Evidence that GH115 α-glucuronidase activity, which is required to degrade plant biomass, is dependent on conformational flexibility

Article abstract of DOI:10.1074/jbc.M113.525295

The microbial degradation of the plant cell wall is an important biological process that is highly relevant to environmentally significant industries such as the bioenergy and biorefining sectors. A major component of the wall is glucuronoxylan, a β1,4-linked xylose polysaccharide that is decorated with α-linked glucuronic and/or methylglucuronic acid (GlcA/MeGlcA). Recently three members of a glycoside hydrolase family, GH115, were shown to hydrolyze MeGlcA side chains from the internal regions of xylan, an activity that has not previously been described. Here we show that a dominant member of the human microbiota, Bacteroides ovatus, contains a GH115 enzyme, BoAgu115A, which displays glucuronoxylan α-(4-Omethyl)-glucuronidase activity. The enzyme is significantly more active against substrates in which the xylose decorated with GlcA/MeGlcA is flanked by one or more xylose residues. The crystal structure of BoAgu115A revealed a four-domain protein in which the active site, comprising a pocket that abuts a cleft-like structure, is housed in the second domain that adopts a TIM barrel-fold. The third domain, a five-helical bundle, and the C-terminal β-sandwich domain make inter-chain contacts leading to protein dimerization. Informed by the structure of the enzyme in complex with GlcA in its open ring form, in conjunction with mutagenesis studies, the potential substrate binding and catalytically significant amino acids were identified. Based on the catalytic importance of residues located on a highly flexible loop, the enzyme is required to undergo a substantial conformational change to form a productive Michaelis complex with glucuronoxylan.

Full text of DOI:10.1074/jbc.M113.525295

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 1, pp. 53–64, January 3, 2014
Author’s Choice
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Evidence That GH115 ␣-Glucuronidase Activity, Which Is
Required to Degrade Plant Biomass, Is Dependent on
Conformational Flexibility*
Received for publication, October 10, 2013, and in revised form, November 4, 2013 Published, JBC Papers in Press, November 8, 2013, DOI 10.1074/jbc.M113.525295
Artur Rogowski‡, Arnaud Baslé‡, Cristiane S. Farinas§1, Alexandra Solovyova‡, Jennifer C. Mortimer§, Paul Dupree§,
Harry J. Gilbert‡2, and David N. Bolam‡3
From the ‡Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH
United Kingdom and the §Department of Biochemistry, University of Cambridge, Tennis Court Road,
Cambridge CB2 1QW, United Kingdom
Background: The structure of GH115 glucuronidases that remove glucuronic acid from xylan chains is unknown.
Results: Bacteroides ovatus GH115 glucuronidase is a dimeric enzyme that contains a flexible active site pocket.
Conclusion: The assembly of the catalytic apparatus of the glucuronidase requires substantial conformational changes.
Significance: Conformational changes are highly unusual in glycoside hydrolases.
The microbial degradation of the plant cell wall is an impor-
tant biological process that is highly relevant to environmentally
significant industries such as the bioenergy and biorefining
sectors. A major component of the wall is glucuronoxylan,
a ␤1,4-linked xylose polysaccharide that is decorated with
␣-linked glucuronic and/or methylglucuronic acid (GlcA/
MeGlcA). Recently three members of a glycoside hydrolase fam-
ily, GH115, were shown to hydrolyze MeGlcA side chains from
the internal regions of xylan, an activity that has not previously
been described. Here we show that a dominant member of the
human microbiota, Bacteroides ovatus, contains a GH115
enzyme, BoAgu115A, which displays glucuronoxylan ␣-(4-O-
methyl)-glucuronidase activity. The enzyme is significantly
more active against substrates in which the xylose decorated
with GlcA/MeGlcA is flanked by one or more xylose residues.
The crystal structure of BoAgu115A revealed a four-domain
protein in which the active site, comprising a pocket that abuts a
cleft-like structure, is housed in the second domain that adopts
a TIM barrel-fold. The third domain, a five-helical bundle, and
the C-terminal ␤-sandwich domain make inter-chain contacts
leading to protein dimerization. Informed by the structure of
the enzyme in complex with GlcA in its open ring form, in con-
junction with mutagenesis studies, the potential substrate bind-
ing and catalytically significant amino acids were identified.
Based on the catalytic importance of residues located on a highly
flexible loop, the enzyme is required to undergo a substantial
conformational change to form a productive Michaelis complex
with glucuronoxylan.
The microbial degradation of plant biomass in soil is critical
to the carbon cycle and is of increasing importance in environ-
mentally significant industries, particularly the bioenergy and
biorefining sectors, where the plant cell wall is the key substrate
(1). Furthermore, dietary plant glycans that are not degraded by
mammalian intestinal enzymes are the major nutrient available
to the microbial communities of the fore-stomach or rumen in
ruminants, and the large bowel in monogastric mammals (2). In
humans the fermentation of dietary glycans by the microbes of
the large bowel (defined as the microbiota) contributes up to
10% of daily calories (3). The microbiota also has a significant
impact on the host through interactions with the immune sys-
tem, which in turn can affect human health (see Ref. 4 for
review). Plant cell walls present a highly complex substrate, and
thus microorganisms that utilize these composite structures
express an array of catabolic enzymes, primarily glycoside
hydrolases, but also carbohydrate esterases and polysaccharide
lyases, which degrade the structural polysaccharides into their
component monosaccharides (see Refs. 5 and 6 for review).
These enzymes are grouped into sequence-based families in the
CAZy database (glycoside hydrolase and carbohydrate esterase
families are defined as GH and CE, respectively) (7).
Xylan, the major matrix polysaccharide of plant cell walls,
consists of ␤1,4-linked D-xylose residues decorated at O2
and/or O3 with L-arabinofuranose and acetyl groups, and
exclusively at O2 with ␣-D-glucuronic acid (GlcA) or 4-O-
methyl-GlcA (MeGlcA) (Fig. 1) (8). These decorated regions
make the polymer backbone recalcitrant to attack by most
endo-xylanases and also block the action of xylosidases. To
overcome this bottleneck, the side chains are removed by spe-
cific debranching enzymes, acetyl esterases, ␣-L-arabinofura-
* This work was supported by Biotechnology and Biological Sciences
Research Council (BBSRC) Grant BB/G016240/1 BBSRC Sustainable Bioen-
ergy Centre (BSBEC) Cell Wall Sugars Programme and Wellcome Trust nosidases, and ␣-glucuronidases (reviewed in Refs. 5 and 6).
Grant WT097907MA.
The majority of ␣-glucuronidases that hydrolyze the GlcA and
Author’s Choice—Final version full access.
