	I-chemical
-	I-chemical
Araf	I-chemical
]-	I-chemical
β	I-chemical
-	I-chemical
1	I-chemical
,	I-chemical
4	I-chemical
-	I-chemical
Xylp	I-chemical
showed	O
that	O
the	O
Araf	B-chemical
decoration	O
linked	O
O3	O
to	O
the	O
xylose	B-chemical
in	O
the	O
active	B-site
site	I-site
is	O
located	O
in	O
the	O
pocket	B-site
(−	O
2	B-site
*	I-site
subsite	I-site
)	O
that	O
abuts	O
onto	O
the	O
catalytic	B-site
center	I-site
.	O

The	O
−	B-site
2	I-site
*	I-site
subsite	I-site
can	O
also	O
bind	O
to	O
Xylp	B-chemical
and	O
Arap	B-chemical
,	O
explaining	O
why	O
the	O
enzyme	O
can	O
utilize	O
xylose	B-chemical
and	O
arabinose	B-chemical
as	O
specificity	O
determinants	O
.	O

Alanine	B-experimental_method
substitution	I-experimental_method
of	O
Glu68	B-residue_name_number
,	O
Tyr92	B-residue_name_number
,	O
or	O
Asn139	B-residue_name_number
,	O
which	O
interact	O
with	O
arabinose	B-chemical
and	O
xylose	B-chemical
side	O
chains	O
at	O
the	O
−	B-site
2	I-site
*	I-site
subsite	I-site
,	O
abrogates	O
catalytic	O
activity	O
.	O

Distal	O
to	O
the	O
active	B-site
site	I-site
,	O
the	O
xylan	B-chemical
backbone	O
makes	O
limited	O
apolar	O
contacts	O
with	O
the	O
enzyme	O
,	O
and	O
the	O
hydroxyls	O
are	O
solvent	B-protein_state
-	I-protein_state
exposed	I-protein_state
.	O

This	O
explains	O
why	O
CtXyl5A	B-protein
is	O
capable	O
of	O
hydrolyzing	O
xylans	B-chemical
that	O
are	O
extensively	O
decorated	O
and	O
that	O
are	O
recalcitrant	O
to	O
classic	O
endo	B-protein_type
-	I-protein_type
xylanase	I-protein_type
attack	O
.	O

The	O
plant	B-taxonomy_domain
cell	O
wall	O
is	O
an	O
important	O
biological	O
substrate	O
.	O

This	O
complex	O
composite	O
structure	O
is	O
depolymerized	O
by	O
microorganisms	B-taxonomy_domain
that	O
occupy	O
important	O
highly	O
competitive	O
ecological	O
niches	O
,	O
whereas	O
the	O
process	O
makes	O
an	O
important	O
contribution	O
to	O
the	O
carbon	O
cycle	O
.	O

Given	O
that	O
the	O
plant	B-taxonomy_domain
cell	O
wall	O
is	O
the	O
most	O
abundant	O
source	O
of	O
renewable	O
organic	O
carbon	O
on	O
the	O
planet	O
,	O
this	O
macromolecular	O
substrate	O
has	O
substantial	O
industrial	O
potential	O
.	O

An	O
example	O
of	O
the	O
chemical	O
complexity	O
of	O
the	O
plant	B-taxonomy_domain
cell	O
wall	O
is	O
provided	O
by	O
xylan	B-chemical
,	O
which	O
is	O
the	O
major	O
hemicellulosic	O
component	O
.	O

This	O
polysaccharide	B-chemical
comprises	O
a	O
backbone	O
of	O
β	B-chemical
-	I-chemical
1	I-chemical
,	I-chemical
4	I-chemical
-	I-chemical
d	I-chemical
-	I-chemical
xylose	I-chemical
residues	O
in	O
their	O
pyranose	B-chemical
configuration	O
(	O
Xylp	B-chemical
)	O
that	O
are	O
decorated	O
at	O
O2	O
with	O
4	B-chemical
-	I-chemical
O	I-chemical
-	I-chemical
methyl	I-chemical
-	I-chemical
d	I-chemical
-	I-chemical
glucuronic	I-chemical
acid	I-chemical
(	O
GlcA	B-chemical
)	O
and	O
at	O
O2	O
and	O
/	O
or	O
O3	O
with	O
α	B-chemical
-	I-chemical
l	I-chemical
-	I-chemical
arabinofuranose	I-chemical
(	O
Araf	B-chemical
)	O
residues	O
,	O
whereas	O
the	O
polysaccharide	B-chemical
can	O
also	O
be	O
extensively	O
acetylated	O
.	O

In	O
addition	O
,	O
the	O
Araf	B-chemical
side	O
chain	O
decorations	O
can	O
also	O
be	O
esterified	O
to	O
ferulic	B-chemical
acid	I-chemical
that	O
,	O
in	O
some	O
species	O
,	O
provide	O
a	O
chemical	O
link	O
between	O
hemicellulose	B-chemical
and	O
lignin	B-chemical
.	O

The	O
precise	O
structure	O
of	O
xylans	B-chemical
varies	O
between	O
plant	B-taxonomy_domain
species	O
,	O
in	O
particular	O
in	O
different	O
tissues	O
and	O
during	O
cellular	O
differentiation	O
.	O

In	O
specialized	O
plant	B-taxonomy_domain
tissues	O
,	O
such	O
as	O
the	O
outer	O
layer	O
of	O
cereal	B-taxonomy_domain
grains	O
,	O
xylans	B-chemical
are	O
extremely	O
complex	O
,	O
and	O
side	O
chains	O
may	O
comprise	O
a	O
range	O
of	O
other	O
sugars	B-chemical
including	O
l	B-chemical
-	I-chemical
and	I-chemical
d	I-chemical
-	I-chemical
galactose	I-chemical
and	O
β	B-chemical
-	I-chemical
and	I-chemical
α	I-chemical
-	I-chemical
Xylp	I-chemical
units	O
.	O

Indeed	O
,	O
in	O
these	O
cereal	B-taxonomy_domain
brans	O
,	O
xylans	B-chemical
have	O
very	O
few	O
backbone	O
Xylp	B-chemical
units	O
that	O
are	O
undecorated	O
,	O
and	O
the	O
side	O
chains	O
can	O
contain	O
up	O
to	O
six	O
sugars	B-chemical
.	O

Reflecting	O
the	O
chemical	O
and	O
physical	O
complexity	O
of	O
the	O
plant	B-taxonomy_domain
cell	O
wall	O
,	O
microorganisms	B-taxonomy_domain
that	O
utilize	O
these	O
composite	O
structures	O
express	O
a	O
large	O
number	O
of	O
polysaccharide	B-protein_type
-	I-protein_type
degrading	I-protein_type
enzymes	I-protein_type
,	O
primarily	O
glycoside	B-protein_type
hydrolases	I-protein_type
,	O
but	O
also	O
polysaccharide	B-protein_type
lyases	I-protein_type
,	O
carbohydrate	B-protein_type
esterases	I-protein_type
,	O
and	O
lytic	B-protein_type
polysaccharide	I-protein_type
monooxygenases	I-protein_type
.	O

These	O
carbohydrate	B-protein_type
active	I-protein_type
enzymes	I-protein_type
are	O
grouped	O
into	O
sequence	O
-	O
based	O
families	O
in	O
the	O
CAZy	O
database	O
.	O

With	O
respect	O
to	O
xylan	B-chemical
degradation	O
,	O
the	O
backbone	O
of	O
simple	O
xylans	B-chemical
is	O
hydrolyzed	O
by	O
endo	B-protein_type
-	I-protein_type
acting	I-protein_type
xylanases	I-protein_type
,	O
the	O
majority	O
of	O
which	O
are	O
located	O
in	O
glycoside	B-protein_type
hydrolase	I-protein_type
(	O
GH	B-protein_type
)	O
5	B-protein_type
families	O
GH10	B-protein_type
and	O
GH11	B-protein_type
,	O
although	O
they	O
are	O
also	O
present	O
in	O
GH8	B-protein_type
.	O

The	O
extensive	O
decoration	O
of	O
the	O
xylan	B-chemical
backbone	O
generally	O
restricts	O
the	O
capacity	O
of	O
these	O
enzymes	O
to	O
attack	O
the	O
polysaccharide	B-chemical
prior	O
to	O
removal	O
of	O
the	O
side	O
chains	O
by	O
a	O
range	O
of	O
α	B-protein_type
-	I-protein_type
glucuronidases	I-protein_type
,	O
α	B-protein_type
-	I-protein_type
arabinofuranosidases	I-protein_type
,	O
and	O
esterases	B-protein_type
.	O

Two	O
xylanases	B-protein_type
,	O
however	O
,	O
utilize	O
the	O
side	O
chains	O
as	O
essential	O
specificity	O
determinants	O
and	O
thus	O
target	O
decorated	O
forms	O
of	O
the	O
hemicellulose	B-chemical
.	O

The	O
GH30	B-protein_type
glucuronoxylanases	B-protein_type
require	O
the	O
Xylp	B-chemical
bound	B-protein_state
at	I-protein_state
the	O
−	B-site
2	I-site
to	O
contain	O
a	O
GlcA	B-chemical
side	O
chain	O
(	O
the	O
scissile	O
bond	O
targeted	O
by	O
glycoside	B-protein_type
hydrolases	I-protein_type
is	O
between	O
subsites	B-site
−	I-site
1	I-site
and	I-site
+	I-site
1	I-site
,	O
and	O
subsites	B-site
that	O
extend	O
toward	O
the	O
non	O
-	O
reducing	O
and	O
reducing	O
ends	O
of	O
the	O
substrate	O
are	O
assigned	O
increasing	O
negative	O
and	O
positive	O
numbers	O
,	O
respectively	O
).	O

The	O
GH5	B-protein_type
arabinoxylanase	B-protein_type
(	O
CtXyl5A	B-protein
)	O
derived	O
from	O
Clostridium	B-species
thermocellum	I-species
displays	O
an	O
absolute	O
requirement	O
for	O
xylans	B-chemical
that	O
contain	O
Araf	B-chemical
side	O
chains	O
.	O

In	O
this	O
enzyme	O
,	O
the	O
key	O
specificity	O
determinant	O
is	O
the	O
Araf	B-chemical
appended	O
to	O
O3	O
of	O
the	O
Xylp	B-chemical
bound	B-protein_state
in	I-protein_state
the	O
active	B-site
site	I-site
(−	O
1	B-site
subsite	I-site
).	O

The	O
reaction	O
products	O
generated	O
from	O
arabinoxylans	B-chemical
,	O
however	O
,	O
suggest	O
that	O
Araf	B-chemical
can	O
be	O
accommodated	O
at	O
subsites	B-site
distal	O
to	O
the	O
active	B-site
site	I-site
.	O

CtXyl5A	B-protein
is	O
a	O
multimodular	O
enzyme	O
containing	O
,	O
in	O
addition	O
to	O
the	O
GH5	B-protein_type
catalytic	B-structure_element
module	I-structure_element
(	O
CtGH5	B-structure_element
);	O
three	O
non	B-structure_element
-	I-structure_element
catalytic	I-structure_element
carbohydrate	I-structure_element
binding	I-structure_element
modules	I-structure_element
(	O
CBMs	B-structure_element
)	O
belonging	O
to	O
families	O
6	B-protein_type
(	O
CtCBM6	B-structure_element
),	O
13	B-protein_type
(	O
CtCBM13	B-structure_element
),	O
and	O
62	B-protein_type
(	O
CtCBM62	B-structure_element
);	O
fibronectin	B-protein_type
type	I-protein_type
3	I-protein_type
(	O
Fn3	B-structure_element
)	O
domain	O
;	O
and	O
a	O
C	O
-	O
terminal	O
dockerin	B-structure_element
domain	O
Fig	O
.	O
1	O
.	O

Previous	O
studies	O
of	O
Fn3	B-structure_element
domains	O
have	O
indicated	O
that	O
they	O
might	O
function	O
as	O
ligand	B-structure_element
-	I-structure_element
binding	I-structure_element
modules	I-structure_element
,	O
as	O
a	O
compact	O
form	O
of	O
peptide	O
linkers	O
or	O
spacers	O
between	O
other	O
domains	O
,	O
as	O
cellulose	B-structure_element
-	I-structure_element
disrupting	I-structure_element
modules	I-structure_element
,	O
or	O
as	O
proteins	O
that	O
help	O
large	O
enzyme	O
complexes	O
remain	O
soluble	O
.	O

