Functional association of the catalytic and ancillary modules dictates enzymatic activity in a

21 Pages·2012·1.52 MB·English

by  Ed Bayer

JBC Papers in Press. Published on January 23, 2012 as Manuscript M111.314286
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M111.314286
1

Functional
 association
 of
 the
 catalytic
 and
 ancillary
 modules
 dictates

enzymatic
 activity
 in
 a
 glycoside
 hydrolase
 family-­‐43
 β-­‐xylosidase*

Sarah Moraïsa,b, Orly Salama-Albera, Yoav Baraka,c, Yitzhak Hadarb, David B.
Wilsond, Raphael Lamede, Yuval Shohamf and Edward A. Bayera

a Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel;
b Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of
Jerusalem, P.O. Box 12, Rehovot, 76100, Israel;
c Chemical Research Support, The Weizmann Institute of Science, Rehovot 76100 Israel;
d Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY14853, USA
D
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e Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69978 n
lo
a
d
Israel; e
d
fro
f Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa m
h
ttp
://w
w

  w
*Running title: Modular association in a family-GH43 β-xylosidase .jb
c
.o
rg
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To whom correspondence should be addressed: Edward A. Bayer, Department of Biological gu
e
s
Chemistry, The Weizmann Institute of Science, Rehovot, Israel. Tel: [phone]. Fax: (+972)- t o
n
A
8-946-8256. Email: [email protected] p
ril 5, 2
0
Keywords: GH43, hemicellulase, Thermobifida fusca. 19

Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
2

Background: Thermobifida fusca β-xylosidase INTRODUCTION
Xyl43A contains a catalytic and an ancillary
module. Xylan is the major constituent of
Results: Enzymatic activity is lost when the hemicellulose, which represents 20 to 30% of
modules are expressed independently, but restored lignocellulosic plant biomass (1) and represents an
upon specific non-covalent reassociation. important industrial target for degradation [e.g.
Conclusion: The catalytic and ancillary modules bleaching in the pulp and paper industry (2)].
of Xyl43A behave as a single functional entity. Xylan is a linear polysaccharide, consisting of β-
Significance: Two phylogenetically distinct 1,4 linked D-xylose units with a large variety of
modular components of an enzyme evolved side-chain substituents. Consequently, the
together to form an enzymatically active unit. contribution of multiple hemicellulases is required
for complete hydrolysis of xylan by synergistically
complementary enzymes.
The well-studied aerobic thermophilic soil
SUMMARY bacterium, Thermobifida fusca, can use cellulose
and xylan as sole carbon sources (3,4). For this
β-Xylosidases are hemicellulases that purpose, the bacterium produces a set of six D
o
hydrolyze short xylo-oligosaccharides into cellulases and several hemicellulases such as w
n
xylose units, thus complementing endoxylanase xylanases, a β-xylosidase, an α-L- loa
d
degradation of the hemicellulose component of arabinofuranosidase and an acetyl xylan esterase ed
lignocellulosic substrates. Here, we describe the (5,6). Two T. fusca endoxylanases (Xyn11A and fro
m
cloning, characterization and kinetic analysis of Xyn10B) have been expressed and fully h
ttp
a glycoside hydrolase family-43 β-xylosidase characterized (7,8). A third endoxylanase, ://w
(Xyl43A) from the aerobic cellulolytic Xyn10A, was characterized from a related species w
w
bacterium, Thermobifida fusca. Temperature Thermomonospora alba (9), and a nearly identical .jb
c
and pH optima of 55-60°C and 5.5 to 6, gene coding for this enzyme was found in T. fusca. .org
respectively, were determined. The apparent Due to their potential industrial applications, the b/
y
Km value was 0.55 mM, using p-nitrophenyl cloning and characterization of other thermostable gu
e
xylopyranoside as substrate, and the catalytic xylanases deserve particular attention. We have st o
n
constant (k ) was 6.72 s-1. T. fusca Xyl43A therefore investigated in this communication the A
cat p
contains a catalytic module at the N-terminus putative hemicellulase encoded by the T. fusca ril 5
and an ancillary module (termed herein gene Tfu 1616 which was related in sequence to , 2
0
Module-A) of undefined function at the C- the family-GH43 glycoside hydrolases (GH43), 19
terminus. We expressed the two recombinant the members of which are known to possess β-
modules independently in Escherichia coli and xylosidase (EC 3.2.1.37), β-1,3-xylosidase (EC
examined their remaining catalytic activity and 3.2.1.72), α-L-arabinofuranosidase (EC 3.2.1.55),
binding properties. The separation of the two arabinanase (EC 3.2.1.99), xylanase (EC 3.2.1.8),
Xyl43A modules caused the complete loss of or galactan 1,3-β-galactosidase (EC 3.2.1.145)
enzymatic activity, whereas potent binding to activities (http://www.cazy.org/) (10). To date,
xylan was fully maintained in the catalytic over 1500 bacterial genes have been associated
module and partially in the ancillary Module-A. with this family. Over fifty bacterial enzymes from
Non-denaturing gel electrophoresis revealed a this family have been examined for their
specific non-covalent coupling of the two enzymatic activities, but only a part of them have
modules, thereby restoring enzymatic activity been fully characterized, the results of which were
to 66.7% (relative to the wild-type enzyme). published in the primary literature. Among them,
Module-A contributes a phenylalanine residue β-xylosidases (11-16) endoxylanases (17,18),
that functions as an essential part of the active arabinanases (19-22), arabinofuranosidases (23-
site, and the two juxtaposed modules function 25) and galactanases (26) were described.
as a single functional entity.
3