1 Present address: Embrapa Instrumentation, Rua XV de Novembro1452, CEP
MeGlcA decorations are located in GH67 (9, 10). These
enzymes, however, do not attack glucuronoxylan, they only
remove uronic acid from the non-reducing end of glucurono-
xylooligosaccharides (9, 10). The structure of several GH67
enzymes show that O4 of the xylose decorated with GlcA is
13560–970, São Carlos, SP, Brazil.
2 To whom correspondence may be addressed. E-mail: harry.gilbert@
ncl.ac.uk.
3 To whom correspondence may be addressed. E-mail: david.bolam@
ncl.ac.uk.
JANUARY 3, 2014•VOLUME 289•NUMBER 1
JOURNAL OF BIOLOGICAL CHEMISTRY 53

GH115 ␣-Glucuronidase
restriction enzyme sites. The amplified DNA was cloned into
the Escherichia coli expression vector pET28a (Novagen) such
that the encoded recombinant proteins contain an N-terminal
His6 tag. The recombinant proteins were produced in E. coli
BL21 DE3 (Novagen) cells, harboring appropriate pET-based
plasmids, and cultured in LB broth containing kanamycin (50
␮g/ml) at 37 °C. Cells were grown to mid-exponential phase
(A600 of 0.6), at which point isopropyl ␤-D-thiogalactopyrano-
side was added to a final concentration of 1 mM, and the cul-
tures were incubated for a further 16 h at 16 °C. The cells were
harvested by centrifugation, and His6-tagged recombinant pro-
tein was purified from cell-free extracts by immobilized metal
affinity chromatography (IMAC) using standard methodology
(18). For crystallographic studies BoAgu115A was further puri-
fied by size exclusion chromatography using a Superdex 75 col-
umn. All proteins were purified to electrophoretic homogene-
ity as judged by SDS-PAGE.
FIGURE 1. Schematic of xylan. The schematic shows a basic xylan structure, the
enzyme classes that hydrolyze the various linkages, and their respective major
CAZy families. Simple glucuronoxylans predominate in the walls of dicots,
whereas monocots usually contain more complex glucuronoarabinoxylans.
pointing at the surface of the enzymes, explaining why these
␣-glucuronidases can only target uronic acids attached to the
non-reducing end of xylan chains (10, 11). Recently, two micro-
bial eukaryotic enzymes from GH115 were shown to be
␣-glucuronidases that could cleave GlcA and MeGlcA from
both the non-reducing end and internal xylose units of xylan
and xylo-oligosaccharides (12, 13), displaying a single displace-
ment acid base-assisted (inverting) mechanism, which results
in inversion of the anomeric configuration of the cleaved uronic
acid (14). The two enzymes had a higher specific activity against
glucurono-xylooligosaccharides compared with glucuronoxy-
lan, although no kinetic parameters for these enzymes were
reported. As no crystal structure for a GH115 enzyme is avail-
able, the structural basis for the specificity of these ␣-glucuroni-
dases is unknown.
Mutagenesis—Site-directed mutagenesis was conducted using
the PCR-based QuikChange site-directed mutagenesis kit
(Stratagene) according to the manufacturer’s instructions,
using the plasmid encoding BoAgu115A as the template and
appropriate primer pairs.
Nomenclature of Oligosaccharides—The oligosaccharides
are defined as follows: for homopolymers of ␤1,4-linked xylose
residues either the full name of the saccharide is used or the
degree of polymerization is indicated as a subscript (e.g. the
tetrasaccharide of xylose is denoted as xylotetraose or X4).
Glucurono-xylooligosaccharides are identified by their sequence
(unless otherwise stated) in which X is an undecorated xylose and
U is a xylose containing an ␣1,2-linked [Me]GlcA (e.g. UXXX is
xylotetraose in which the xylose at the non-reducing end contains
an ␣1,2-linked [Me]GlcA).
To date, GH115 contains 237 members of which the majority
(177) are bacterial, although only a single prokaryotic enzyme
(from Streptomyces pristinaespiralis) has been shown to display
␣-glucuronidase activity (15). Bacteroidetes is a major phylum
in the human gut microbiota. These organisms, exemplified by
Bacteroides ovatus, are capable of degrading an extensive range
of human and plant glycans (16). This glycan-degrading capac-
ity is orchestrated by physically linked and co-regulated genes
that are known as polysaccharide utilization loci (see Ref. 17 for
review). The genome of B. ovatus contains seven open reading
frames that are annotated as GH115s, two of which are in a
polysaccharide utilization locus that is up-regulated during
growth on complex xylans (16). Here we report the biochemical
characterization and structure of one of these enzymes,
BACOVA_03449, defined henceforth as BoAgu115A. The
enzyme was shown to be a glucuronoxylan-specific ␣-glucu-
ronidase that displayed a strong preference for uronic acids that
decorate internal xylose units. The enzyme displays the same
core-fold as GH67 ␣-glucuronidases, however, the active site
residues are not conserved in the two enzymes. Mutagenesis
and structural data indicate that a significant conformational
change is required to assemble the catalytic apparatus in
BoAgu115A.
Enzyme Assays—Substrates consisted of glucurono-xylans or
glucurono-xylooligosaccharides. The glucurono-xylooligosac-
charides UX and UXXX were purchased from Megazyme Inter-
national (County Wicklow, Ireland), whereas UXX and XUXX
were made as follows: 5 g of birch wood xylan (Sigma) was
digested to completion with either CjXyn10A (19) or
NpXyn11A (20) xylanases in 50 mM sodium phosphate, 12 mM
citrate buffer, pH 6.5, containing 1 mg/ml of BSA (PC buffer) to
completion. The glucurono-xylooligosaccharides generated by
CjXyn10A, UXX, or NpXyn11A, XUXX, were purified from
neutral xylooligosaccharides using Dowex chromatography (9).
The structure of the products were confirmed by incubation
with CjGlcA67A, which generated xylotriose from the
CjXyn10A product but no undecorated xylooligosaccharide
from the NpXyn11A product, whereas BoAgu115A released
xylotriose and xylotetraose from glucurono-xylooligosaccha-
rides generated by the GH10 and GH11 xylanases, respectively.