The	O
dockerin	B-structure_element
domain	O
recruits	O
the	O
enzyme	O
into	O
the	O
cellulosome	B-complex_assembly
,	O
a	O
multienzyme	O
plant	B-taxonomy_domain
cell	O
wall	O
degrading	O
complex	O
presented	O
on	O
the	O
surface	O
of	O
C	B-species
.	I-species
thermocellum	I-species
.	O

CtCBM6	B-structure_element
stabilizes	O
CtGH5	B-structure_element
,	O
and	O
CtCBM62	B-structure_element
binds	O
to	O
d	B-chemical
-	I-chemical
galactopyranose	I-chemical
and	O
l	B-chemical
-	I-chemical
arabinopyranose	I-chemical
.	O

The	O
function	O
of	O
the	O
CtCBM13	B-structure_element
and	O
Fn3	B-structure_element
modules	O
remains	O
unclear	O
.	O

This	O
report	O
exploits	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
mature	B-protein_state
CtXyl5A	B-protein
lacking	B-protein_state
its	O
C	O
-	O
terminal	O
dockerin	B-structure_element
domain	O
(	O
CtXyl5A	B-mutant
-	I-mutant
Doc	I-mutant
),	O
and	O
the	O
enzyme	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
ligands	B-chemical
,	O
to	O
explore	O
the	O
mechanism	O
of	O
substrate	O
specificity	O
.	O

The	O
data	O
show	O
that	O
the	O
plasticity	O
in	O
substrate	O
recognition	O
enables	O
the	O
enzyme	O
to	O
hydrolyze	O
highly	O
complex	O
xylans	B-chemical
that	O
are	O
not	O
accessible	O
to	O
classical	O
GH10	B-protein_type
and	O
GH11	B-protein_type
endo	B-protein_type
-	I-protein_type
xylanases	I-protein_type
.	O

Molecular	O
architecture	O
of	O
GH5_34	B-protein_type
enzymes	O
.	O

Modules	O
prefaced	O
by	O
GH	B-structure_element
,	O
CBM	B-structure_element
,	O
or	O
CE	B-structure_element
are	O
modules	O
in	O
the	O
indicated	O
glycoside	B-protein_type
hydrolase	I-protein_type
,	O
carbohydrate	B-structure_element
binding	I-structure_element
module	I-structure_element
,	O
or	O
carbohydrate	B-protein_type
esterase	I-protein_type
families	O
,	O
respectively	O
.	O

Laminin_3_G	B-structure_element
domain	O
belongs	O
to	O
the	O
concanavalin	B-protein_type
A	I-protein_type
lectin	I-protein_type
superfamily	I-protein_type
,	O
and	O
FN3	B-structure_element
denotes	O
a	O
fibronectin	B-structure_element
type	I-structure_element
3	I-structure_element
domain	I-structure_element
.	O

Segments	O
labeled	O
D	O
are	O
dockerin	B-structure_element
domains	O
.	O

Substrate	O
Specificity	O
of	O
CtXyl5A	B-protein

Previous	O
studies	O
showed	O
that	O
CtXyl5A	B-protein
is	O
an	O
arabinoxylan	B-protein_type
-	I-protein_type
specific	I-protein_type
xylanase	I-protein_type
that	O
generates	O
xylooligosaccharides	B-chemical
with	O
an	O
arabinose	B-chemical
linked	O
O3	O
to	O
the	O
reducing	O
end	O
xylose	B-chemical
.	O

The	O
enzyme	O
is	O
active	O
against	O
both	O
wheat	B-taxonomy_domain
and	O
rye	B-taxonomy_domain
arabinoxylans	B-chemical
(	O
abbreviated	O
as	O
WAX	B-chemical
and	O
RAX	B-chemical
,	O
respectively	O
).	O

It	O
was	O
proposed	O
that	O
arabinose	B-chemical
decorations	O
make	O
productive	O
interactions	O
with	O
a	O
pocket	B-site
(−	O
2	B-site
*)	I-site
that	O
is	O
abutted	O
onto	O
the	O
active	B-site
site	I-site
or	O
−	B-site
1	I-site
subsite	I-site
.	O

Arabinose	B-chemical
side	O
chains	O
of	O
the	O
other	O
backbone	O
xylose	B-chemical
units	O
in	O
the	O
oligosaccharides	B-chemical
generated	O
by	O
CtXyl5A	B-protein
were	O
essentially	O
random	O
.	O

These	O
data	O
suggest	O
that	O
O3	O
,	O
and	O
possibly	O
O2	O
,	O
on	O
the	O
xylose	B-chemical
residues	O
at	O
subsites	B-site
distal	O
to	O
the	O
active	B-site
site	I-site
and	O
−	B-site
2	I-site
*	I-site
pocket	I-site
are	O
solvent	B-protein_state
-	I-protein_state
exposed	I-protein_state
,	O
implying	O
that	O
the	O
enzyme	O
can	O
access	O
highly	O
decorated	O
xylans	B-chemical
.	O

To	O
test	O
this	O
hypothesis	O
,	O
the	O
activity	O
of	O
CtXyl5A	B-protein
against	O
xylans	B-chemical
from	O
cereal	B-taxonomy_domain
brans	O
was	O
assessed	O
.	O

CtXyl5a	B-protein
was	O
incubated	B-experimental_method
with	O
a	O
range	O
of	O
xylans	B-chemical
for	O
16	O
h	O
at	O
60	O
°	O
C	O
,	O
and	O
the	O
limit	O
products	O
were	O
visualized	O
by	O
TLC	B-experimental_method
.	O

These	O
xylans	B-chemical
are	O
highly	O
decorated	O
not	O
only	O
with	O
Araf	B-chemical
and	O
GlcA	B-chemical
units	O
but	O
also	O
with	O
l	B-chemical
-	I-chemical
Gal	I-chemical
,	O
d	B-chemical
-	I-chemical
Gal	I-chemical
,	O
and	O
d	B-chemical
-	I-chemical
Xyl	I-chemical
.	O

Indeed	O
,	O
very	O
few	O
xylose	B-chemical
units	O
in	O
the	O
backbone	O
of	O
bran	O
xylans	B-chemical
lack	O
side	O
chains	O
.	O

The	O
data	O
presented	O
in	O
Table	O
1	O
showed	O
that	O
CtXyl5A	B-protein
was	O
active	O
against	O
corn	B-taxonomy_domain
bran	O
xylan	B-chemical
(	O
CX	B-chemical
).	O

In	O
contrast	O
typical	O
endo	B-protein_type
-	I-protein_type
xylanases	I-protein_type
from	O
GH10	B-protein_type
and	O
GH11	B-protein_type
were	O
unable	O
to	O
attack	O
CX	B-chemical
,	O
reflecting	O
the	O
lack	B-protein_state
of	I-protein_state
undecorated	O
xylose	B-chemical
units	O
in	O
the	O
backbone	O
(	O
the	O
active	B-site
site	I-site
of	O
these	O
enzymes	O
can	O
only	O
bind	B-protein_state
to	I-protein_state
non	O
-	O
substituted	O
xylose	B-chemical
residues	O
).	O

The	O
limit	O
products	O
generated	O
by	O
CtXyl5A	B-protein
from	O
CX	B-chemical
consisted	O
of	O
an	O
extensive	O
range	O
of	O
oligosaccharides	B-chemical
.	O

These	O
data	O
support	O
the	O
view	O
that	O
in	O
subsites	B-site
out	O
with	O
the	O
active	B-site
site	I-site
the	O
O2	O
and	O
O3	O
groups	O
of	O
the	O
bound	O
xylose	B-chemical
units	O
are	O
solvent	B-protein_state
-	I-protein_state
exposed	I-protein_state
and	O
will	O
thus	O
tolerate	O
decoration	O
.	O

Kinetics	B-evidence
of	O
GH5_34	B-protein_type
arabinoxylanases	B-protein_type

Enzyme	O
Variant	O
kcat	B-evidence
/	O
Km	B-evidence
WAX	B-chemical
RAX	B-chemical
CX	B-chemical
min	O
−	O
1mg	O
−	O
1ml	O
CtXyl5A	B-protein
CtGH5	B-structure_element
-	I-structure_element
CBM6	I-structure_element
-	I-structure_element
CBM13	I-structure_element
-	I-structure_element
Fn3	I-structure_element
-	I-structure_element
CBM62	I-structure_element
800	O
ND	O
460	O
CtXyl5A	B-protein
CtGH5	B-structure_element
-	I-structure_element
CBM6	I-structure_element
-	I-structure_element
CBM13	I-structure_element
-	I-structure_element
Fn3	I-structure_element
1	O
,	O
232	O
ND	O
659	O
CtXyl5A	B-protein
CtGH5	B-structure_element
-	I-structure_element
CBM6	I-structure_element
-	I-structure_element
CBM13	I-structure_element
1	O
,	O
307	O
ND	O
620	O
CtXyl5A	B-protein
CtGH5	B-structure_element
-	I-structure_element
CBM6	I-structure_element
488	O
ND	O
102	O
CtXyl5A	B-protein
CtGH5	B-structure_element
-	I-structure_element
CBM6	I-structure_element
:	O
E68A	B-mutant
NA	O
NA	O
NA	O
CtXyl5A	B-protein
CtGH5	B-structure_element
-	I-structure_element
CBM6	I-structure_element
:	O
Y92A	B-mutant
NA	O
NA	O
NA	O
CtXyl5A	B-protein
CtGH5	B-structure_element
-	I-structure_element
CBM6	I-structure_element
:	O
N135A	B-mutant
260	O
ND	O
ND	O
CtXyl5A	B-protein
CtGH5	B-structure_element
-	I-structure_element
CBM6	I-structure_element
:	O
N139A	B-mutant
NA	O
NA	O
NA	O
AcGH5	B-protein
Wild	B-protein_state
type	I-protein_state
628	O
1	O
,	O
641	O
289	O
GpGH5	B-protein
Wild	B-protein_state
type	I-protein_state
2	O
,	O
600	O
9	O
,	O
986	O
314	O
VbGH5	B-protein
Wild	B-protein_state
type	I-protein_state
ND	O
ND	O
ND	O
VbGH5	B-protein
D45A	B-mutant
102	O
203	O
23	O

To	O
explore	O
whether	O
substrate	O
bound	B-protein_state
only	I-protein_state
at	I-protein_state
−	B-site
2	I-site
*	I-site
and	O
−	B-site
1	I-site
in	O
the	O
negative	B-site
subsites	I-site
was	O
hydrolyzed	O
by	O
CtXyl5A	B-protein
,	O
the	O
limit	O
products	O
of	O
CX	B-chemical
digested	O
by	O
the	O
arabinoxylanase	B-protein_type
were	O
subjected	O
to	O
size	B-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
using	O
a	O
Bio	O
-	O
Gel	O
P	O
-	O
2	O
,	O
and	O
the	O
smallest	O
oligosaccharides	B-chemical
(	O
largest	O
elution	O
volume	O
)	O
were	O
chosen	O
for	O
further	O
study	O
.	O

HPAEC	B-experimental_method
analysis	O
of	O
the	O
smallest	O
oligosaccharide	B-chemical
fraction	O
(	O
pool	O
4	O
)	O
contained	O
two	O
species	O
with	O
retention	O
times	O
of	O
14	O
.	O
0	O
min	O
(	O
oligosaccharide	B-chemical
1	O
)	O
and	O
20	O
.	O
8	O
min	O
(	O
oligosaccharide	B-chemical
2	O
)	O
(	O
Fig	O
.	O
2	O
).	O

Positive	B-experimental_method
mode	I-experimental_method
electrospray	I-experimental_method
mass	I-experimental_method
spectrometry	I-experimental_method
showed	O
that	O
pool	O
4	O
contained	O
exclusively	O
molecular	O
ions	O
with	O
a	O
m	O
/	O
z	O
=	O
305	O
[	O
M	O
+	O
Na	O
]+,	O
which	O
corresponds	O
to	O
a	O
pentose	B-chemical
-	O
pentose	B-chemical
disaccharide	B-chemical
(	O
molecular	O
mass	O
=	O
282	O
Da	O
)	O
as	O
a	O
sodium	O
ion	O
adduct	O
,	O
whereas	O
a	O
dimer	O
of	O
the	O
disaccharide	B-chemical
with	O
a	O
sodium	O
adduct	O
(	O
m	O
/	O
z	O
=	O
587	O
[	O
2M	O
+	O
Na	O
]+)	O
was	O
also	O
evident	O
.	O

The	O
monosaccharide	O
composition	O
of	O
pool	O
4	O
determined	O
by	O
TFA	B-experimental_method
hydrolysis	I-experimental_method
contained	O
xylose	B-chemical
and	O
arabinose	B-chemical
in	O
a	O
3	O
:	O
1	O
ratio	O
.	O