The present work reports the cloning of the Tfu (ordered from Syntezza Bioscience Ltd.,
1616 gene of T. fusca and the characterization of Jerusalem), and PCR reactions were performed
the protein. The gene was expressed in E. coli, and using Pfu UltraII DNA polymerase (Aligent
the protein was purified to near homogeneity. Data Technologies, Santa Clara, CA).
on physico-chemical parameters and enzyme
Protein expression and purification—
activity of the intact Xyl43A are provided. The
Plasmids containing genes coding fo rthe full-
two modules of the protein (a GH43 catalytic
length Xyl43A enzyme, the GH43 modul,e
module and an associated ancilalry module —
ancillary Module-A, and mutants Xyl43A(F518A)
Module-A) were produced as separate protein
and Module-A(F518A) were expressed in E. coli
entities; the catalytic and binding activities were
BL21 (lDE3) pLysS cells, and the expressed His-
studied for both the separated modules as well as
tagged proteins were purified on a Ni-NTA
their in-vitro combined form.
column (Qiagen), as reported earlier (27). A gel

filtration purification step was conducted for
Experimental procedures
association of the GH43 module and the Module-

A, using an AKTA-prime System and a Hiload
Cloning—Xyl43A (Tfu 1616, GenBank
16/60 Superdex 75 column (GE Healthcare). SDS-
accession number: AAZ555651) was cloned from
PAGE was employed to test the purity of the
Thermobifida fusca YX genomic DNA. Primers
recombinant proteins (12% acrylamide gels). The D
o
were designed with the program Oligo Primer w
fractions with the highest degree of purity were n
Analysis Software version 5.1 and ordered at the lo
pooled, and the concentrations of the recombinant ad
Weizmann Institute of Sciences facility (N- ed
terminal primer: 5’- proteins were estimated by absorbance at 280 nm fro
based on their amino acid composition using the m
TCATGACATATGCACCATCAC h
Protparam program ttp
CATCACCATACTTCTCCCCAAGTCACGTCC (http://web.expasy.org/protparam/). Proteins were ://w
T-3’; C-terminal primer 5’- w
stored in 50% (v/v) glycerol at -20°C. w
TGATTGCTCGAGTTAGGAGGGGGACTGAG Substrates—Microcrystalline cellulose .jbc
GCCGGTA-3’ (NdeI and XhoI sites in bold). The .o
(Avicel) was purchased from FMC Biopolymer rg
PCR product was inserted into and ligated into b/
(Philadelphia, PA, USA) and was used for the y
linearized pET21a to form pXyl43A. g
preparation of phosphoric acid swollen cellulose ue
GH43 was cloned using Xyl43A WT forward (PASC 7.5 mg ml-1 pH 7). Insoluble xylan was st o
primer and 5’- prepared by boiling oat-spelt xylan (Sigma Chem. n A
p
TGAGCGG-3C’T CasT aC GreAveGrsCeT pAriCmGeGr C(XChAoIC GsiGteG iTn GbColGd) Canod., rSetc. oLvoeuriinsg, MthOe ) pfeolrl e3t0 bmyi nc einnt rdiifsutgilaletido nw; attheirs ril 5, 2
0
and Module-A using 5’- 1
was followed by 3 cycles of washes with distilled 9
TTAAGCCATATGCAGCCGTCAGAGACCGA
water to remove soluble sugars, and determination
CCACTTCGACGA-3’ and 5’-
of dry weight (28). Birchwood xylan, beechwood
TTATGTCTCGAGGGAGGGGGACTGAGGCC
xylan, chitin, p-nitrophenyl-β-D-glucopyranoside,
GGT-3’ (NdeI and XhoI sites in bold).
p-nitrophenyl-α-L-arabinofuranoside, p-
PCR reactions were performed using ABgene
nitrophenyl-β-D-cellobioside, p-nitrophenyl-β-D-
Reddymix x2 (Advanced Biotechnologies Ltd.,
xylopyranoside (pNPX) and xylobiose were
Epsom, UK). DNA samples were purified using a
HiYieldTM Gel/PCR Fragments Extraction Kit purchased from Sigma Chemical Co. (St. Louis
MO). Debranched linear arabinan was purchased
(Real Biotech Corporation, RBC, Banqiao City,
from Megazyme International, Ltd. (Wicklow,
Taiwan).
Ireland). Hatched wheat straw (0.2–0.8 mm) was
PCR mutagenesis was performed for the provided by Valagro (Poitiers, France) and treated
preparation of Xyl43A(F518A) and Module- as described previously (29,30).
A(F518A) plasmids using phosphorylated primers
pH studies—The β-xylosidase activity over a
5’-
pH range of 3.5 to 10 was investigated using
GGTGCCACGGGAGCGTTCCTCGGCCTGTG
citrate buffer from 3.5 to 8, Tris buffer from 7 to 9,
GG-3’ and 5’-CCACATGATGGGGTCGTTGC-3’
and glycine/NaOH buffer from 9 to 10. Xyl43A
4