The deduced structure of the two glucurono-xylooligosaccha-
rides is entirely consistent with the glucuronoxylan binding
mode of the two xylanases (21, 22). Enzyme assays, in which
polysaccharides or glucurono-xylooligosaccharides were the
EXPERIMENTAL PROCEDURES
Cloning, Expression, and Purification of GH115 Enzymes— substrates, were carried out in PC buffer at 37 °C using enzyme
The open reading frame encoding mature forms (and truncated purified to electrophoretic homogeneity by immobilized metal
derivatives of BoAgu115A) of the seven B. ovatus GH115 pro- ion affinity chromatography. The concentration of enzyme var-
teins were amplified from genomic DNA (B. ovatus ATCC ied from 10 nM for the wild type glucuronidase to 10 ␮M for the
8483) by PCR using primers that introduce NheI and XhoI least active variants of BoAgu115A. For kinetic assays, glucu-
54 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 289•NUMBER 1•JANUARY 3, 2014

GH115 ␣-Glucuronidase
TABLE 1
ronic acid was detected using the ␣-D-glucuronidase assay kit in
Data statistics and refinement details
which the uronic acid released is oxidized to glucarate with the
ϩ
03449
Selenomethionine
concomitant reduction of NAD to NADH, which was moni-
Native
Ligand
tored continuously at 340 nm and quantified using a molar
extinction coefficient of 6220 MϪ1 cmϪ1. The molar concentra-
tion of the GlcA/MeGlcA in the glucuronoxylans was quanti-
fied by digesting 100 ␮g of the polysaccharides to completion
with BoAgu115A. To measure the activity of the BoAgu115A
mutants the xylotetraose reaction product released from
XUXX was monitored by high performance anion-exchange
chromatography (HPAEC) as described previously (18). The
reaction was carried out in 20 mM sodium phosphate buffer, pH
7.0, at a substrate concentration (1 mM) that was well below the
KM. Thus, the initial rate of hydrolysis of the glucurono-xylo-
oligosaccharides gives a direct readout of kcat/KM (19). The glu-
curonidase-catalyzed reactions were also subjected to Polysac-
charide Analysis using Carbohydrate gel Electrophoresis
(PACE)4 as follows: a alcohol-insoluble residue was prepared
from mature Arabidopsis thaliana wild type and gux1gux2
stems as well as wild type willow, barley, sugar cane, and Mis-
canthus stems, as previously described (23). Alcohol-insoluble
residue (500 ␮g) was pre-treated with 20 ␮l of 4 M NaOH for 1 h,
neutralized with HCl, and ammonium acetate buffer, pH 6.0,
added to a final concentration of 0.1 M and a final volume of 500
␮l. The Arabidopsis alcohol-insoluble residue was digested to
completion with xylanases (CjXyn10A and NpXyn11A), a
glucuronoxylanase (BoGH30; Bacova_03432), and GH67 and
GH115 ␣-glucuronidases (CjGlcA67A and BoAgu115A) as
stated in the text and then dried in vacuum, whereas the other
xylans were just digested with BoAgu115A. Released mono-
and oligosaccharides were labeled with 8-aminonaphthalene-
1,3,6-trisulfonic acid (Invitrogen) and separated by gel electro-
phoresis as previously described (24). Xylooligosaccharides
(Xyl1–6, Megazyme Int.) were derivatized alongside each set of
samples and run as standards within each gel.
Data statisticsa
Beamline
IO2
IO2
IO4-1
Date
18/12/10
0.9795
27/02/11
0.9795
16/10/11
0.9173
2.14
Wavelength (Å)
Resolution (Å)
65.52-2.65
67.05-3.00
(3.16-3.00)
P212121
(2.79-2.65)
(2.26-2.14)
Space group
P212121
P212121
Unit cell parameters
a (Å)
75.39
76.13
72.04
b (Å)
131.68
199.40
132.75
201.14
130.29
190.17
90.0,90.0,90.0
1,784,954
48
c (Å)
␣, ␤, ␥ (°)
90.0,90.0,90.0 90.0,90.0,90.0
Unit cell volume (Å3)
Solvent content (%)
No. of measured reflections
2,093,534
56
2,032,772
54
219,395
1,240,973
41,710
272,938
93,249
94.4 (91.5)
2.9 (2.7)
9.6 (42.5)
9.3 (2.5)
NA
No. of independent reflections 56,839
Completeness (%)
Redundancy
97.5 (94.5)
100.0 (100.0)
29.8 (29.9)
19.8 (57.3)
20.2 (8)
3.9 (3.8)
R
merge (%)
12.6 (49.7)
8.5 (2.4)
͗I͘/͗␴(I)͘
Anomalous completeness (%) NA
100.0 (100.0)
15.5 (15.4)
Anomalous redundancy
NA
NA
Refinement statisticsa
R
work (%)
20.34
26.59
NAb
NA
17.40
21.91
Rfreec (%)
No. of non-H atoms
No. of protein, atoms
No. of water molecules
No. of other solvent atoms
No. of ligand atoms
Root mean square deviation
from ideal values
Bond length (Å)
12888
23
NA
NA
NA
NA
12695
602
13
2
N/A
13
0.09
1.35
NA
NA
0.10
1.62
Angle distance (Å)
Average B factor (Å2)
Protein
31.5
NA
NA
NA
NA
NA
NA
26.9
36.9
24.8
37.3
99.81
Ligand
Solvent water
22.5
26.5
Solute ions (sodium)
Ramachandran plot,d residues 99.63
in allowed and most
favored regions (%)
Protein Data Bank codes
4C90
4C91
a Values in parentheses are for the highest resolution shell.
b N/A not applicable.
c
5% of the randomly selected reflections excluded from refinement.
d Calculated using MOLPROBITY.
Crystallization, Data Collection, Structure Solution, and
Refinement—SeMet-BoAgu115A and native BoAgu115A, at 10
mg/ml, were crystallized from 19% PEG3350, 0.2 M sodium cit-
rate, pH 5.5. For both selenomethionine and native enzyme,
crystals were harvested in a solution containing the mother
liquor supplemented with 15% (v/v) PEG 400 or Paratone N oil
as cryoprotectant and flash frozen in liquid nitrogen. Native
crystals were soaked with 300 mM GlcA to obtain structures of
BoAgu115A in complex with the uronic acid product.
Phases were extended to an initial native dataset at 2.65 Å and a
starting model was built using Buccaneer (29). The model
underwent recursive cycles of model building in COOT (30)
and refinement in REFMAC (31). The native model was used as
the search model for molecular replacement in Molrep to solve
the ligand datasets (32). Solvent molecules were added using
COOT and checked manually. Five percent of the observations
were randomly selected for the Rfree set. The models were vali-
dated using Molprobity (33). The data statistics and refinement
details are reported in Table 1.
Diffraction data were collected at the Diamond Light Source,
Didcot, United Kingdom, on beamlines I04–1 (ligand, ␭ ϭ
0.9163 Å) and I02 (native and selenomethionine, ␭ ϭ 0.9795 Å)
at a temperature of 100 K. Data were processed and integrated
with iMosflm (25) and scaled using SCALA (26). For all data-
sets, the space groups were determined to be P212121 with two
molecules in the asymmetric unit (giving a Matthews coeffi-
cient of about 2.7 Å3 DaϪ1 and a solvent content of about 50%).
Heavy atom sites were found and initial phases were calculated
using the HKL2MAP interface for the ShelxC/D/E pipeline
(27). All other computing used the CCP4 suite of programs (28).