This	O
suggests	O
that	O
the	O
two	O
oligosaccharides	B-chemical
consist	O
of	O
two	O
disaccharides	B-chemical
:	O
one	O
consisting	O
of	O
two	O
xylose	B-chemical
residues	O
and	O
the	O
other	O
consisting	O
of	O
an	O
arabinose	B-chemical
linked	O
to	O
a	O
xylose	B-chemical
.	O

Treatment	O
of	O
pool	O
4	O
with	O
the	O
nonspecific	B-protein_type
arabinofuranosidase	I-protein_type
,	O
CjAbf51A	B-protein
,	O
resulted	O
in	O
the	O
loss	O
of	O
oligosaccharide	B-chemical
2	O
and	O
the	O
production	O
of	O
both	O
xylose	B-chemical
and	O
arabinose	B-chemical
,	O
indicative	O
of	O
a	O
disaccharide	B-chemical
of	O
xylose	B-chemical
and	O
arabinose	B-chemical
.	O

Incubation	O
of	O
pool	O
4	O
with	O
a	O
β	B-protein_type
-	I-protein_type
1	I-protein_type
,	I-protein_type
3	I-protein_type
-	I-protein_type
xylosidase	I-protein_type
(	O
XynB	B-protein
)	O
converted	O
oligosaccharide	B-chemical
1	O
into	O
xylose	B-chemical
,	O
demonstrating	O
that	O
this	O
molecule	O
is	O
the	O
disaccharide	B-chemical
β	B-chemical
-	I-chemical
1	I-chemical
,	I-chemical
3	I-chemical
-	I-chemical
xylobiose	I-chemical
.	O

This	O
view	O
is	O
supported	O
by	O
the	O
inability	O
of	O
a	O
β	B-protein_type
-	I-protein_type
1	I-protein_type
,	I-protein_type
4	I-protein_type
-	I-protein_type
specific	I-protein_type
xylosidase	I-protein_type
to	O
hydrolyze	O
oligosaccharide	B-chemical
1	O
or	O
oligosaccharide	B-chemical
2	O
(	O
data	O
not	O
shown	O
).	O

The	O
crucial	O
importance	O
of	O
occupancy	O
of	O
the	O
−	B-site
2	I-site
*	I-site
pocket	I-site
for	O
catalytic	O
competence	O
is	O
illustrated	O
by	O
the	O
inability	O
of	O
the	O
enzyme	O
to	O
hydrolyze	O
linear	O
β	B-chemical
-	I-chemical
1	I-chemical
,	I-chemical
4	I-chemical
-	I-chemical
xylooligosaccharides	I-chemical
.	O

The	O
generation	O
of	O
Araf	B-chemical
-	I-chemical
Xylp	I-chemical
and	O
Xyl	B-chemical
-	I-chemical
β	I-chemical
-	I-chemical
1	I-chemical
,	I-chemical
3	I-chemical
-	I-chemical
Xyl	I-chemical
as	O
reaction	O
products	O
demonstrates	O
that	O
occupancy	O
of	O
the	O
−	B-site
2	I-site
subsite	I-site
is	O
not	O
essential	O
for	O
catalytic	O
activity	O
,	O
which	O
is	O
in	O
contrast	O
to	O
all	O
endo	B-protein_type
-	I-protein_type
acting	I-protein_type
xylanases	I-protein_type
where	O
this	O
subsite	B-site
plays	O
a	O
critical	O
role	O
in	O
enzyme	O
activity	O
.	O

Indeed	O
,	O
the	O
data	O
demonstrate	O
that	O
−	B-site
2	I-site
*	I-site
plays	O
a	O
more	O
important	O
role	O
in	O
productive	O
substrate	O
binding	O
than	O
the	O
−	B-site
2	I-site
subsite	I-site
.	O

Unfortunately	O
,	O
the	O
inability	O
to	O
generate	O
highly	O
purified	O
(	B-chemical
Xyl	I-chemical
-	I-chemical
β	I-chemical
-	I-chemical
1	I-chemical
,	I-chemical
4	I-chemical
)	I-chemical
n	I-chemical
-[	I-chemical
β	I-chemical
-	I-chemical
1	I-chemical
,	I-chemical
3	I-chemical
-	I-chemical
Xyl	I-chemical
/	I-chemical
Ara	I-chemical
]-	I-chemical
Xyl	I-chemical
oligosaccharides	B-chemical
from	O
arabinoxylans	B-chemical
prevented	O
the	O
precise	O
binding	O
energies	O
at	O
the	O
negative	O
subsites	O
to	O
be	O
determined	O
.	O

Identification	O
of	O
the	O
disaccharide	B-chemical
reaction	O
products	O
generated	O
from	O
CX	B-chemical
.	O

The	O
smallest	O
reaction	O
products	O
were	O
purified	O
by	O
size	B-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
and	O
analyzed	O
by	O
HPAEC	B-experimental_method
(	O
A	O
)	O
and	O
positive	O
mode	O
ESI	B-experimental_method
-	I-experimental_method
MS	I-experimental_method
(	O
B	O
),	O
respectively	O
.	O

The	O
samples	O
were	O
treated	O
with	O
a	O
nonspecific	B-protein_type
arabinofuranosidase	I-protein_type
(	O
CjAbf51A	B-protein
)	O
and	O
a	O
GH3	B-protein_type
xylosidase	I-protein_type
(	O
XynB	B-protein
)	O
that	O
targeted	O
β	O
-	O
1	O
,	O
3	O
-	O
xylosidic	O
bonds	O
.	O

X	O
,	O
xylose	B-chemical
;	O
A	O
,	O
arabinose	B-chemical
.	O

The	O
m	O
/	O
z	O
=	O
305	O
species	O
denotes	O
a	O
pentose	B-chemical
disaccharide	B-chemical
as	O
a	O
sodium	O
adduct	O
[	O
M	O
+	O
Na	O
]+,	O
whereas	O
the	O
m	O
/	O
z	O
=	O
587	O
signal	O
corresponds	O
to	O
an	O
ESI	B-experimental_method
-	I-experimental_method
MS	I-experimental_method
dimer	O
of	O
the	O
pentose	B-chemical
disaccharide	B-chemical
also	O
as	O
a	O
sodium	O
adduct	O
[	O
2M	O
+	O
Na	O
]+.	O

Crystal	B-evidence
Structure	I-evidence
of	O
the	O
Catalytic	B-structure_element
Module	I-structure_element
of	O
CtXyl5A	B-protein
in	B-protein_state
Complex	I-protein_state
with	I-protein_state
Ligands	B-chemical

To	O
understand	O
the	O
structural	O
basis	O
for	O
the	O
biochemical	O
properties	O
of	O
CtXyl5A	B-protein
,	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
enzyme	O
with	O
ligands	O
that	O
occupy	O
the	O
substrate	B-site
binding	I-site
cleft	I-site
and	O
the	O
critical	O
−	B-site
2	I-site
*	I-site
subsite	I-site
were	O
sought	O
.	O

The	O
data	O
presented	O
in	O
Fig	O
.	O
3A	O
show	O
the	O
structure	B-evidence
of	O
the	O
CtXyl5A	B-protein
derivative	O
CtGH5	B-structure_element
-	I-structure_element
CtCBM6	I-structure_element
in	B-protein_state
complex	I-protein_state
with	I-protein_state
arabinose	B-chemical
bound	B-protein_state
in	I-protein_state
the	O
−	B-site
2	I-site
*	I-site
pocket	I-site
.	O

Interestingly	O
,	O
the	O
bound	B-protein_state
arabinose	B-chemical
was	O
in	O
the	O
pyranose	B-chemical
conformation	O
rather	O
than	O
in	O
its	O
furanose	B-chemical
form	O
found	O
in	O
arabinoxylans	B-chemical
.	O

O1	O
was	O
facing	O
toward	O
the	O
active	B-site
site	I-site
−	B-site
1	I-site
subsite	I-site
,	O
indicative	O
of	O
the	O
bound	B-protein_state
arabinose	B-chemical
being	O
in	O
the	O
right	O
orientation	O
to	O
be	O
linked	O
to	O
the	O
xylan	B-chemical
backbone	O
via	O
an	O
α	O
-	O
1	O
,	O
3	O
linkage	O
.	O

As	O
discussed	O
on	O
below	O
,	O
the	O
axial	O
O4	O
of	O
the	O
Arap	B-chemical
did	O
not	O
interact	O
with	O
the	O
−	B-site
2	I-site
*	I-site
subsite	I-site
,	O
suggesting	O
that	O
the	O
pocket	B-site
might	O
be	O
capable	O
of	O
binding	O
a	O
xylose	B-chemical
molecule	O
.	O

Indeed	O
,	O
soaking	B-experimental_method
apo	B-protein_state
crystals	B-evidence
with	O
xylose	B-chemical
showed	O
that	O
the	O
pentose	B-chemical
sugar	B-chemical
also	O
bound	B-protein_state
in	I-protein_state
the	O
−	B-site
2	I-site
*	I-site
subsite	I-site
in	O
its	O
pyranose	B-chemical
conformation	O
(	O
Fig	O
.	O
3B	O
).	O

These	O
crystal	B-evidence
structures	I-evidence
support	O
the	O
biochemical	O
data	O
presented	O
above	O
showing	O
that	O
the	O
enzyme	O
generated	O
β	B-chemical
-	I-chemical
1	I-chemical
,	I-chemical
3	I-chemical
-	I-chemical
xylobiose	I-chemical
from	O
CX	B-chemical
,	O
which	O
would	O
require	O
the	O
disaccharide	B-chemical
to	O
bind	O
at	O
the	O
−	B-site
1	I-site
and	I-site
−	I-site
2	I-site
*	I-site
subsites	I-site
.	O

A	O
third	O
product	O
complex	O
was	O
generated	O
by	O
co	B-experimental_method
-	I-experimental_method
crystallizing	I-experimental_method
the	O
nucleophile	B-protein_state
inactive	I-protein_state
mutant	B-protein_state
CtGH5E279S	B-mutant
-	O
CtCBM6	B-structure_element
with	O
a	O
WAX	B-chemical
-	O
derived	O
oligosaccharide	B-chemical
(	O
Fig	O
.	O
3C	O
).	O

The	O
data	O
revealed	O
a	O
pentasaccharide	B-chemical
bound	B-protein_state
to	I-protein_state
the	O
enzyme	O
,	O
comprising	O
β	B-chemical
-	I-chemical
1	I-chemical
,	I-chemical
4	I-chemical
-	I-chemical
xylotetraose	I-chemical
with	O
an	O
Araf	B-chemical
linked	O
α	O
-	O
1	O
,	O
3	O
to	O
the	O
reducing	O
end	O
xylose	B-chemical
.	O

The	O
xylotetraose	B-chemical
was	O
positioned	O
in	O
subsites	B-site
−	I-site
1	I-site
to	I-site
−	I-site
4	I-site
and	O
the	O
Araf	B-chemical
in	O
the	O
−	B-site
2	I-site
*	I-site
pocket	I-site
.	O

Analysis	O
of	O
the	O
three	O
structures	B-evidence
showed	O
that	O
O1	O
,	O
O2	O
,	O
O3	O
,	O
and	O
the	O
endocyclic	O
oxygen	O
occupied	O
identical	O
positions	O
in	O
the	O
Arap	B-chemical
,	O
Araf	B-chemical
,	O
and	O
Xylp	B-chemical
ligands	O
bound	B-protein_state
in	I-protein_state
the	O
−	B-site
2	I-site
*	I-site
subsite	I-site
and	O
thus	O
made	O
identical	O
interactions	O
with	O
the	O
pocket	B-site
.	O

O1	O
makes	O
a	O
polar	B-bond_interaction
contact	I-bond_interaction
with	O
Nδ2	O
of	O
Asn139	B-residue_name_number
,	O
O2	O
is	O
within	O
hydrogen	B-bond_interaction
bonding	I-bond_interaction
distance	O
with	O
Oδ1	O
of	O
Asn139	B-residue_name_number
and	O
the	O
backbone	O
N	O
of	O
Asn135	B-residue_name_number
,	O
and	O
O3	O
interacts	O
with	O
the	O
N	O
of	O
Gly136	B-residue_name_number
and	O
Oϵ2	O
of	O
Glu68	B-residue_name_number
.	O