was assayed at 50°C for 7 min in a 700 µl reaction buffer pH 6.0 containing 12 mM CaCl and 2 mM
2
mixture containing 0.05 µM enzyme, 100 mM EDTA were mixed with 0.5 mg of insoluble xylan
buffer, 1 mg/ml bovine serum albumin (BSA) and (or 10 mg of microcrystalline cellulose). Tubes
5 mM pNPX. After 7 min, the tubes were placed were mixed gently at 4 °C for 1 h and centrifuged
on ice, the reaction was terminated by adding 200 at 14000 rpm for 1 min. The supernatant fluids
µl of 1 M NaOH, and the optical density was (containing the unbound fraction of the proteins)
measured at 420 nm. were transferred to new tubes and supplemented
with SDS-containing buffer. The pellets
Temperature dependence and stability—The
(containing the bound fraction of the proteins)
influence of temperature upon activity was assayed
were washed once by resuspension in 100 µl of the
as follows: the release of p-nitrophenol by 0.05
above-described buffer, centrifuged, and
µM Xyl43A at temperatures ranging from 30 to
resuspended in 60 µl of SDS-containing buffer.
80°C was measured at 420 nm after 7 min reaction
Boiled samples were subjected to SDS-PAGE
and 15 min cooling on ice-water in 700 µl final
(12% gels) for analysis. BSA served as a negative
volume (100 mM buffer citrate pH 6, 1 mg/ml
control for binding specificity.
BSA, 5 mM pNPX). The effect of temperature on
the stability of Xyl43A was determined by Thin Layer Chromatography (TLC)—The
incubating the enzyme without substrate over a 15- hydrolysis of xylobiose by Xyl43A was monitored
min period for 50 h at 50, 60 and 70°C at pH 6. by TLC analysis. The xylobiose hydrolysis Do
w
Similarly, the purified GH43 catalytic module, and reaction included 5 mg xylobiose, 0.2 mM Xyl43A nlo
Module-A were incubated at 50°C over the same and 100 mM citrate buffer, pH 6. The reaction was ad
e
d
time period, either before or after combining the performed at 50°C, at different time points (5 min, fro
GH43:Module-A at 1:0.8 molar ratio. After adding 15 min, 30 min and 2 h), samples were taken, and m
h
substrates, 7-min reactions (as specified above) the reaction was terminated by adding Hg2+ to a ttp
were carried out at 50°C. The optical density at final concentration of 1 mM (which completely ://w
w
420 nm was then measured after 15-min inhibits the enzyme). Chromatography was carried w
incubation on ice. out on silica gel 60 plates (Merck, Germany), .jbc
.o
using butanol/ethanol/water (3:2:2) as a rg
Kinetic studies—Temperature-equilibrated b/
reactions of 180 µl final volume (100 mM buffer developing solvent. Spots were visualized by y g
u
charring with 0.2% orcinol (in methanol and 20% e
c5i0t°raCte) pwHe r6e, 1in mitiga/tmedl BbSyA ,a d0d tion g1 51 m0 Mµ lp NoPf Xt haet phosphoric acid in methanol, 1:1 vol/vol) and st on A
eanbzsoyrmbaen c(fei nwala sc omnocnenittorraetido na t o4f 200 .0n5m µ aMt )1. 4-Tshece heatiMngu flotirp l2e mainm aint o1-5a0c°iCd . sequence alignment— pril 5, 2
0
intervals for 10 min, using an ELISA plate reader The Clustal W2 program 1
9
(BioTek Synergy HT, Winooski, VT USA). (http://www.ebi.ac.uk/Tools/msa/clustalw2/) was
Absorbancies (420 nm) were then converted to employed to analyze the amino-acid sequence-
molarities using an extinction coefficient of 3365 based alignment between the characterized β-
M-1·cm-1 for the absorbance of p-nitrophenol in xylosidases in GH family 43 (11-16) and T. fusca
citrate buffer pH 6 at 50 °C. Xyl43A. This alignment served to identify the
homologous conserved residues between
Effect of salts and chemicals—Various salts
and chemicals were added at a final concentration Geobacillus stearothermophilus and T. fusca β-
of 1 mM to the standard enzymatic reaction xylosidases that appear to be involved in the
mixtures in order to check potential inhibitory interface between the catalytic and the C-terminal
effects on enzymatic activity. Module-A of Xyl43A.
Binding to insoluble polysaccharides—The
binding of the proteins to insoluble xylan and RESULTS
microcrystalline cellulose was determined
qualitatively using SDS-PAGE. In a final volume Sequence analysis and production of Xyl43A
of 100 µl, 10 µg (5 µg for cellulose binding (Tfu1616). The Xyl43A gene is part of the
hemicellulolytic system of Thermobifida fusca. It
experiments) of pure protein in 50 mM citrate
5