Analytical Ultracentrifugation—Sedimentation velocity exper-
iments were carried out in a Beckman Coulter (Palo Alto, CA)
ProteomeLab XL-I analytical ultracentrifuge using interference
optics. All analytical ultracentrifugation runs were carried out
at the rotation speed of 48,000 rpm and experimental temper-
ature of 20 °C; the velocity scans were taken 1 s apart, 600 scans
in total. The sample volume was 400 ␮l. The rate protein sedi-
mentation was used to calculate the Mr of the glucuronidase as
described previously (34).
RESULTS AND DISCUSSION
4 The abbreviation used is: PACE, polysaccharide analysis using carbohydrate
gel electrophoresis.
BoAgu115A Is a Xylan-specific ␣-Glucuronidase—The genome
of B. ovatus encodes seven proteins that are members of GH115.
JANUARY 3, 2014•VOLUME 289•NUMBER 1
JOURNAL OF BIOLOGICAL CHEMISTRY 55

GH115 ␣-Glucuronidase
FIGURE 2. The release of GlcA and MeGlcA from different xylans by BoAgu115A. In panel A, wild type (WT) Arabidopsis or the mutant gux1gux2 (which does
not decorate xylans with [Me]GlcA) were treated with BoAgu115A. In panel B, the xylans extracted from the four plants were subjected to PACE in the absence
(Ϫ) or presence (ϩ) of BoAgu115A. *, non-enzyme dependent product. In panel C, wild type Arabidopsis was treated with a GH30 glucuronoxylanase alone
(WTϩBoGH30), priortotreatmentwithBoAgu115A(WT ϩBoGH30ϩBoAgu115A), orsubsequenttotreatmentwithBoAgu115A(WTϩBoAgu115AϩBoGH30).
The glucurono-xylooligosaccharides displayed in panel C are as follows: UX3, XXUX; UX4, XXXUX; UX5, XXXXUX; UX6, XXXXXUX.
To explore their potential enzymatic activities, six of the seven response to xylans, which may point to specificities that are not
B. ovatus GH115 proteins were expressed in E. coli in soluble related to the hemicellulose.
form (BACOVA_00249 could not be expressed in E. coli), puri-
Biochemical and Biophysical Properties of BoAgu115A—To
fied by IMAC to electrophoretic homogeneity, and their activ- explore the catalytic properties of BoAgu115A in more detail
ity against glucuronoxylans was explored using PACE. The data the activity of the enzyme against glucurono-xylooligosaccha-
showed that BoAgu115A released glucuronic acid (GlcA) and rides and pre-treated glucuronoxylan was evaluated using
4-O-methyl-GlcA (MeGlcA) from wild type Arabidopsis glucu- PACE. When Arabidopsis glucuronoxylan was treated with the
ronoxylan, but not from an Arabidopsis mutant completely GH30 glucuronoxylan-specific xylanase, BACOVA_03432, a
lacking MeGlcA and GlcA decorations on xylan (gux1gux2) range of products were observed with the most prominent spe-
(23) (Fig. 2A). The release of both GlcA and MeGlcA demon- cies having a degree of polymerization of 7 to 11. The GH30
strate that the enzyme can accommodate the 4-O-methyl sub- enzyme was inactive against the hemicellulose that had been
stitution of GlcA, a trait shared with the other family of pre-treated with BoAgu115A (Fig. 2C), indicating that glucu-
␣-glucuronidases, GH67. The enzyme could also remove ronidase can remove [Me]GlcA from all locations within the
MeGlcA from glucuronoxylans present in bioenergy-relevant hemicellulose.
dicots such as willow, and from the more complex glucurono-
Glucurono-xylooligosaccharides were generated by digest-
arabinoxylans derived from monocots such as barley, sugar ing Arabidopsis glucuronoxylan to completion (i.e. the product
cane, and Miscanthus (Fig. 2B). Thus, BoAgu115A is a ␣-glucu- profile remains stable) with GH10 or GH11 xylanases, which
ronidase that targets the uronic acids that decorate xylans. generated UXX and XUXX as the limit products, respectively
None of the other B. ovatus GH115 proteins exhibited measur- (35). The data showed that BoAgu115A hydrolyzed UXX and
able catalytic activity against a range of plant polysaccharides XUXX (Fig. 3A), indicating that the enzyme cleaves GlcA from
that contain uronic acids including glucuronoxylans, homoga- both non-reducing terminal and internal xylose residues, con-
lacturonic acid, and rhamnogalacturonan I and II (data not sistent with its activity against xylan, whereas the GH67 ␣-glu-
shown). The lack of activity of one of these B. ovatus curonidase CjGlcA67A released [Me]GlcA from UXX, but not
enzymes, BACOVA_03434, against glucuronoxylan is sur- from XUXX (data not shown), consistent with its capacity to
prising as it is closely related to BoAgu115A (54% identity), remove the uronic acid only when it decorates the non-reduc-
and is encoded by a gene within the same xylan-activated poly- ing terminal xylose (9, 11).
saccharide utilization locus. In contrast, BACOVA_00492,
When BoAgu115A was assayed against a mixture of termi-
BACOVA_00982, BACOVA_02173, and BACOVA_02777 are nally substituted aldouronic acids the activity of the enzyme
more distantly related to BoAgu115A and are not expressed in followed the order UXXXϾUXXϾϾUX (Fig. 3B), demonstrat-
56 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 289•NUMBER 1•JANUARY 3, 2014

GH115 ␣-Glucuronidase
FIGURE 3. Activity of BoAgu115A against glucurono-xylooligosaccharides. In panel A, wild type (WT) Arabidopsis glucuronoxylan was treated with either a
GH10 or GH11 xylanase that produce terminal or internally substituted glucurono-xylooligosaccharides, respectively, as their end products. The enzymes were
inactivatedbyheatingandthensubjectedtoPACEbeforeandaftertreatmentwithBoAgu115A.InpanelB,amixtureofUX,UXX,andUXXXwereincubatedwith
BoAgu115A and the product generated over time was monitored by the release of the appropriate xylooligosaccharide by high performance anion-exchange
chromatography with pulsed amperometric detection (HPAEC-PAD).