Although	O
O4	O
of	O
Arap	B-chemical
does	O
not	O
make	O
a	O
direct	O
interaction	O
with	O
the	O
enzyme	O
,	O
O4	O
and	O
O5	O
of	O
Xylp	B-chemical
and	O
Araf	B-chemical
,	O
respectively	O
,	O
form	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
with	O
Oϵ1	O
of	O
Glu68	B-residue_name_number
.	O

Finally	O
Tyr92	B-residue_name_number
makes	O
apolar	O
parallel	B-bond_interaction
interactions	I-bond_interaction
with	O
the	O
pyranose	B-chemical
or	O
furanose	B-chemical
rings	O
of	O
the	O
three	O
sugars	O
.	O

Representation	O
of	O
the	O
residues	O
involved	O
in	O
the	O
ligands	O
recognition	O
at	O
the	O
−	B-site
2	I-site
*	I-site
subsite	I-site
.	O

Interacting	O
residues	O
are	O
represented	O
as	O
stick	O
in	O
blue	O
,	O
and	O
the	O
catalytic	B-site
residues	I-site
and	O
the	O
mutated	B-experimental_method
glutamate	B-residue_name
(	O
into	O
a	O
serine	B-residue_name
)	O
are	O
in	O
magenta	O
.	O

A	O
,	O
CtGH5	B-structure_element
-	I-structure_element
CBM6	I-structure_element
in	B-protein_state
complex	I-protein_state
with	I-protein_state
an	O
arabinopyranose	B-chemical
.	O

B	O
,	O
CtGH5	B-structure_element
-	I-structure_element
CBM6	I-structure_element
in	B-protein_state
complex	I-protein_state
with	I-protein_state
a	O
xylopyranose	B-chemical
.	O

C	O
,	O
CtGH5E279S	B-mutant
-	O
CBM6	B-structure_element
in	B-protein_state
complex	I-protein_state
with	I-protein_state
a	O
pentasaccharide	B-chemical
(	O
β1	B-chemical
,	I-chemical
4	I-chemical
-	I-chemical
xylotetraose	I-chemical
with	O
an	O
l	B-chemical
-	I-chemical
Araf	I-chemical
linked	O
α1	O
,	O
3	O
to	O
the	O
reducing	O
end	O
xylose	B-chemical
).	O

The	O
xylan	B-chemical
backbone	O
is	O
shown	O
transparently	O
for	O
more	O
clarity	O
.	O

Densities	B-evidence
shown	O
in	O
blue	O
are	O
RefMac	O
maximum	B-evidence
-	I-evidence
likelihood	I-evidence
σA	I-evidence
-	I-evidence
weighted	I-evidence
2Fo	I-evidence
−	I-evidence
Fc	I-evidence
at	I-evidence
1	I-evidence
.	I-evidence
5	I-evidence
σ	I-evidence
.	O

The	O
importance	O
of	O
the	O
interactions	O
between	O
the	O
ligands	O
and	O
the	O
side	O
chains	O
of	O
the	O
residues	O
in	O
the	O
−	B-site
2	I-site
*	I-site
pocket	I-site
were	O
evaluated	O
by	O
alanine	B-experimental_method
substitution	I-experimental_method
of	O
these	O
amino	O
acids	O
.	O

The	O
mutants	B-protein_state
E68A	B-mutant
,	O
Y92A	B-mutant
,	O
and	O
N139A	B-mutant
were	O
all	O
inactive	B-protein_state
(	O
Table	O
1	O
),	O
demonstrating	O
the	O
importance	O
of	O
the	O
interactions	O
of	O
these	O
residues	O
with	O
the	O
substrate	O
and	O
reinforcing	O
the	O
critical	O
role	O
the	O
−	B-site
2	I-site
*	I-site
subsite	I-site
plays	O
in	O
the	O
activity	O
of	O
the	O
enzyme	O
.	O

N135A	B-mutant
retained	O
wild	B-protein_state
type	I-protein_state
activity	O
because	O
the	O
O2	O
of	O
the	O
sugars	O
interacts	O
with	O
the	O
backbone	O
N	O
of	O
Asn135	B-residue_name_number
and	O
not	O
with	O
the	O
side	O
chain	O
.	O

Because	O
the	O
hydroxyls	O
of	O
Xylp	B-chemical
or	O
Araf	B-chemical
in	O
the	O
−	B-site
2	I-site
*	I-site
pocket	I-site
are	O
not	O
solvent	B-protein_state
-	I-protein_state
exposed	I-protein_state
,	O
the	O
active	B-site
site	I-site
of	O
the	O
arabinoxylanase	B-protein_type
can	O
only	O
bind	O
to	O
xylose	B-chemical
residues	O
that	O
contain	O
a	O
single	O
xylose	B-chemical
or	O
arabinose	B-chemical
O3	O
decoration	O
.	O

This	O
may	O
explain	O
why	O
the	O
kcat	B-evidence
/	O
Km	B-evidence
for	O
CtXyl5A	B-protein
against	O
WAX	B-chemical
was	O
2	O
-	O
fold	O
higher	O
than	O
against	O
CX	B-chemical
(	O
Table	O
1	O
).	O

WAX	B-chemical
is	O
likely	O
to	O
have	O
a	O
higher	O
concentration	O
of	O
single	O
Araf	B-chemical
decorations	O
compared	O
with	O
CX	B-chemical
and	O
thus	O
contain	O
more	O
substrate	O
available	O
to	O
the	O
arabinoxylanase	B-protein_type
.	O

In	O
the	O
active	B-site
site	I-site
of	O
CtXyl5A	B-protein
the	O
α	B-chemical
-	I-chemical
d	I-chemical
-	I-chemical
Xylp	I-chemical
,	O
which	O
is	O
in	O
its	O
relaxed	O
4C1	O
conformation	O
,	O
makes	O
the	O
following	O
interactions	O
with	O
the	O
enzyme	O
(	O
Fig	O
.	O
4	O
,	O
A	O
–	O
C	O
):	O
O1	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
with	O
the	O
Nδ1	O
of	O
His253	B-residue_name_number
and	O
Oϵ2	O
of	O
Glu171	B-residue_name_number
(	O
catalytic	O
acid	O
-	O
base	O
)	O
and	O
makes	O
a	O
possible	O
weak	O
polar	B-bond_interaction
contact	I-bond_interaction
with	O
the	O
OH	O
of	O
Tyr255	B-residue_name_number
and	O
Oγ	O
of	O
Ser279	B-residue_name_number
(	O
mutation	O
of	O
the	O
catalytic	O
nucleophile	O
);	O
O2	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
with	O
Nδ2	O
of	O
Asn170	B-residue_name_number
and	O
OH	O
of	O
Tyr92	B-residue_name_number
.	O

O3	O
(	O
O1	O
of	O
the	O
Araf	B-chemical
at	O
the	O
−	B-site
2	I-site
*	I-site
subsite	I-site
)	O
makes	O
a	O
polar	B-bond_interaction
contact	I-bond_interaction
with	O
Nδ2	O
of	O
Asn139	B-residue_name_number
;	O
the	O
endocyclic	O
oxygen	O
hydrogens	B-bond_interaction
bonds	I-bond_interaction
with	O
the	O
OH	O
of	O
Tyr255	B-residue_name_number
.	O

The	O
Xylp	B-chemical
in	O
the	O
active	B-site
site	I-site
makes	O
strong	O
parallel	B-bond_interaction
apolar	I-bond_interaction
interactions	I-bond_interaction
with	O
Phe310	B-residue_name_number
.	O

Substrate	O
recognition	O
in	O
the	O
active	B-site
site	I-site
is	O
conserved	B-protein_state
between	O
CtXyl5A	B-protein
and	O
the	O
closest	O
GH5	B-protein_type
structural	O
homolog	O
,	O
the	O
endoglucanase	B-protein_type
BaCel5A	B-protein
(	O
PDB	O
code	O
1qi2	O
)	O
as	O
noted	O
previously	O
.	O

Comparison	O
of	O
the	O
ligand	O
recognition	O
at	O
the	O
distal	O
negative	B-site
subsites	I-site
between	O
CtGH5E279S	B-mutant
-	O
CBM6	B-structure_element
,	O
the	O
cellulase	B-protein_type
BaCel5A	B-protein
,	O
and	O
the	O
xylanase	B-protein_type
GH10	B-protein_type
.	O

A	O
–	O
C	O
show	O
CtGH5E279S	B-mutant
-	O
CBM6	O
is	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
a	O
pentasaccharide	B-chemical
(	O
β1	B-chemical
,	I-chemical
4	I-chemical
-	I-chemical
xylotetraose	I-chemical
with	O
an	O
l	B-chemical
-	I-chemical
Araf	I-chemical
linked	O
α1	O
,	O
3	O
to	O
the	O
reducing	O
end	O
xylose	B-chemical
).	O

A	O
,	O
Poseview	O
representation	O
highlighting	O
the	O
hydrogen	B-bond_interaction
bonding	I-bond_interaction
and	O
the	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
that	O
occur	O
in	O
the	O
negative	B-site
subsites	I-site
.	O

C	O
,	O
density	B-evidence
of	O
the	O
ligand	O
shown	O
in	O
blue	O
is	O
RefMac	O
maximum	B-evidence
-	I-evidence
likelihood	I-evidence
σA	I-evidence
-	I-evidence
weighted	I-evidence
2Fo	I-evidence
−	I-evidence
Fc	I-evidence
at	I-evidence
1	I-evidence
.	I-evidence
5	I-evidence
σ	I-evidence
.	O

D	O
and	O
E	O
display	O
BaCel5A	B-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
deoxy	B-chemical
-	I-chemical
2	I-chemical
-	I-chemical
fluoro	I-chemical
-	I-chemical
β	I-chemical
-	I-chemical
d	I-chemical
-	I-chemical
cellotrioside	I-chemical
(	O
PDB	O
code	O
1qi2	O
),	O
and	O
F	O
and	O
G	O
show	O
CmXyn10B	B-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
a	O
xylotriose	B-chemical
(	O
PDB	O
code	O
1uqy	O
).	O

B	O
,	O
D	O
,	O
and	O
F	O
are	O
surface	O
representations	O
(	O
CtGH5E279S	B-mutant
-	O
CBM6	O
in	O
gray	O
,	O
BaCel5A	B-protein
in	O
cyan	O
,	O
and	O
the	O
xylanase	B-protein_type
GH10	B-protein_type
in	O
light	O
brown	O
).	O

The	O
black	O
dashes	O
represent	O
the	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
.	O

The	O
capacity	O
of	O
CtXyl5A	B-protein
to	O
act	O
on	O
the	O
highly	O
decorated	O
xylan	B-chemical
CX	B-chemical
indicates	O
that	O
O3	O
and	O
possibly	O
O2	O
of	O
the	O
backbone	O
Xylp	B-chemical
units	O
are	O
solvent	B-protein_state
-	I-protein_state
exposed	I-protein_state
.	O

This	O
is	O
consistent	O
with	O
the	O
interaction	O
of	O
the	O
xylotetraose	B-chemical
backbone	O
with	O
the	O
enzyme	O
distal	O
to	O
the	O
active	B-site
site	I-site
.	O

A	O
surface	O
representation	O
of	O
the	O
enzyme	O
(	O
Fig	O
.	O
4B	O
)	O
shows	O
that	O
O3	O
and	O
O2	O
of	O
xylose	B-chemical
units	O
at	O
subsites	B-site
−	I-site
2	I-site
to	I-site
−	I-site
4	I-site
are	O
solvent	B-protein_state
-	I-protein_state
exposed	I-protein_state
and	O
are	O
thus	O
available	O
for	O
decoration	O
.	O

Indeed	O
,	O
these	O
pyranose	B-chemical
sugars	B-chemical
make	O
very	O
weak	O
apolar	B-bond_interaction
interactions	I-bond_interaction
with	O
the	O
arabinoxylanase	B-protein_type
.	O

At	O
−	B-site
2	I-site
,	O
Xylp	B-chemical
makes	O
planar	B-bond_interaction
apolar	I-bond_interaction
interactions	I-bond_interaction
with	O
the	O
Araf	B-chemical
bound	B-protein_state
to	I-protein_state
the	O
−	B-site
2	I-site
*	I-site
subsite	I-site
(	O
Fig	O
.	O
4C	O
).	O