encodes a 550 amino-acid protein with a Pro 334 and Gln 335, where there seemed to be
calculated molecular weight of 61686 Da. The little or no interactions between the linker and the
amino-acid sequence of the Xyl43A protein was modules in the predicted model. The two modules
subjected to BLAST analysis (blastp, nr database, were therefore cloned separately (each with an N-
http://blast.ncbi.nlm.nih.gov/) and showed terminal His tag), expressed and purified by metal
significant homology (from 44% to 59% sequence ion affinity chromatography. The resultant GH43
identity) to β -xylosidases and α-L- module and ancillary Module-A exhibited their

arabinofuranosidases that belong to GH family 43. expected molecular masses (35.8 kDa and 25.9
The ORF has a G+C content of 68.4%, not kDa, respectively) in the SDS-PAGE gel and
significantly different from that (67%) of the purity above 95% was estimated (data not shown).
whole genome of T. fusca, which is typical of A schematic representation of the recombinant
many thermophilic organisms (31). The Xyl43A proteins expressed in this study is shown in Fig. 2.
gene does not include any recognizable signal Substrate specificity. The activity of the
peptide as determined by the SignalP server recombinant enzyme was tested on a variety of
(http://www.cbs.dtu.dk/services/SignalP/). Thus, substrates. No enzymatic activity was detected on
the protein is assumed to remain intracellularly in xylans (from birch wood, beech wood and oat-
T. fusca. SDS-PAGE analysis of the purified spelt, both soluble and insoluble), cellulosic
Xyl43A protein revealed a single protein band of substrates (Avicel and PASC), chitin, wheat straw, D
o
about 60 kDa, which is in good agreement with the arabinan p-nitrophenyl-β-D-glucopyranoside, p- w
n
calculated molecular mass. A typical Xyl43A nitrophenyl-β-D-cellobioside and p-nitrophenyl-α- loa
d
preparation yielded 31.2 mg/L of purified protein. L-arabinofuranoside (pNPA). Detectable levels of ed
Bioinformatic analysis of the protein revealed hydrolysis were only observed for p-nitrophenyl- fro
m
the presence of a GH43 catalytic module located β-D-xylopyranoside (pNPX) and xylobiose, http
between residues 16 and 319 and a second module confirming that the enzyme belongs to the β- ://w
of undefined function (ancillary module, Module- xylosidase class. Residual activity (kcat/Km of ww
A) located between residues
 338
 and
 550
 in the 0.75 -s1·mM-1 versus 12.25 for pNPX) was .jb
c
C-terminal part of the protein. In order to evaluate observed with pNPA, due to the structural .org
the binding properties and the enzymatic activity similarity between β-D-xylopyranoside and by/
of both N-terminal (catalytic core) and C-terminal α-L-arabinofuranoside. These results gue
(ancillary) modules, we produced two distinct support the premise that the conserved arginine st o
n
chimaeras, GH43 and Module-A. Since there is (position 288 in T. fusca Xyl43A) allows the A
p
45% identity and 60% similarity between the distinction between β-xylosidase and ril 5
previously characterized GH43 from G. arabinofuranosidase classes (32). This arginine is , 20
stearothermophilus (15,32), its structure was used 1
believed to be conserved only in β-xylosidases. 9
and compared to the predicted structure of T. fusca
Neither the GH43 nor Module-A alone had
Xyl43A, obtained using Swiss Model
measurable activities on any substrates (including
(http://swissmodel.expasy.org/) (Fig. 1). The
xylans and arabinoxylan).
quality of the model was estimated using the
pH studies. The optimal pH range of Xyl43A
ProSA-web site
was 5.5 to 6 for β-xylosidase activity (Fig. 3A)
(https://prosa.services.came.sbg.ac.at/prosa.php).
with more than 70% activity retained at pH 4.5 and
The Z-score of the model was -6.57, which is well
pH 7. Xyl43A is more stable at alkaline pH than at
within the range of scores typically found for
acidic pH and is drastically inactivated at pH 4. It
native proteins of similar size.
is indeed known that changes in pH affects ionic
The model was especially helpful for
or electric charge of active site amino acids (that
determining the linker region of T. fusca Xyl43A.