TABLE 2
ing that the enzyme contains at least two positive subsites
Kinetics of wild type BoAgu115A against glucuronoxylans and glucu-
downstream of the ϩ1 subsite (ϩ2R and ϩ3R). Positive sub-
rono-xylooligosaccharides
sites bind to the xylan backbone with the ϩ1 subsite housing
Data are averages and S.D. from three independent experiments.
the xylose linked to the cleaved GlcA, and subsites that bind
Substrate
kcat
min
Km
mM
kcat/Km
successively to sugars toward the reducing end of the xylan
chain are labeled ϩR2, ϩR3 etc., whereas the subsites that bind
to xylose toward the non-reducing end are defined as ϩNR2,
ϩNR3 etc. (nomenclature described in Ref. 36). It was also evi-
dent that BoAgu115A was significantly more active against
XUXX than UXX (Table 2), indicating that the glucuronidase
contains at least one subsite upstream of the ϩ1 subsite
(ϩ2NR). Against glucuronoxylans from birch and beech the Km
Ϫ1
Ϫ1
minϪ1 mM
XUXX
UXX
3233 (Ϯ187) 4.5 (Ϯ0.54)
718
1106 (Ϯ71)
696 (Ϯ38)
665 (Ϯ24)
19.5 (Ϯ1.90) 57
Beech wood xylan
Birch wood xylan
0.4 (Ϯ0.06)
0.3 (Ϯ0.04)
0.9 (Ϯ0.15)
1.1 (Ϯ0.15)
1740
2217
930
Beech wood xylan /GH30a 837 (Ϯ77)
Birch wood xylan/GH30a
862 (Ϯ64)
784
a The polysaccharide was pre-digested to completion with a GH30 glucuronoxy-
lan-specific xylanase.
of BoAgu115A is ϳ10-fold lower than XUXX, whereas the kcat olution of 2.65 Å, whereas the ligand structure of the enzyme
of the enzyme is ϳ5-fold less against the polysaccharide com- was determined by molecular replacement at a resolution of
pared with the oligosaccharide. Thus, although the ␣-glucuron- 2.14 Å (Table 1). The final model of BoAgu115A consists of one
idase has a higher catalytic efficiency against xylan than XUXX, dimeric molecule in the asymmetric unit, with each protomer
its lower kcat likely reflects tighter binding of the deglucurony- consisting of residues 33–856, with an average B factor of 31.5
lated xylan chains (i.e. reaction product) to the positive subsites, (Table 1). The final crystallographic R value is 20.34, with an
compared with the xylooligosaccharides released from the
Rfree of 26.59 for the native apo model, 17.40 and 21.91, respec-
glucurono-xylooligosaccharides, resulting in slow product tively, for the ligand bound model. BoAgu115A is an ␣/␤ glob-
release and hence turnover rate. Thus, it is possible that the ular protein with overall dimensions of about 100 ϫ 70 ϫ 50 Å
xylan binding site of BoAgu115A may be able to bind more than per protomer. The enzyme consists of four distinct domains,
four xylose residues.
which are connected by extended loops (Fig. 5A). The N-termi-
The pH optimum of BoAgu115A was ϳ7.0 (Fig. 4A) and nal domain, residues 33–196 (all residues are identified by their
when subjected to analytical ultracentrifugation migrated with position in the full-length protein), comprises six ␤-strands that
a sedimentation coefficient of 8.78 Ϯ 0.03 S, which equates to a lay on top of two parallel ␣-helices. The second domain (amino
molecular mass of 199 Ϯ 6.7 kDa (Fig. 4B). Given that the 824- acids 197–482) displays a (␤/␣)8-fold (TIM barrel). This domain
amino acid recombinant form of BoAgu115A has a molecular deviates slightly from a canonical TIM barrel; there is an addi-
mass of 85 kDa, these data indicate that the glucuronidase is a tional helix between ␤-strands ␤-1 and ␤-2, and between ␤-3
dimer in solution, consistent with its crystal structure (see and ␤-4, and the helices protruding from ␤-5 and ␤-6 are
below).
unusually short. The two ␣-helices of the N-terminal domain
Structure of BoAgu115A—The crystal structure of the native make extensive contacts with the ␣-helices extending from ␤-1
BoAgu115A was solved by extending the phase information and ␤-2 of the TIM-barrel of the second domain. The third
from an initial selenomethionine SAD at 3.0 Å to a native res- domain, extending from residues 488–641, comprises a five-
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GH115 ␣-Glucuronidase
FIGURE4. BoAgu115Aisaneutralactingdimeric ␣-glucuronidase. InpanelA, theenzymewasassayedagainst1mM XUXXin50mM sodiumacetate, sodium
HEPES, and sodium phosphate buffers at the different pH values. Panel B shows the analytical ultracentrifugation of BoAgu115A.
FIGURE 5. Structure of BoAgu115A. Panel A is a schematic of a protomer of BoAgu115A showing the four distinct domains of the enzyme: the N-terminal
␤-strand/␣-helix domain (blue), the core TIM barrel catalytic domain (green), the five-helical bundle domain (yellow), and the C-terminal ␤-sandwich domain
(red). Panel B depicts the enzyme in its dimeric butterfly-like form with protomers 1 and 2 colored green and light blue, respectively. Panel C shows a surface
representation of the GH115 dimer. The relative orientation of the dimer and color of the protomers is as indicated in B. Both panels B and C show the GlcA
bound form (sugar shown in stick format, carbons colored salmon pink).
helical bundle. The C-terminal domain, amino acids residues following order: ␤-sheet 1 (concave surface), ␤-1, ␤-8, ␤-3, and
673–844, displays a canonical ␤-sandwich-fold consisting of ␤-6; ␤-sheet 2 (convex surface), ␤-2, ␤-7, ␤-4, and ␤-5. In gen-
two ␤-sheets each containing five anti-parallel ␤-strands in the eral the ␤-strands are connected by loops, however, the loop
58 JOURNAL OF BIOLOGICAL CHEMISTRY
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GH115 ␣-Glucuronidase
FIGURE 6. The active site of BoAgu115A. Panel A shows the three-dimensional position of the amino acids (carbons colored green in the structure of the
BoAgu115A-GlcA complex and cyan in the apo structure) of BoAgu115A that make polar interactions (indicated by black dotted lines) with bound GlcA (shown
in stick format with carbons numbered and colored salmon pink) in its open ring conformation, or polar side contacts with other amino acids (indicated by
magenta dotted lines). The distance in Å of these interactions are indicated. The red dotted line is a potential polar contact between Asp-332 and GlcA. The
three-dimensional position of Arg-328, Asp-332, and Glu-375 are derived only from the apo structure of BoAgu115A as the loop containing the aspartate and
arginine and the side chain of the glutamate are disordered in the BoAgu115-GlcA complex. The secondary structural elements shown in green and cyan,
correspond to residues Glu-320 to Glu-345 of BoAgu115A-GlcA and apo BoAgu115A, respectively. The selected amino acids are shown in stick format with the
carbons colored green (BoAgu115A-GlcA) or cyan (apo BoAgu115A) with all oxygens and nitrogens colored red and blue, respectively. Panel B shows the
electron density map (2Fo Ϫ Fc) of GlcA at 1.3 ␴. The electron density is shown in dark blue, protomer 1 is displays as a schematic in green and the atoms are
colored as described in panel A.
connecting ␤-1 and ␤-2 contains a pair of anti-parallel between GlcA and the enzyme are as follows (Fig. 6): O3 makes
␤-strands, whereas there is a short ␣-helix in the loop connect- a polar contact with N⑀-1 of Trp-249. O6A and O6B of the
ing ␤-5 with ␤-6. The two protomers in BoAgu115A display a uronic acid carboxylate form polar contacts with the OH of
“butterfly” like structure in which the two subunits make sev- Tyr-420 and N⑀-2 of His-422, respectively, the endocyclic oxy-
eral interactions through the helical bundle domains. In addi- gen interacts with the N of Val-426, and O1 makes a hydrogen
tion, the C-terminal ␤-sandwich domain of protomer-1 makes bond with Asp-206. O4 is pointing into a hydrophobic open
extensive interactions with the TIM-barrel domain of protomer-2 channel, comprising the side chains of Phe-203, Met-248, and
and vice versa (Fig. 5, B and C).