Xylp	B-chemical
at	O
subsites	B-site
−	I-site
2	I-site
and	I-site
−	I-site
3	I-site
,	O
respectively	O
,	O
make	O
weak	O
hydrophobic	B-bond_interaction
contact	I-bond_interaction
with	O
Val318	B-residue_name_number
,	O
the	O
−	B-site
3	I-site
Xylp	B-chemical
makes	O
planar	B-bond_interaction
apolar	I-bond_interaction
interactions	I-bond_interaction
with	O
Ala137	B-residue_name_number
,	O
whereas	O
the	O
xylose	B-chemical
at	O
−	B-site
4	I-site
forms	O
parallel	B-bond_interaction
apolar	I-bond_interaction
contacts	I-bond_interaction
with	O
Trp69	B-residue_name_number
.	O

Comparison	O
of	O
the	O
distal	O
negative	B-site
subsites	I-site
of	O
CtXyl5A	B-protein
with	O
BaCel5A	B-protein
and	O
a	O
typical	O
GH10	B-protein_type
xylanase	B-protein_type
(	O
CmXyn10B	B-protein
,	O
PDB	O
code	O
1uqy	O
)	O
highlights	O
the	O
paucity	O
of	O
interactions	O
between	O
the	O
arabinoxylanase	B-protein_type
and	O
its	O
substrate	O
out	O
with	O
the	O
active	B-site
site	I-site
(	O
Fig	O
.	O
4	O
).	O

Thus	O
,	O
the	O
cellulase	B-protein_type
contains	O
three	O
negative	B-site
subsites	I-site
and	O
the	O
sugars	B-chemical
bound	B-protein_state
in	I-protein_state
the	O
−	B-site
2	I-site
and	I-site
−	I-site
3	I-site
subsites	I-site
make	O
a	O
total	O
of	O
9	O
polar	B-bond_interaction
interactions	I-bond_interaction
with	O
the	O
enzyme	O
(	O
Fig	O
.	O
4	O
,	O
D	O
and	O
E	O
).	O

The	O
GH10	B-protein_type
xylanase	B-protein_type
also	O
contains	O
a	O
−	B-site
2	I-site
subsite	I-site
that	O
,	O
similar	O
to	O
the	O
cellulase	B-protein_type
,	O
makes	O
numerous	O
interactions	O
with	O
the	O
substrate	O
(	O
Fig	O
.	O
4	O
,	O
F	O
and	O
G	O
).	O

The	O
Influence	O
of	O
the	O
Modular	O
Architecture	O
of	O
CtXyl5A	B-protein
on	O
Catalytic	O
Activity	O

CtXyl5A	B-protein
,	O
in	O
addition	O
to	O
its	O
catalytic	B-structure_element
module	I-structure_element
,	O
contains	O
three	O
CBMs	B-structure_element
(	O
CtCBM6	B-structure_element
,	O
CtCBM13	B-structure_element
,	O
and	O
CtCBM62	B-structure_element
)	O
and	O
a	O
fibronectin	B-structure_element
domain	I-structure_element
(	O
CtFn3	B-structure_element
).	O

A	O
previous	O
study	O
showed	O
that	O
although	O
the	O
CBM6	B-structure_element
bound	B-protein_state
in	I-protein_state
an	O
exo	B-protein_state
-	I-protein_state
mode	I-protein_state
to	O
xylo	B-chemical
-	I-chemical
and	I-chemical
cellulooligosaccharides	I-chemical
,	O
the	O
primary	O
role	O
of	O
this	O
module	O
was	O
to	O
stabilize	O
the	O
structure	O
of	O
the	O
GH5	B-protein_type
catalytic	B-structure_element
module	I-structure_element
.	O

To	O
explore	O
the	O
contribution	O
of	O
the	O
other	O
non	B-structure_element
-	I-structure_element
catalytic	I-structure_element
modules	I-structure_element
to	O
CtXyl5A	B-protein
function	O
,	O
the	O
activity	O
of	O
a	O
series	O
of	O
truncated	B-protein_state
derivatives	O
of	O
the	O
arabinoxylanase	B-protein_type
were	O
assessed	O
.	O

The	O
data	O
in	O
Table	O
1	O
show	O
that	O
removal	B-experimental_method
of	I-experimental_method
CtCBM62	B-structure_element
caused	O
a	O
modest	O
increase	O
in	O
activity	O
against	O
both	O
WAX	B-chemical
and	O
CX	B-chemical
,	O
whereas	O
deletion	B-experimental_method
of	I-experimental_method
the	O
Fn3	B-structure_element
domain	O
had	O
no	O
further	O
impact	O
on	O
catalytic	O
performance	O
.	O

Truncation	B-experimental_method
of	O
CtCBM13	B-structure_element
,	O
however	O
,	O
caused	O
a	O
4	O
–	O
5	O
-	O
fold	O
reduction	O
in	O
activity	O
against	O
both	O
substrates	O
.	O

Members	O
of	O
CBM13	B-structure_element
have	O
been	O
shown	O
to	O
bind	O
to	O
xylans	B-chemical
,	O
mannose	B-chemical
,	O
and	O
galactose	B-chemical
residues	O
in	O
complex	B-chemical
glycans	I-chemical
,	O
hinting	O
that	O
the	O
function	O
of	O
CtCBM13	B-structure_element
is	O
to	O
increase	O
the	O
proximity	O
of	O
substrate	O
to	O
the	O
catalytic	B-structure_element
module	I-structure_element
of	O
CtXyl5A	B-protein
.	O

Binding	B-experimental_method
studies	I-experimental_method
,	O
however	O
,	O
showed	O
that	O
CtCBM13	B-structure_element
displayed	O
no	O
affinity	O
for	O
a	O
range	O
of	O
relevant	O
glycans	B-chemical
including	O
WAX	B-chemical
,	O
CX	B-chemical
,	O
xylose	B-chemical
,	O
mannose	B-chemical
,	O
galactose	B-chemical
,	O
and	O
birchwood	B-chemical
xylan	I-chemical
(	O
BX	B-chemical
)	O
(	O
data	O
not	O
shown	O
).	O

It	O
would	O
appear	O
,	O
therefore	O
,	O
that	O
CtCBM13	B-structure_element
makes	O
a	O
structural	O
contribution	O
to	O
the	O
function	O
of	O
CtXyl5A	B-protein
.	O

Crystal	B-evidence
Structure	I-evidence
of	O
CtXyl5A	B-mutant
-	I-mutant
D	I-mutant

To	O
explore	O
further	O
the	O
role	O
of	O
the	O
non	B-structure_element
-	I-structure_element
catalytic	I-structure_element
modules	I-structure_element
in	O
CtXyl5A	B-protein
the	O
crystal	B-evidence
structure	I-evidence
of	O
CtXyl5A	B-protein
extending	O
from	O
CtGH5	B-structure_element
to	O
CtCBM62	B-structure_element
was	O
sought	O
.	O

To	O
obtain	O
a	O
construct	O
that	O
could	O
potentially	O
be	O
crystallized	B-experimental_method
,	O
the	O
protein	O
was	O
generated	O
without	B-protein_state
the	O
C	O
-	O
terminal	O
dockerin	B-structure_element
domain	O
because	O
it	O
is	O
known	O
to	O
be	O
unstable	O
and	O
prone	O
to	O
cleavage	O
.	O

Using	O
this	O
construct	O
(	O
CtXyl5A	B-mutant
-	I-mutant
D	I-mutant
)	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
arabinoxylanase	B-protein_type
was	O
determined	O
by	O
molecular	B-experimental_method
replacement	I-experimental_method
to	O
a	O
resolution	O
of	O
2	O
.	O
64	O
Å	O
with	O
Rwork	B-evidence
and	O
Rfree	B-evidence
at	O
23	O
.	O
7	O
%	O
and	O
27	O
.	O
8	O
%,	O
respectively	O
.	O

The	O
structure	B-evidence
comprises	O
a	O
continuous	O
polypeptide	O
extending	O
from	O
Ala36	B-residue_range
to	I-residue_range
Trp742	I-residue_range
displaying	O
four	O
modules	O
GH5	B-structure_element
-	I-structure_element
CBM6	I-structure_element
-	I-structure_element
CBM13	I-structure_element
-	I-structure_element
Fn3	I-structure_element
.	O

Although	O
there	O
was	O
some	O
electron	B-evidence
density	I-evidence
for	O
CtCBM62	B-structure_element
,	O
it	O
was	O
not	O
sufficient	O
to	O
confidently	O
build	O
the	O
module	O
(	O
Fig	O
.	O
5	O
).	O

Further	O
investigation	O
of	O
the	O
crystal	B-evidence
packing	I-evidence
revealed	O
a	O
large	O
solvent	B-site
channel	I-site
adjacent	O
to	O
the	O
area	O
the	O
CBM62	B-structure_element
occupies	O
.	O

We	O
postulate	O
that	O
the	O
reason	O
for	O
the	O
poor	O
electron	B-evidence
density	I-evidence
is	O
due	O
to	O
the	O
CtCBM62	B-structure_element
being	O
mobile	B-protein_state
compared	O
with	O
the	O
rest	O
of	O
the	O
protein	O
.	O

The	O
structures	B-evidence
of	O
CtGH5	B-structure_element
and	O
CtCBM6	B-structure_element
have	O
been	O
described	O
previously	O
.	O

Surface	O
representation	O
of	O
the	O
tetra	O
-	O
modular	O
arabinoxylanase	B-protein_type
and	O
zoom	O
view	O
on	O
the	O
CtGH5	B-structure_element
loop	B-structure_element
.	O

The	O
blue	O
module	O
is	O
the	O
CtGH5	B-structure_element
catalytic	B-structure_element
domain	I-structure_element
,	O
the	O
green	O
module	O
corresponds	O
to	O
the	O
CtCBM6	B-structure_element
,	O
the	O
yellow	O
module	O
is	O
the	O
CtCBM13	B-structure_element
,	O
and	O
the	O
salmon	O
module	O
is	O
the	O
fibronectin	B-structure_element
domain	I-structure_element
.	O

The	O
CtGH5	B-structure_element
loop	B-structure_element
is	O
stabilized	O
between	O
the	O
CtCBM6	B-structure_element
and	O
the	O
CtCBM13	B-structure_element
modules	O
.	O

CtCBM13	B-structure_element
extends	O
from	O
Gly567	B-residue_range
to	I-residue_range
Pro648	I-residue_range
.	O

Typical	O
of	O
CBM13	B-protein_type
proteins	O
CtCBM13	B-structure_element
displays	O
a	O
β	B-structure_element
-	I-structure_element
trefoil	I-structure_element
fold	I-structure_element
comprising	O
the	O
canonical	O
pseudo	O
3	O
-	O
fold	O
symmetry	O
with	O
a	O
3	B-structure_element
-	I-structure_element
fold	I-structure_element
repeating	I-structure_element
unit	I-structure_element
of	O
40	B-residue_range
–	I-residue_range
50	I-residue_range
amino	I-residue_range
acid	I-residue_range
residues	O
characteristic	O
of	O
the	O
Ricin	B-protein_type
superfamily	I-protein_type
.	O

Each	O
repeat	B-structure_element
contains	O
two	O
pairs	O
of	O
antiparallel	B-structure_element
β	I-structure_element
-	I-structure_element
strands	I-structure_element
.	O

A	O
Dali	B-experimental_method
search	I-experimental_method
revealed	O
structural	O
homologs	O
from	O
the	O
CBM13	B-protein_type
family	O
with	O
an	O
root	B-evidence
mean	I-evidence
square	I-evidence
deviation	I-evidence
less	O
than	O
2	O
.	O
0	O
Å	O
and	O
sequence	O
identities	O
of	O
less	O
than	O
20	O
%	O
that	O
include	O
the	O
functionally	O
relevant	O
homologs	O
C	B-species
.	I-species
thermocellum	I-species
exo	B-protein_type
-	I-protein_type
β	I-protein_type
-	I-protein_type
1	I-protein_type
,	I-protein_type
3	I-protein_type
-	I-protein_type
galactanase	I-protein_type
(	O
PDB	O
code	O
3vsz	O
),	O
Streptomyces	B-species
avermitilis	I-species
β	B-protein_type
-	I-protein_type
l	I-protein_type
-	I-protein_type
arabinopyranosidase	I-protein_type
(	O
PDB	O
code	O
3a21	O
),	O
Streptomyces	B-species
lividans	I-species
xylanase	B-protein
10A	I-protein
(	O
PDB	O
code	O
,	O
1mc9	O
),	O
and	O
Streptomyces	B-species
olivaceoviridis	I-species
E	I-species
-	I-species
86	I-species
xylanase	B-protein
10A	I-protein
(	O
PDB	O
code	O
1v6v	O
).	O