participate in substrate binding and catalysis) and
In G. stearothermophilus, the linker extends from
either enhances and stabilizes interactions with the
Val 319 to Asp 329; by analogy, we deduced that
substrate or breaks intra- and intermolecular
the linker region in T. fusca Xyl43A would extend
bonds, changing the shape of the enzyme and,
similarly from Leu 328 to Glu 338. The most
therefore, affecting its efficacy (33). Activity
appropriate juncture between the two modules was
profiles of glycosidases versus pH are typically
determined to be the bond between the adjacent
6

bell-shaped, reflecting the ionization state of, at length Xyl43A exhibited strong binding capacity
least, two essential amino-acids residues. The for insoluble xylan, whereas weak binding to
expected pK values of the general base and microcrystalline cellulose was observed (Fig. 4).
a
general-acid ionizable groups in the enzyme As expected, the bulk of the BSA negative control
(derived from the pH-dependency graph) are 4.5 was found in the unbound fraction, and the
and 8, respectively, close to the values obtained for respective insoluble xylan and microcrystalline
G. stearothermophilus XynB3 (15). cellulose positive controls were located in the
bound fractions. The GH43 catalytic module alone
Thermostability. The hydrolysis rate
demonstrated full binding capacity for insoluble
increased from 30°C to 60°C, and the temperature
xylan whereas Module-A was found in bound and
coefficient (Q10) was calculated to be 1.3. An
unbound fractions in almost equal proportions.
Arrhenius plot exhibited a straight line from 30°C
Thus it seems that xylan-binding capacity of
to 60°C, typical of a single rate-limited thermally
Xyl43A is due mainly to the residues present in
activated process, indicating an increase in enzyme
the catalytic module and the ancillary Module-A
activity with increasing temperature. The
plays a minor role in the binding interaction (Fig.
activation energy and the pre-exponential factor
4).
were determined to be 18.9 KJ.  mol−1 and 390,
Re-association of GH43 and Module-A. Non-
respectively. Under the reaction conditions the
covalent association of the separately expressed
temperature optimum was found to be 55-60°C for D
o
and purified GH43 and Module-A components at w
β-xylosidase activity (Fig. 3B); however the n
room temperature was analyzed by non-denaturing lo
enzyme underwent rapid inactivation at 60°C (Fig. ad
PAGE. The catalytic module was mixed with ed
3C). Purified Xyl43A demonstrated moderate increasing amounts of the helper module in molar fro
thermostability, even after 50 h at 50°C, 70% of m
ratios ranging from 1:0.2 to 1:1.8, and the mobility h
the β-xylosidase activity remained. Incubation at ttp
60°C resulted in about 50% loss of activities after pattern of each mixture was compared with that of ://w
each module alone and with that of the full-length w
only 2 h and almost no activity after 24 h. w
Xyl43A enzyme. Mixtures of GH43 and Module- .jb
Treatment at 70°C resulted in complete c
A resulted in complex formation (Fig. 5A). The .o
inactivation of the enzyme in less than 15 min rg
mobility pattern of the complex was nearly b/
(Fig. 3C). identical to that of wild-type Xyl43A, revealing a y g
u
e
Kinetic constants. The kinetic constants of specific interaction between the catalytic and st o
Xyl43A on pNPX were determined by running the ancillary modules. The intensity of the band, n A
p
r3eDac).t ioAnt a5t 0d°iCff eraenndt sautb sptHra te6 ,c otnhcee nrterlaetaiosen s o(fF ipg-. cMoorrdeuslpeo-And, ingi ntcor eathseed intuernatcilt iona of 1:G0.H84 3 raatnido ril 5, 2
0
nitrophenol was linear with time and proportional (GH43:Module-A), whereas the intensity of the 1
9
to enzyme concentration. The apparent Km value bands of each of the individual modules decreased.
was 0.55 mM pNPX while the k was 6.72 ·s-1. At this ratio, the Module-A band interacted
cat
Effect of various chemicals. The effects of completely with GH43. A relatively small part of
different chemicals on Xyl43A activity are shown the latter fails to form the complex, probably due
in Table 1. The enzyme retained almost full to incorrect folding of a portion of the GH43