Trp-245, which likely make apolar contacts with the methyl
Identifying the Active Site of BoAgu115A—Inspection of the group of MeGlcA. Unlike the vast majority of glycoside hydro-
surface of the BoAgu115A protomers revealed a cleft-like struc- lases, the active site of BoAgu115A does not make typical par-
ture in the (␤/␣)8 domain that extends over the central ␤-barrel, allel stacking interactions between the planar faces of aromatic
on the opposite surface to the N-terminal domain. The center residues and the sugar ring, although edge on edge apolar inter-
of the cleft, which may accommodate the xylan backbone, abuts actions between the sugar and two aromatic residues, Trp-249
onto a deep pocket that likely comprises the active site. This and Tyr-420, and the aliphatic side chain of Val-426 were evi-
hypothesis was confirmed when BoAgu115A was co-crystal- dent. The O␦-2 of Asp-332 in the apo structure (the amino acid
lized with GlcA, which revealed electron density for the uronic is not visible in the structure of the BoAgu115A-GlcA complex,
acid in the proposed active site pocket (Figs. 5, B and C, 6, and discussed in detail below), is in an appropriate position to make
7). The sugar was found to be in its ring open conformation, polar contacts with O2 of GlcA, whereas O␦-1 of Asp-206
which is highly unusual for hexaose aldehyde sugars. It is pos- forms a polar contact with O1 and may therefore act as the
sible that the specific interactions between the uronic acid and catalytic acid (Fig. 6).
the enzyme encourage GlcA to adopt an open chain conforma-
To explore further the significance of the interactions
tion after glycosidic bond cleavage. The direct interactions between GlcA and BoAgu115A, and the function of other resi-
JANUARY 3, 2014•VOLUME 289•NUMBER 1
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GH115 ␣-Glucuronidase
FIGURE 7. The topology of the xylan binding cleft of BoAgu115A. A and B show a solvent-exposed surface representation of the substrate binding cleft of
protomer 1 of theapo (A)andGlcAbound(B)formsof BoAgu115A. In bothpanels theleft-hand structures show a side on view down theputativexylan binding
cleft (indicated with an arrow), and the right-hand structures provide a bird’s eye view of the xylan binding cleft. In A and B, protomer 1 is colored light blue,
protomer 2 is green, and the proximal wall (Trp-249) of the active site pocket is colored red. The flexible loop extending from Met-327 to Asn-346 is shown in
cyan in B but not in A (the loop was too disordered to be modeled in the BoAgu115A-GlcA complex). C shows the position of the amino acids that interact
(Lys-374, Glu-375) with residues in the flexible loop (Asp-328, Asp-332) in stick format with the carbons (and schematic of the overall fold) colored cyan (apo
BoAgu115A) or green (BoAgu115A-GlcA complex), the carbons of GlcA are colored salmon pink, and all oxygens and nitrogens colored red and blue, respec-
tively. NotethesidechainsofLys-374, Glu-375, andGlu-782are toodisordered intheapo structure tobe modeled. The polar contactsbetween the aminoacids
are shown by dotted lines and the distance in Å of these interactions are indicated.
dues in the active site of the enzyme, targeted amino acids were were shown to make a significant contribution to the activity
substituted with alanine and the activity of the enzyme variants of the enzyme; the putative function of these residues is dis-
determined. The data, presented in Table 3 and Fig. 6, showed cussed below. Substituting amino acids near the putative
that mutating the residues that interact with the carboxylate of xylan binding cleft/active site generally caused a modest
GlcA, His-422 and Tyr-420, resulted in a ϳ105- and ϳ103-fold reduction (5–100-fold) in the activity of the glucuronidase
reduction in activity, respectively, indicating that this compo- (see below), and mutating acid residues located in the other
nent of the substrate is an important specificity determinant. pocket/cleft-like structure on the enzyme (E162A, W169A,
O3 appears to interact with Trp-249 as the W249A mutation D192A, and D478A) did not affect activity (Table 3). These
caused a ϳ3000-fold reduction in catalytic activity. In addition data are consistent with the view that the pocket located in
to the residues that interact directly with the bound GlcA, sev- the cleft-like structure in the TIM-barrel domain comprises
eral other amino acids in, or within the vicinity of, the active site the active site of BoAgu115A.
60 JOURNAL OF BIOLOGICAL CHEMISTRY
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GH115 ␣-Glucuronidase
TABLE 3
Activity of mutants of BoAgu115A
BoAgu115A derivative
ever, that the distance between Asp-332 and Glu-375 (in the
apo structure) and the O1 of GlcA (in the structure of
BoAgu115A in complex with GlcA); 5.4 Å and 11.8 Å, respec-
tively, are greater than the normal ϳ3.5 Å between the catalytic
base and the anomeric carbon of the glycone sugar in inverting
glycoside hydrolases (Fig. 6). It is possible that BoAgu115A dis-
plays a “Grotthus”-style mechanism in which a remote amino
acid activates the active site nucleophilic water via a string of
solvent molecules (37), as proposed for some inverting glyco-
side hydrolases (37, 38). In such a mechanism the distance
between the catalytic base and the sugar is not restricted to 3.5
Å. Both Asp-332 and Glu-375, however, are on highly mobile
loops that have high B-factors. Indeed, in the BoAgu115A-GlcA
structure the loop that contains Asp-332 (Met-327 to Asn-346)
was too disordered to be modeled, whereas Glu-375 could not
be built past the ␤-carbon (Fig. 6). Thus, as discussed below, in
the Michaelis catalytic complex Glu-375 and Asp-332 may
adopt conformations, not observed in the apo and product
structures, which reveal their true function in substrate binding
and catalysis.