The	O
Fn3	B-structure_element
module	O
displays	O
a	O
typical	O
β	B-structure_element
-	I-structure_element
sandwich	I-structure_element
fold	I-structure_element
with	O
the	O
two	O
sheets	B-structure_element
comprising	O
,	O
primarily	O
,	O
three	O
antiparallel	B-structure_element
strands	I-structure_element
in	O
the	O
order	O
β1	B-structure_element
-	I-structure_element
β2	I-structure_element
-	I-structure_element
β5	I-structure_element
in	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
1	I-structure_element
and	O
β4	B-structure_element
-	I-structure_element
β3	I-structure_element
-	I-structure_element
β6	I-structure_element
in	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
2	I-structure_element
.	O

Although	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
2	I-structure_element
presents	O
a	O
cleft	B-site
-	O
like	O
topology	O
,	O
typical	O
of	O
endo	B-protein_type
-	I-protein_type
binding	I-protein_type
CBMs	I-protein_type
,	O
the	O
surface	O
lacks	O
aromatic	O
residues	O
that	O
play	O
a	O
key	O
role	O
in	O
ligand	O
recognition	O
,	O
and	O
in	O
the	O
context	O
of	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
enzyme	B-protein
,	O
the	O
cleft	B-site
abuts	O
into	O
CtCBM13	B-structure_element
and	O
thus	O
would	O
not	O
be	O
able	O
to	O
accommodate	O
an	O
extended	O
polysaccharide	B-chemical
chain	O
(	O
see	O
below	O
).	O

In	O
the	O
structure	B-evidence
of	O
CtXyl5A	B-mutant
-	I-mutant
D	I-mutant
,	O
the	O
four	O
modules	B-structure_element
form	O
a	O
three	O
-	O
leaf	O
clover	O
-	O
like	O
structure	O
(	O
Fig	O
.	O
5	O
).	O

Between	O
the	O
interfaces	B-site
of	O
CtGH5	B-structure_element
-	I-structure_element
CBM6	I-structure_element
-	I-structure_element
CBM13	I-structure_element
there	O
are	O
a	O
number	O
of	O
interactions	O
that	O
maintain	O
the	O
modules	O
in	O
a	O
fixed	O
position	O
relative	O
to	O
each	O
other	O
.	O

The	O
interaction	O
of	O
CtGH5	B-structure_element
and	O
CtCBM6	B-structure_element
,	O
which	O
buries	O
a	O
substantial	O
apolar	B-site
solvent	I-site
-	I-site
exposed	I-site
surface	I-site
of	O
the	O
two	O
modules	O
,	O
has	O
been	O
described	O
previously	O
.	O

The	O
polar	B-bond_interaction
interactions	I-bond_interaction
between	O
these	O
two	O
modules	O
comprise	O
14	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
and	O
5	O
salt	B-bond_interaction
bridges	I-bond_interaction
.	O

The	O
apolar	B-bond_interaction
and	I-bond_interaction
polar	I-bond_interaction
interactions	I-bond_interaction
between	O
these	O
two	O
modules	O
likely	O
explaining	O
why	O
they	O
do	O
not	O
fold	O
independently	O
compared	O
with	O
other	O
glycoside	B-protein_type
hydrolases	I-protein_type
that	O
contain	O
CBMs	B-structure_element
.	O

CtCBM13	B-structure_element
acts	O
as	O
the	O
central	B-structure_element
domain	I-structure_element
,	O
which	O
interacts	B-protein_state
with	I-protein_state
CtGH5	B-structure_element
,	O
CtCBM6	B-structure_element
,	O
and	O
CtFn3	B-structure_element
via	O
2	O
,	O
5	O
,	O
and	O
4	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
,	O
respectively	O
,	O
burying	O
a	O
surface	O
area	O
of	O
∼	O
450	O
,	O
350	O
,	O
and	O
500	O
Å2	O
,	O
respectively	O
,	O
to	O
form	O
a	O
compact	B-protein_state
heterotetramer	B-oligomeric_state
.	O

With	O
respect	O
to	O
the	O
CtCBM6	B-site
-	I-site
CBM13	I-site
interface	I-site
,	O
the	O
linker	B-structure_element
(	O
SPISTGTIP	B-structure_element
)	O
between	O
the	O
two	O
modules	B-structure_element
,	O
extending	O
from	O
Ser514	B-residue_name_number
to	O
Pro522	B-residue_name_number
,	O
adopts	O
a	O
fixed	B-protein_state
conformation	I-protein_state
.	O

Such	O
sequences	O
are	O
normally	O
extremely	O
flexible	O
;	O
however	O
,	O
the	O
two	O
Ile	B-residue_name
residues	O
make	O
extensive	O
apolar	B-bond_interaction
contacts	I-bond_interaction
within	O
the	O
linker	B-structure_element
and	O
with	O
the	O
two	O
CBMs	B-structure_element
,	O
leading	O
to	O
conformational	O
stabilization	O
.	O

The	O
interactions	O
between	O
CtGH5	B-structure_element
and	O
the	O
two	O
CBMs	B-structure_element
,	O
which	O
are	O
mediated	O
by	O
the	O
tip	O
of	O
the	O
loop	B-structure_element
between	O
β	B-structure_element
-	I-structure_element
7	I-structure_element
and	O
α	B-structure_element
-	I-structure_element
7	I-structure_element
(	O
loop	B-structure_element
7	I-structure_element
)	O
of	O
CtGH5	B-structure_element
,	O
not	O
only	O
stabilize	O
the	O
trimodular	B-structure_element
clover	I-structure_element
-	O
like	O
structure	O
but	O
also	O
make	O
a	O
contribution	O
to	O
catalytic	O
function	O
.	O

Central	O
to	O
the	O
interactions	O
between	O
the	O
three	O
modules	B-structure_element
is	O
Trp285	B-residue_name_number
,	O
which	O
is	O
intercalated	B-bond_interaction
between	I-bond_interaction
the	O
two	O
CBMs	B-structure_element
.	O

The	O
Nϵ	O
of	O
this	O
aromatic	O
residue	O
makes	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
with	O
the	O
backbone	O
carbonyl	O
of	O
Val615	B-residue_name_number
and	O
Gly616	B-residue_name_number
in	O
CtCBM13	B-structure_element
,	O
and	O
the	O
indole	O
ring	O
makes	O
several	O
apolar	B-bond_interaction
contacts	I-bond_interaction
with	O
CtCBM6	B-structure_element
(	O
Pro440	B-residue_name_number
,	O
Phe489	B-residue_name_number
,	O
Gly491	B-residue_name_number
,	O
and	O
Ala492	B-residue_name_number
)	O
(	O
Fig	O
.	O
5	O
).	O

Indeed	O
,	O
loop	B-structure_element
7	I-structure_element
is	O
completely	B-protein_state
disordered	I-protein_state
in	O
the	O
truncated	B-protein_state
derivative	O
of	O
CtXyl5A	B-protein
comprising	O
CtGH5	B-structure_element
and	O
CtCBM6	B-structure_element
,	O
demonstrating	O
that	O
the	O
interactions	O
with	O
CtCBM13	B-structure_element
stabilize	O
the	O
conformation	O
of	O
this	O
loop	B-structure_element
.	O

Although	O
the	O
tip	O
of	O
loop	B-structure_element
7	I-structure_element
does	O
not	O
directly	O
contribute	O
to	O
the	O
topology	O
of	O
the	O
active	B-site
site	I-site
,	O
it	O
is	O
only	O
∼	O
12	O
Å	O
from	O
the	O
catalytic	O
nucleophile	O
Glu279	B-residue_name_number
.	O

Thus	O
,	O
any	O
perturbation	O
of	O
the	O
loop	B-structure_element
(	O
through	O
the	O
removal	B-experimental_method
of	O
CtCBM13	B-structure_element
)	O
is	O
likely	O
to	O
influence	O
the	O
electrostatic	O
and	O
apolar	O
environment	O
of	O
the	O
catalytic	O
apparatus	O
,	O
which	O
could	O
explain	O
the	O
reduction	O
in	O
activity	O
associated	O
with	O
the	O
deletion	B-experimental_method
of	O
CtCBM13	B-structure_element
.	O

Similar	O
to	O
the	O
interactions	O
between	O
CtCBM6	B-structure_element
and	O
CtCBM13	B-structure_element
,	O
there	O
are	O
extensive	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
between	O
CtCBM13	B-structure_element
and	O
CtFn3	B-structure_element
,	O
resulting	O
in	O
very	O
little	O
flexibility	O
between	O
these	O
modules	B-structure_element
.	O

As	O
stated	O
above	O
,	O
the	O
absence	B-protein_state
of	I-protein_state
CtCBM62	B-structure_element
in	O
the	O
structure	B-evidence
suggests	O
that	O
the	O
module	B-structure_element
can	O
adopt	O
multiple	O
positions	O
with	O
respect	O
to	O
the	O
rest	O
of	O
the	O
protein	O
.	O

The	O
CtCBM62	B-structure_element
,	O
by	O
binding	B-protein_state
to	I-protein_state
its	O
ligands	O
(	O
d	B-chemical
-	I-chemical
Galp	I-chemical
and	O
l	B-chemical
-	I-chemical
Arap	I-chemical
)	O
in	O
plant	B-taxonomy_domain
cell	O
walls	O
,	O
may	O
be	O
able	O
to	O
recruit	O
the	O
enzyme	O
onto	O
its	O
target	O
substrate	O
.	O

Xylans	B-chemical
are	O
not	O
generally	O
thought	O
to	O
contain	O
such	O
sugars	B-chemical
.	O

d	B-chemical
-	I-chemical
Galp	I-chemical
,	O
however	O
,	O
has	O
been	O
detected	O
in	O
xylans	B-chemical
in	O
the	O
outer	O
layer	O
of	O
cereal	B-taxonomy_domain
grains	O
and	O
in	O
eucalyptus	B-taxonomy_domain
trees	I-taxonomy_domain
,	O
which	O
are	O
substrates	O
used	O
by	O
CtXyl5A	B-protein
.	O

Thus	O
,	O
CtCBM62	B-structure_element
may	O
direct	O
the	O
enzyme	O
to	O
particularly	O
complex	O
xylans	B-chemical
containing	O
d	B-chemical
-	I-chemical
Galp	I-chemical
at	O
the	O
non	O
-	O
reducing	O
termini	O
of	O
the	O
side	O
chains	O
,	O
consistent	O
with	O
the	O
open	B-protein_state
substrate	B-site
binding	I-site
cleft	I-site
of	O
the	O
arabinoxylanase	B-protein_type
that	O
is	O
optimized	O
to	O
bind	O
highly	O
decorated	O
forms	O
of	O
the	O
hemicellulose	B-chemical
.	O

In	O
general	O
CBMs	B-structure_element
have	O
little	O
influence	O
on	O
enzyme	O
activity	O
against	O
soluble	O
substrates	O
but	O
have	O
a	O
significant	O
impact	O
on	O
glycans	B-chemical
within	O
plant	B-taxonomy_domain
cell	O
walls	O
.	O

Thus	O
,	O
the	O
role	O
of	O
CBM62	B-structure_element
will	O
likely	O
only	O
be	O
evident	O
against	O
insoluble	O
composite	O
substrates	O
.	O

Exploring	O
GH5	B-protein_type
Subfamily	I-protein_type
34	I-protein_type

CtXyl5A	B-protein
is	O
a	O
member	O
of	O
a	O
seven	O
-	O
protein	O
subfamily	O
of	O
GH5	B-protein_type
,	O
GH5_34	B-protein_type
.	O

Four	O
of	O
these	O
proteins	O
are	O
distinct	O
,	O
whereas	O
the	O
other	O
three	O
members	O
are	O
essentially	O
identical	O
(	O
derived	O
from	O
different	O
strains	O
of	O
C	B-species
.	I-species
thermocellum	I-species
).	O

To	O
investigate	O
further	O
the	O
substrate	O
specificity	O
within	O
this	O
subfamily	O
,	O
recombinant	O
forms	O
of	O
three	O
members	O
of	O
GH5_34	B-protein_type
that	O
were	O
distinct	O
from	O
CtXyl5A	B-protein
were	O
generated	O
.	O

AcGH5	B-protein
has	O
a	O
similar	O
molecular	O
architecture	O
to	O
CtXyl5A	B-protein
with	O
the	O
exception	O
of	O
an	O
additional	O
carbohydrate	B-structure_element
esterase	I-structure_element
family	I-structure_element
6	I-structure_element
module	I-structure_element
at	O
the	O
C	O
terminus	O
(	O
Fig	O
.	O
1	O
).	O