Catalytic activitya
k
Ϫ1
cat/Km minϪ1 mM
Wild type
E162A
780.0
ϳ780.0b
ϳ780.0b
ϳ780.0b
ϳ780.0b
198.0
152.0
123.0
44.0
10.8
8.0
D192A
W169A
D478A
E785A
Y788A
E782A
N205A
Y425A
K374A
Y792A
7.6
D396N
5.4
N398A
3.7
D206A
2.5
N462A
1.8
Y373A
1.1
Y420A
0.8
H275A
0.3
W249A
0.28
0.17
0.01
0.01
NDc
ND
E375A
H422A
R328A
H275A/H422A
D332A
In enzymes and non-catalytic binding proteins that interact
with glucuronic acid, arginines are often important specificity
determinates, making bidentate hydrogen bonds/salt bridges
with the carboxylate group (9, 39). It is possible that in
BoAgu115A Arg-328 contributes to substrate binding by also
interacting with the carboxylate of the glucuronic acid sub-
strate, a view supported by the observation that the R328A
mutation caused ϳ105-fold decrease in activity (Table 3). Arg-
328 (and Glu-375) in the apo structure, however, are too distant
from the hub of the active site to play a direct role in enzyme
⌬C-terminal (1–526)
⌬C-terminal (1–639)
⌬C-terminal (1–665)
ND
ND
ND
a Wild type and mutants of BoAgu115A were assayed using 1 mM XUXX as the
substrate in 20 mM sodium phosphate buffer, pH 7.0, containing 1 mg mlϪ1 of
BSA. At this substrate concentration the initial rate provides a direct readout of
the catalytic efficiency of the enzyme.
b ϳ780; the activity of the mutants of residues from the other cleft/pocket-like
structure on the enzyme was estimated from a single time point reaction.
c ND, no activity could be determined using an assay that can detect activity that is
ϳ105-fold less than the wild type enzyme.
The Possible Assembly of the Catalytic Apparatus of function (Fig. 6). Thus, for the arginine to contribute to sub-
BoAgu115A—GH115 enzymes are inverting glycoside hydro- strate binding the loop containing this amino acid (and Asp-
lase (14) and thus glycosidic bond cleavage is catalyzed by an 332) would be required to undergo a conformational change.
acid base-assisted single displacement mechanism. In such a Another possibility is that the role of Arg-328 is to stabilize the
mechanism the catalytic Brønsted base, typically a carboxylate, conformation of Asp-332, which may function as the catalytic
activates a water molecule that attacks the anomeric carbon of base, whereas the primary role of Glu-375 could be to orientate
the glycone sugar, whereas the catalytic acid, also a carboxylic the guanidino group of the arginine toward the aspartate. To
acid residue, donates a proton to the glycosidic oxygen promot- conclude, the above structural and mutagenesis data indicate
ing leaving group departure (14). As stated above, Asp-206, that the assembly of the Michaelis complex in BoAgu115A
based on its interaction with O1 of GlcA, may comprise the requires the repositioning of several flexible elements, most
catalytic acid. Support for this view is provided by the apolar notably the loop carrying Arg-328 and Asp-332. Indeed, close
environment of the aspartate; the carboxylate amino acid is in inspection of the apo structure reveals that the conformation of
close proximity with Trp-249 and Val-426, promoting an ele- this loop, which forms a component of the xylan binding cleft,
vated pKa. Furthermore, O␦-2 of Asp-206 is within hydrogen in addition to part of the active site pocket, likely plays a central
bonding distance with O␦-1 of Asp-424, and thus may function role in both glucuronoxylan binding and departure of the reac-
as the pKa modulator of the putative catalytic acid (Fig. 6). It tion products, both GlcA and the undecorated xylose polymer
should be emphasized, however, that the assignment of Asp- (Fig. 7, A and B). Furthermore, the loop also plays a role in
206 as the catalytic residue must be viewed with some caution stabilizing residues in the putative xylan binding cleft, notably
as the D206A substitution resulted in only a 300-fold reduction Lys-374 and Glu-782, whose side chains are disordered in the
in activity, and, as the bound GlcA is in an open chain form, the GlcA bound structure where the loop is too flexible to be mod-
position of O1 may not reflect the location of the atom when eled (Fig. 7C). Although conformational changes are unusual in
the uronic acid is in a closed pyranose configuration, adopted in glycoside hydrolases, they are not without precedent, exempli-
the substrate.
fied by the substantial movement of the catalytic TIM-barrel
Based on mutagenesis data the other carboxylate amino acids domain induced by substrate binding observed in GH112 gly-
in the vicinity of the active site, Asp-332 and Glu-375, are can- coside phosphorylases (40).
didate catalytic residues; the D332A and E375A mutations
Whether Asp-332, or possibly Glu-375, comprise the actual
caused complete inactivation of the enzyme and a ϳ5000-fold catalytic base is currently unclear, as is the role of Arg-328 in
reduction in activity, respectively (Table 3). It is evident, how- substrate binding, as a Michaelis complex could not be
JANUARY 3, 2014•VOLUME 289•NUMBER 1
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GH115 ␣-Glucuronidase
FIGURE 8. Alignment of GH115 ␣-glucuronidases active against glucuronoxylan. Only the amino acids that comprise the end of the N-terminal domain
and the catalytic TIM-barrel domain of the BoAgu115A are shown. Numbering in each protein is from the full-length native sequence. Identical residues in all
sequences are indicated by an asterisk. Amino acids in BoAgu115A highlighted in green are implicated in the catalytic activity of the enzyme. The flexible loop
in BoAgu115A that contains the key catalytic residues, Arg-328 and Asp-332, and is ordered only in the apo structure, is shown boxed in red. GenBank accession
codes for the other characterized xylan-active GH115s shown are as follows: Streptomyces pristinaespiralis, EDY63299.2; Schizophyllum commune, ADV52250.1;
and Pichia stipitis, ABN67901.2.
obtained. Although the catalytic apparatus of glycoside hydro-
Structural Comparison of GH115 and GH67 ␣-Glucu-
lases generally comprise two carboxylate residues, exceptions ronidases—Structural comparison of BoAgu115A with the PDB
are evident with histidine acting as a catalytic acid-base in a database (using DaliLite version 3) identified the Geobacillus
GH3 N-acetylglucosaminidase (41), and as a catalytic acid in an stearothermophilus GH67 ␣-glucuronidase, AguA (11), as the
inverting GH117 anhydro-L-galactosidase (42). Given that ala- closest, albeit weak, structural homolog with a Z-score of 16.1,
nine substitution of His-422 caused a ϳ105-fold reduction in root mean square deviation of 4.7 Å, and sequence identity of
activity, it is also possible that this residue is a component of the 11% over 470 aligned resides. Despite the weak structural
catalytic apparatus.
homology and sequence similarity, the first three domains of
Sequence alignments showed that the residues that had the the two proteins display the same fold and, intriguingly, the
greatest influence on BoAgu115A activity (mutants of amino location of the active site of the GH115 and GH67 enzymes,
acids that cause Ͼ104-fold reduction in activity, those that endo- and exo-acting xylan-specific ␣-glucuronidase, respec-
interact with GlcA, and the proposed catalytic acid and pKa tively, is conserved, pointing to a distant evolutionary link
modulator, Asp-206 and Asp-424, respectively), are invariant in between the two families (Fig. 9A). It is evident, however, that
the other three GH115 enzymes that were shown to display there is no conservation of the active site residues, although it
␣-glucuronidaseactivity(12, 13, 15)(Fig. 8). Thesedataareentirely should be noted that the catalytic acid in GH67 enzymes is
consistent with the proposed location of the BoAgu115A active positioned on a highly mobile loop that can only be stabilized in
site, and the likely identity of the amino acids that contribute to the presence of ligands (11), which has some resonance with the
catalysis.