The	O
GH5_34	B-protein_type
from	O
Verrucomicrobiae	B-taxonomy_domain
bacterium	B-taxonomy_domain
,	O
VbGH5	B-protein
,	O
contains	O
the	O
GH5	B-structure_element
-	I-structure_element
CBM6	I-structure_element
-	I-structure_element
CBM13	I-structure_element
core	O
structure	O
,	O
but	O
the	O
C	O
-	O
terminal	O
Fn3	B-structure_element
-	I-structure_element
CBM62	I-structure_element
-	I-structure_element
dockerin	I-structure_element
modules	O
,	O
present	O
in	O
CtXyl5A	B-protein
,	O
are	O
replaced	O
with	O
a	O
Laminin_3_G	B-structure_element
domain	I-structure_element
,	O
which	O
,	O
by	O
analogy	O
to	O
homologous	O
domains	O
in	O
other	O
proteins	O
that	O
have	O
affinity	O
for	O
carbohydrates	B-chemical
,	O
may	O
display	O
a	O
glycan	B-chemical
binding	O
function	O
.	O

The	O
Verrucomicobiae	B-taxonomy_domain
enzyme	O
also	O
has	O
an	O
N	O
-	O
terminal	O
GH43	B-protein_type
subfamily	I-protein_type
10	I-protein_type
(	O
GH43_10	B-protein_type
)	O
catalytic	B-structure_element
module	I-structure_element
.	O

The	O
fungal	B-taxonomy_domain
GH5_34	B-protein_type
,	O
GpGH5	B-protein
,	O
unlike	O
the	O
two	O
bacterial	B-taxonomy_domain
homologs	O
,	O
comprises	O
a	O
single	O
GH5	B-protein_type
catalytic	B-structure_element
module	I-structure_element
lacking	O
all	O
of	O
the	O
other	O
accessory	O
modules	O
(	O
Fig	O
.	O
1	O
).	O

GpGh5	B-protein
is	O
particularly	O
interesting	O
as	O
Gonapodya	B-species
prolifera	I-species
is	O
the	O
only	O
fungus	B-taxonomy_domain
of	O
the	O
several	O
hundred	O
fungal	B-taxonomy_domain
genomes	O
that	O
encodes	O
a	O
GH5_34	B-protein_type
enzyme	O
.	O

In	O
fact	O
there	O
are	O
four	O
potential	O
GH5_34	B-protein_type
sequences	O
in	O
the	O
G	B-species
.	I-species
prolifera	I-species
genome	O
,	O
all	O
of	O
which	O
show	O
high	O
sequence	O
homology	O
to	O
Clostridium	B-taxonomy_domain
GH5_34	B-protein_type
sequences	O
.	O

G	B-species
.	I-species
prolifera	I-species
and	O
Clostridium	B-taxonomy_domain
occupy	O
similar	O
environments	O
,	O
suggesting	O
that	O
the	O
GpGH5_34	B-protein
gene	O
was	O
acquired	O
from	O
a	O
Clostridium	B-taxonomy_domain
species	O
,	O
which	O
was	O
followed	O
by	O
duplication	O
of	O
the	O
gene	O
in	O
the	O
fungal	B-taxonomy_domain
genome	O
.	O

The	O
sequence	O
identity	O
of	O
the	O
GH5_34	B-protein_type
catalytic	B-structure_element
modules	I-structure_element
with	O
CtXyl5A	B-protein
ranged	O
from	O
55	O
to	O
80	O
%	O
(	O
supplemental	O
Fig	O
.	O
S1	O
).	O

All	O
the	O
GH5_34	B-protein_type
enzymes	O
were	O
active	O
on	O
the	O
arabinoxylans	B-chemical
RAX	B-chemical
,	O
WAX	B-chemical
,	O
and	O
CX	B-chemical
but	O
displayed	O
no	O
activity	O
on	O
BX	B-chemical
(	O
Table	O
1	O
and	O
Fig	O
.	O
6	O
)	O
and	O
are	O
thus	O
defined	O
as	O
arabinoxylanases	B-protein_type
.	O

The	O
limit	O
products	O
generated	O
by	O
CtXyl5A	B-protein
,	O
AcGH5	B-protein
,	O
and	O
GpGH5	B-protein
comprised	O
a	O
range	O
of	O
oligosaccharides	B-chemical
with	O
some	O
high	O
molecular	O
weight	O
material	O
.	O

The	O
oligosaccharides	B-chemical
with	O
low	O
degrees	O
of	O
polymerization	O
were	O
absent	O
in	O
the	O
VbGH5	B-protein
reaction	O
products	O
.	O

However	O
,	O
the	O
enzyme	O
generated	O
a	O
large	O
amount	O
of	O
arabinose	B-chemical
,	O
which	O
was	O
not	O
produced	O
by	O
the	O
other	O
arabinoxylanases	B-protein_type
.	O

Given	O
that	O
GH43_10	B-protein_type
is	O
predominantly	O
an	O
arabinofuranosidase	B-protein_type
subfamily	O
of	O
GH43	B-protein_type
,	O
the	O
arabinose	B-chemical
generated	O
by	O
VbGH5	B-protein
is	O
likely	O
mediated	O
by	O
the	O
N	O
-	O
terminal	O
catalytic	B-structure_element
module	I-structure_element
(	O
see	O
below	O
).	O

Kinetic	O
analysis	O
showed	O
that	O
AcGH5	B-protein
displayed	O
similar	O
activity	O
to	O
CtXyl5A	B-protein
against	O
both	O
WAX	B-chemical
and	O
RAX	B-chemical
and	O
was	O
2	O
-	O
fold	O
less	O
active	O
against	O
CX	B-chemical
.	O

When	O
initially	O
measuring	O
the	O
activity	O
of	O
wild	B-protein_state
type	I-protein_state
VbGH5	B-protein
against	O
the	O
different	O
substrates	O
,	O
no	O
clear	O
data	O
could	O
be	O
obtained	O
,	O
regardless	O
of	O
the	O
concentration	O
of	O
enzyme	O
used	O
the	O
reaction	O
appeared	O
to	O
cease	O
after	O
a	O
few	O
minutes	O
.	O

We	O
hypothesized	O
that	O
the	O
N	O
-	O
terminal	O
GH43_10	B-protein_type
rapidly	O
removed	O
single	O
arabinose	B-chemical
decorations	O
from	O
the	O
arabinoxylans	B-chemical
depleting	O
the	O
substrate	O
available	O
to	O
the	O
arabinoxylanase	B-protein_type
,	O
explaining	O
why	O
this	O
activity	O
was	O
short	O
lived	O
.	O

To	O
test	O
this	O
hypothesis	O
,	O
the	O
conserved	B-protein_state
catalytic	O
base	O
(	O
Asp45	B-residue_name_number
)	O
of	O
the	O
GH43_10	B-structure_element
module	O
of	O
VbGH5	B-protein
was	O
substituted	B-experimental_method
with	I-experimental_method
alanine	B-residue_name
,	O
which	O
is	O
predicted	O
to	O
inactivate	O
this	O
catalytic	B-structure_element
module	I-structure_element
.	O

The	O
D45A	B-mutant
mutant	B-protein_state
did	O
not	O
produce	O
arabinose	B-chemical
consistent	O
with	O
the	O
arabinofuranosidase	B-protein_type
activity	O
displayed	O
by	O
the	O
GH43_10	B-structure_element
module	O
in	O
the	O
wild	B-protein_state
type	I-protein_state
enzyme	O
(	O
Fig	O
.	O
6	O
).	O

The	O
kinetics	B-evidence
of	O
the	O
GH5_34	B-protein_type
arabinoxylanase	B-protein_type
catalytic	B-structure_element
module	I-structure_element
was	O
now	O
measurable	O
,	O
and	O
activities	O
were	O
determined	O
to	O
be	O
between	O
∼	O
6	O
-	O
and	O
10	O
-	O
fold	O
lower	O
than	O
that	O
of	O
CtXyl5A	B-protein
.	O

Interestingly	O
,	O
the	O
fungal	B-taxonomy_domain
arabinoxylanase	B-protein_type
displays	O
the	O
highest	O
activities	O
against	O
WAX	B-chemical
and	O
RAX	B-chemical
,	O
∼	O
4	O
-	O
and	O
6	O
-	O
fold	O
higher	O
,	O
respectively	O
,	O
than	O
CtXyl5A	B-protein
;	O
however	O
,	O
there	O
is	O
very	O
little	O
difference	O
in	O
the	O
activity	O
between	O
the	O
eukaryotic	B-taxonomy_domain
and	O
prokaryotic	B-taxonomy_domain
enzymes	O
against	O
CX	B-chemical
.	O

Attempts	O
to	O
express	O
individual	O
modules	O
of	O
a	O
variety	O
of	O
truncations	O
of	O
AcGH5	B-protein
and	O
VbGH5	B-protein
were	O
unsuccessful	O
.	O

This	O
may	O
indicate	O
that	O
the	O
individual	O
modules	O
can	O
only	O
fold	O
correctly	O
when	O
incorporated	O
into	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
enzyme	O
,	O
demonstrating	O
the	O
importance	O
of	O
intermodule	O
interactions	O
to	O
maintain	O
the	O
structural	O
integrity	O
of	O
these	O
enzymes	O
.	O

Products	O
profile	O
generated	O
of	O
GH5_34	B-protein_type
enzymes	O
.	O

The	O
enzymes	O
at	O
1	O
μm	O
were	O
incubated	B-experimental_method
with	O
the	O
four	O
different	O
xylans	B-chemical
at	O
1	O
%	O
in	O
50	O
mm	O
sodium	O
phosphate	O
buffer	O
for	O
16	O
h	O
at	O
37	O
°	O
C	O
(	O
GpGH5	B-protein
,	O
VbGH5	B-protein
,	O
and	O
AcGH5	B-protein
)	O
or	O
60	O
°	O
C	O
.	O

The	O
limit	O
products	O
were	O
separated	O
by	O
TLC	B-experimental_method
.	O

The	O
xylooligosaccharide	B-chemical
standards	O
(	O
X	O
)	O
are	O
indicated	O
by	O
their	O
degrees	O
of	O
polymerization	O
.	O

A	O
characteristic	O
feature	O
of	O
enzymes	O
that	O
attack	O
the	O
plant	B-taxonomy_domain
cell	O
wall	O
is	O
their	O
complex	O
molecular	O
architecture	O
.	O

The	O
CBMs	B-structure_element
in	O
these	O
enzymes	O
generally	O
play	O
a	O
role	O
in	O
substrate	O
targeting	O
and	O
are	O
appended	O
to	O
the	O
catalytic	B-structure_element
modules	I-structure_element
through	O
flexible	B-structure_element
linker	I-structure_element
sequences	I-structure_element
.	O

CtXyl5A	B-protein
provides	O
a	O
rare	O
visualization	O
of	O
the	O
structure	B-evidence
of	O
multiple	O
modules	O
within	O
a	O
single	O
enzyme	O
.	O

The	O
central	O
feature	O
of	O
these	O
data	O
is	O
the	O
structural	O
role	O
played	O
by	O
two	O
of	O
the	O
CBMs	B-structure_element
,	O
CtCBM6	B-structure_element
and	O
CtCBM13	B-structure_element
,	O
in	O
maintaining	O
the	O
active	B-protein_state
conformation	O
of	O
the	O
catalytic	B-structure_element
module	I-structure_element
,	O
CtGH5	B-structure_element
.	O

The	O
crystallographic	B-evidence
data	I-evidence
described	O
here	O
are	O
supported	O
by	O
biochemical	O
data	O
showing	O
either	O
that	O
these	O
two	O
modules	O
do	O
not	O
bind	O
to	O
glycans	B-chemical
(	O
CtCBM13	B-structure_element
)	O
or	O
that	O
the	O
recognition	O
of	O
the	O
non	O
-	O
reducing	O
end	O
of	O
xylan	B-chemical
or	O
cellulose	B-chemical
chains	O
(	O
CtCBM6	B-structure_element
)	O
is	O
unlikely	O
to	O
be	O
biologically	O
significant	O
.	O

It	O
should	O
be	O
emphasized	O
,	O
however	O
,	O
that	O
glycan	B-chemical
binding	O
and	O
substrate	O
targeting	O
may	O
only	O
be	O
evident	O
in	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
enzyme	O
acting	O
on	O
highly	O
complex	O
structures	O
such	O
as	O
the	O
plant	B-taxonomy_domain
cell	O
wall	O
,	O
as	O
observed	O
recently	O
by	O
a	O
CBM46	B-structure_element
module	O
in	O
the	O
Bacillus	B-taxonomy_domain
xyloglucanase	B-protein_type
/	O
mixed	B-protein_type
linked	I-protein_type
glucanase	I-protein_type
BhCel5B	B-protein
.	O

CtXyl5A	B-protein
is	O
a	O
member	O
of	O
GH5	B-protein_type
that	O
contains	O
6644	O
members	O
.	O

CtXyl5A	B-protein
is	O
a	O
member	O
of	O
subfamily	O
GH5_34	B-protein_type
.	O

Despite	O
differences	O
in	O
sequence	O
identity	O
all	O
of	O
the	O
homologs	O
were	O
shown	O
to	O
be	O
arabinoxylanases	B-protein_type
.	O

Consistent	O
with	O
the	O
conserved	O
substrate	O
specificity	O
,	O
all	O
members	O
of	O
GH5_34	B-protein_type
contained	O
the	O
specificity	B-site
determinants	I-site
Glu68	B-residue_name_number
,	O
Tyr92	B-residue_name_number
,	O
and	O
Asn139	B-residue_name_number
,	O
which	O
make	O
critical	O
interactions	O
with	O
the	O
xylose	B-chemical
or	O
arabinose	B-chemical
in	O
the	O
−	B-site
2	I-site
*	I-site
subsite	I-site
,	O
which	O
are	O
1	O
,	O
3	O
-	O
linked	O
to	O
the	O
xylose	B-chemical
positioned	O
in	O
the	O
active	B-site
site	I-site
.	O

The	O
presence	O
of	O
a	O
CBM62	B-structure_element
in	O
CtXyl5A	B-protein
and	O
AcGH5	B-protein
suggests	O
that	O
these	O
enzymes	O
target	O
highly	O
complex	O
xylans	B-chemical
that	O
contain	O
d	B-chemical
-	I-chemical
galactose	I-chemical
in	O
their	O
side	O
chains	O
.	O

The	O
absence	B-protein_state
of	I-protein_state
a	O
“	O
non	O
-	O
structural	O
”	O
CBM	B-structure_element
in	O
GpGH5	B-protein
may	O
indicate	O
that	O
this	O
arabinoxylanase	B-protein_type
is	O
designed	O
to	O
target	O
simpler	O
arabinoxylans	B-chemical
present	O
in	O
the	O
endosperm	O
of	O
cereals	B-taxonomy_domain
.	O

Although	O
the	O
characterization	O
of	O
all	O
members	O
of	O
GH5_34	B-protein_type
suggests	O
that	O
this	O
subfamily	O
is	O
monospecific	O
,	O
differences	O
in	O
specificity	O
are	O
observed	O
in	O
other	O
subfamilies	O
of	O
GHs	B-protein_type
including	O
GH43	B-protein_type
and	O
GH5	B-protein_type
.	O

Thus	O
,	O
as	O
new	O
members	O
of	O
GH5_34	B-protein_type
are	O
identified	O
from	O
genomic	O
sequence	O
data	O
and	O
subsequently	O
characterized	O
,	O
the	O
specificity	O
of	O
this	O
family	O
may	O
require	O
reinterpretation	O
.	O

An	O
intriguing	O
feature	O
of	O
VbGH5	B-protein
is	O
that	O
the	O
limited	O
products	O
generated	O
by	O
this	O
enzymes	O
are	O
much	O
larger	O
than	O
those	O
produced	O
by	O
the	O
other	O
arabinoxylanases	B-protein_type
.	O

This	O
suggests	O
that	O
although	O
arabinose	B-chemical
decorations	O
contribute	O
to	O
enzyme	O
specificity	O
(	O
VbGH5	B-protein
is	O
not	O
active	O
on	O
xylans	B-chemical
lacking	O
arabinose	B-chemical
side	O
chains	O
),	O
the	O
enzyme	O
requires	O
other	O
specificity	O
determinants	O
that	O
occur	O
less	O
frequently	O
in	O
arabinoxylans	B-chemical
.	O

This	O
has	O
some	O
resonance	O
with	O
a	O
recently	O
described	O
GH98	B-protein_type
xylanase	B-protein_type
that	O
also	O
exploits	O
specificity	O
determinants	O
that	O
occur	O
infrequently	O
and	O
are	O
only	O
evident	O
in	O
highly	O
complex	O
xylans	B-chemical
(	O
e	O
.	O
g	O
.	O
CX	B-chemical
).	O

To	O
conclude	O
,	O
this	O
study	O
provides	O
the	O
molecular	O
basis	O
for	O
the	O
specificity	O
displayed	O
by	O
arabinoxylanases	B-protein_type
.	O

Substrate	O
specificity	O
is	O
dominated	O
by	O
the	O
pocket	B-site
that	O
binds	O
single	O
arabinose	B-chemical
or	O
xylose	B-chemical
side	O
chains	O
.	O

The	O
open	B-protein_state
xylan	B-site
binding	I-site
cleft	I-site
explains	O
why	O
the	O
enzyme	O
is	O
able	O
to	O
attack	O
highly	O
decorated	O
forms	O
of	O
the	O
hemicellulose	B-chemical
.	O

It	O
is	O
also	O
evident	O
that	O
appending	O
additional	O
catalytic	B-structure_element
modules	I-structure_element
and	O
CBMs	B-structure_element
onto	O
the	O
core	O
components	O
of	O
these	O
enzymes	O
generates	O
bespoke	O
arabinoxylanases	B-protein_type
with	O
activities	O
optimized	O
for	O
specific	O
functions	O
.	O

The	O
specificities	O
of	O
the	O
arabinoxylanases	B-protein_type
described	O
here	O
are	O
distinct	O
from	O
the	O
classical	O
endo	B-protein_type
-	I-protein_type
xylanases	I-protein_type
and	O
thus	O
have	O
the	O
potential	O
to	O
contribute	O
to	O
the	O
toolbox	O
of	O
biocatalysts	O
required	O
by	O
industries	O
that	O
exploit	O
the	O
plant	B-taxonomy_domain
cell	O
wall	O
as	O
a	O
sustainable	O
substrate	O
.	O

Data	B-evidence
collection	I-evidence
and	I-evidence
refinement	I-evidence
statistics	I-evidence

CtXyl5A	B-mutant
-	I-mutant
D	I-mutant
GH5	B-complex_assembly
-	I-complex_assembly
CBM6	I-complex_assembly
-	I-complex_assembly
Arap	I-complex_assembly
GH5	B-complex_assembly
-	I-complex_assembly
CBM6	I-complex_assembly
-	I-complex_assembly
Xylp	I-complex_assembly
GH5	B-complex_assembly
-	I-complex_assembly
CBM6	I-complex_assembly
-	I-complex_assembly
(	I-complex_assembly
Araf	I-complex_assembly
-	I-complex_assembly
Xylp4	I-complex_assembly
)	I-complex_assembly
Data	O
collection	O
Source	O
ESRF	O
-	O
ID14	O
-	O
1	O
Diamond	O
I04	O
–	O
1	O
Diamond	O
I24	O
Diamond	O
I02	O
Wavelength	O
(	O
Å	O
)	O
0	O
.	O
9334	O
0	O
.	O
9173	O
0	O
.	O
9772	O
0	O
.	O
9791	O
Space	O
group	O
P21212	O
P212121	O
P212121	O
P212121	O
Cell	O
dimensions	O
a	O
,	O
b	O
,	O
c	O
(	O
Å	O
)	O
147	O
.	O
4	O
,	O
191	O
.	O
7	O
,	O
50	O
.	O
7	O
67	O
.	O
1	O
,	O
72	O
.	O
4	O
,	O
109	O
.	O
1	O
67	O
.	O
9	O
,	O
72	O
.	O
5	O
,	O
109	O
.	O
5	O
76	O
.	O
3	O
,	O
123	O
.	O
2	O
,	O
125	O
.	O
4	O
α	O
,	O
β	O
,	O
γ	O
(°)	O
90	O
,	O
90	O
,	O
90	O
90	O
,	O
90	O
,	O
90	O
90	O
,	O
90	O
,	O
90	O
90	O
,	O
90	O
,	O
90	O
No	O
.	O
of	O
measured	O
reflections	O
244	O
,	O
475	O
(	O
29	O
,	O
324	O
)	O
224	O
,	O
842	O
(	O
11	O
,	O
281	O
)	O
152	O
,	O
004	O
(	O
4	O
,	O
996	O
)	O
463	O
,	O
237	O
(	O
23	O
,	O
068	O
)	O
No	O
.	O
of	O
independent	O
reflections	O
42246	O
(	O
5	O
,	O
920	O
)	O
63	O
,	O
523	O
(	O
3	O
,	O
175	O
)	O
42	O
,	O
716	O
(	O
2	O
,	O
334	O
)	O
140	O
,	O
288	O
(	O
6	O
,	O
879	O
)	O
Resolution	O
(	O
Å	O
)	O
50	O
.	O
70	O
–	O
2	O
.	O
64	O
(	O
2	O
.	O
78	O
–	O
2	O
.	O
64	O
)	O
44	O
.	O
85	O
–	O
1	O
.	O
65	O
(	O
1	O
.	O
68	O
–	O
1	O
.	O
65	O
)	O
45	O
.	O
16	O
–	O
1	O
.	O
90	O
(	O
1	O
.	O
94	O
–	O
1	O
.	O
90	O
)	O
48	O
.	O
43	O
–	O
1	O
.	O
65	O
(	O
1	O
.	O
68	O
–	O
1	O
.	O
65	O
)	O
Rmerge	O
(%)	O
16	O
.	O
5	O
(	O
69	O
.	O
5	O
)	O
6	O
.	O
7	O
(	O
65	O
.	O
1	O
)	O
2	O
.	O
8	O
(	O
8	O
.	O
4	O
)	O
5	O
.	O
7	O
(	O
74	O
.	O
9	O
)	O
CC1	O
/	O
2	O
0	O
.	O
985	O
(	O
0	O
.	O
478	O
)	O
0	O
.	O
998	O
(	O
0	O
.	O
594	O
)	O
0	O
.	O
999	O
(	O
0	O
.	O
982	O
)	O
0	O
.	O
998	O
(	O
0	O
.	O
484	O
)	O
I	O
/	O
σI	O
8	O
.	O
0	O
(	O
2	O
.	O
0	O
)	O
13	O
(	O
1	O
.	O
6	O
)	O
26	O
.	O
6	O
(	O
8	O
.	O
0	O
)	O
11	O
.	O
2	O
(	O
1	O
.	O
6	O
)	O
Completeness	O
(%)	O
98	O
.	O
5	O
(	O
96	O
.	O
4	O
)	O
98	O
.	O
5	O
(	O
99	O
.	O
4	O
)	O
98	O
.	O
6	O
(	O
85	O
.	O
0	O
)	O
98	O
.	O
8	O
(	O
99	O
.	O
4	O
)	O
Redundancy	O
5	O
.	O
8	O
(	O
5	O
.	O
0	O
)	O
3	O
.	O
5	O
(	O
3	O
.	O
6	O
)	O
3	O
.	O
6	O
(	O
2	O
.	O
1	O
)	O
3	O
.	O
3	O
(	O
3	O
.	O
4	O
)	O
Refinement	O
Rwork	B-evidence
/	O
Rfree	B-evidence
23	O
.	O
7	O
/	O
27	O
.	O
8	O
12	O
.	O
2	O
/	O
17	O
.	O
0	O
12	O
.	O
9	O
/	O
16	O
.	O
1	O
14	O
.	O
5	O
/	O
19	O
.	O
9	O
No	O
.	O
atoms	O
Protein	O
5446	O
3790	O
3729	O
7333	O
Ligand	O
19	O
20	O
20	O
92	O
Water	O
227	O
579	O
601	O
923	O
B	O
-	O
factors	O
Protein	O
41	O
.	O
6	O
17	O
.	O
8	O
15	O
.	O
8	O
21	O
.	O
0	O
Ligand	O
65	O
.	O
0	O
19	O
.	O
4	O
24	O
.	O
2	O
39	O
.	O
5	O
Water	O
35	O
.	O
4	O
38	O
.	O
5	O
32	O
.	O
2	O
37	O
.	O
6	O
R	O
.	O
m	O
.	O
s	O
deviations	O
Bond	O
lengths	O
(	O
Å	O
)	O
0	O
.	O
008	O
0	O
.	O
015	O
0	O
.	O
012	O
0	O
.	O
012	O
Bond	O
angles	O
(°)	O
1	O
.	O
233	O
1	O
.	O
502	O
1	O
.	O
624	O
1	O
.	O
554	O
Protein	O
Data	O
Bank	O
code	O
5G56	O
5LA0	O
5LA1	O
2LA2	O