mobile loop containing the catalytically significant residues
The Potential Role of the C-terminal Domain of BoAgu115A— Arg-328 and Asp-332 in BoAgu115A (Fig. 9B). In addition, the
Inspection of the dimeric form of BoAgu115A suggests that the topology of the active site of the two enzymes is very different,
C-terminal ␤-sandwich domain of protomer 2 contributes to comprising a deep pocket embedded in a rigid blind canyon
one of the faces of the likely xylan binding cleft of protomer 1 (GH67 AguA) or a more accessible binding cleft (BoAgu115A),
and vice versa (Fig. 7). To explore the role of the C-terminal consistent with the exo and endo specificities of the respective
domain in the activity of the enzyme, alanine substitutions were glucuronidases. In general the catalytic apparatus is conserved
made of Glu-782, Glu-785, Tyr-788, and Tyr-792, whose side in enzymes that are structurally related and display the same
chains line the proposed xylan binding cleft. The 100-fold substrate specificity, but exhibit distinct modes of action; the
reduction in catalytic activity mediated by the Y792A substitu- introduction of exo activity is mediated by the modification of
tion (Table 3) indicated that Tyr-792 makes a significant con- the substrate binding cleft of endo-acting enzymes such that
tribution to xylan binding, whereas removal of the C-terminal the cleft is converted into a blind canyon or a tunnel (reviewed
domain (enzyme truncated at residue Ile-665) resulted in the by Refs. 5 and 6). It is intriguing, therefore, that despite signifi-
complete abrogation of enzyme activity (Table 3), supporting a cant structural conservation, indicating an evolutionary link
key role for this structural element in the topology of the between the two glucuronidase families, the region of the
GH115 catalytic apparatus.
enzymes that are most likely to be highly conserved, the cata-
62 JOURNAL OF BIOLOGICAL CHEMISTRY
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GH115 ␣-Glucuronidase
316–317
2. Hooper, L. V., Midtvedt, T., and Gordon, J. I. (2002) How host-microbial
interactions shape the nutrient environment of the mammalian intestine.
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3. Bergman, E. N. (1990) Energy contributions of volatile fatty acids from the
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4. Kau, A. L., Ahern, P. P., Griffin, N. W., Goodman, A. L., and Gordon, J. I.
(2011) Human nutrition, the gut microbiome and the immune system.
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crumbling down. Recent structural biochemistry of plant polysaccharide
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6. Gilbert, H. J. (2010) The biochemistry and structural biology of plant cell
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and Henrissat, B. (2009) The Carbohydrate-Active EnZymes database
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FIGURE9. ComparisonoftheactivesiteofBoAgu115AwithaGH67␣-glu-
curonidase. Panel A shows an overlay of a protomer of BoAgu115A (green)
with AguA, a GH67 ␣-glucuronidase (magenta; PDB code 1MQQ) from G.
stearothermophilus(11). ThepositionoftheboundGlcAfromBoAgu115Aand
AguA are circled. Panel B shows an overlay of the active site of the two
enzymes depicted in panel A in schematic format with the key catalytic and
sugar binding residues displayed as sticks. The residues in which the carbons
are colored green (apo form of BoAgu115A; see Fig. 6 for the rationale for
showing residues from the two structures) are from the GH115 enzyme and
amino acids with carbons shown in magenta from the GH67 glucuronidase.
The carbons of the GlcA in complex with BoAgu115A and the GlcA bound to
AguA are shown in salmon pink and slate blue, respectively, in both panels. All
oxygens and nitrogens are shown in red and dark blue, respectively.
12. Ryabova, O., Vrsanská, M., Kaneko, S., van Zyl, W. H., and Biely, P. (2009)
A novel family of hemicellulolytic ␣-glucuronidase. FEBS Lett. 583,
1457–1462
13. Tenkanen, M., and Siika-aho, M. (2000) An ␣-glucuronidase of Schizo-
phyllum commune acting on polymeric xylan. J. Biotechnol. 78, 149–161
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character of family GH115 ␣-glucuronidases. FEBS Lett. 584, 4063–4068
15. Fujimoto, Z., Ichinose, H., Biely, P., and Kaneko, S. (2011) Crystallization
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logr. Sect. F Struct. Biol. Cryst. Commun. 67, 68–71
lytic apparatus, is clearly different in GH67 and GH115. The
evolutionary selection pressures that led to such diversity in the
active site of structurally and functionally related enzymes fam-
ilies are currently unclear.
16. Martens, E. C., Lowe, E. C., Chiang, H., Pudlo, N. A., Wu, M., McNulty,
N. P., Abbott, D. W., Henrissat, B., Gilbert, H. J., Bolam, D. N., and
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and Gilbert, H. J. (2003) The modular architecture of Cellvibrio japonicus
mannanases in glycoside hydrolase families 5 and 26 points to differences
in their role in mannan degradation. Biochem. J. 371, 1027–1043
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R. A., Hazlewood, G. P., and Gilbert, H. J. (1998) The topology of the
substrate binding clefts of glycosyl hydrolase family 10 xylanases are not
conserved. J. Biol. Chem. 273, 32187–32199
Conclusions—This report reveals the first structure of a
GH115 enzyme, identifying a likely distant evolutionary link
to GH67, the other major family of ␣-glucuronidases. The
locations of the residues that contribute to activity indicate
that the enzyme undergoes a substantial conformational
change to assemble a functional catalytic apparatus. It is inter-
esting to note that despite complete conservation of the cata-
lytically important amino acids in seven GH115 proteins, only
BoAgu115A was shown to display activity (against glucuron-
oxylan). Although it is possible that the other six B. ovatus
GH115 proteins are not catalytically active, a more likely expla-
nation is that their target uronic acid-containing substrates are
not glucuronoxylan, suggesting that GH115 is a poly-specific
family.
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64 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 289•NUMBER 1•JANUARY 3, 2014

Protein Structure and Folding:
Evidence That GH115 α-Glucuronidase
Activity, Which Is Required to Degrade
Plant Biomass, Is Dependent on
Conformational Flexibility
Artur Rogowski, Arnaud Baslé, Cristiane S.
Farinas, Alexandra Solovyova, Jennifer C.
Mortimer, Paul Dupree, Harry J. Gilbert and
David N. Bolam
J. Biol. Chem. 2014, 289:53-64.
doi: 10.1074/jbc.M113.525295 originally published online November 8, 2013
Access the most updated version of this article at doi: 10.1074/jbc.M113.525295
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