activity in the presence of 1 mM Li+, Na2+, K+, fraction. Up to 25% of the wild-type β-xylosidase
Mg2+, Ca2+, Fe3+, Mn2+, Co2+, Cu2+, and the reagent activity was thus recovered upon association of the
SDS. The addition of EDTA had no effect on the two modules (Fig. 5B).
activity, suggesting that no metals are needed for Gel filtration was used to purify the
the enzyme reactions. Group IIb metals such as reassociated enzyme from the two modules at a
Hg2+ and Ag2+ gave stronger inhibition [mercury 1:0.8 ratio, and the kinetics parameters were
inhibition is indeed well known for most enzymes determined for the purest fraction obtained. The
including cellulases and xylanases, indicating the apparent Km value was 0.6 mM pNPX (similar to
existence of essential thiol groups (7)]. that of the wild-type enzyme) while the k was
cat
Binding to insoluble polysaccharides. The 4.83 s-1. Up to 71.8% of the wild-type β-xylosidase
binding capacity of Xyl43A to insoluble xylan and activity was thus recovered upon association of the
microcrystalline cellulose was investigated. Full- two modules.
7

The moderate thermostability characteristics Phe506 therein. This phenylalanine is the only
observed for the wild-type enzyme (Fig. 3C) raised residue in Module-A, which participates in the
the question as to whether the re-associated active site and presumably plays a role in substrate
Xyl43A would also be stable to heat treatment binding and specificity. In order to further
under the same conditions. Moreover, it was of investigate the role of this amino acid in the
interest to determine whether exposure of the two catalytic activity of T. fusca Xyl43A, the
constitutive modules to heat would result in conserved Phe518 was replaced with Ala.
reduced stability. We therefore subjected the two The kinetic constants of Xyl43A(F518A) for
isolated modules to heat treatment at 50°C both pNPX hydrolysis were determined by interaction
prior to and after re-association. Heat treatment of of the mutant enzyme at different substrate
the two modules separately before re-association concentrations as performed for the wild-type
led to a marked loss of stability (Fig. 5C). About enzyme (data not shown). At 50°C and pH 6, the
60% of the residual activity was obtained after 2 h release of p-nitrophenol was linear with time and
of heat treatment and only 15% remained after 5 h. proportional to enzyme concentration. The
In contrast, re-association of the two modules apparent Km value was 0.98 mM pNPX while the
before heat treatment resulted in increased k was 0.059 s-1. The replacement of the
cat
thermostability: about 80% relative activity phenylalanine by alanine affected the activity of
remained after 5 h of heat treatment and 50% after the enzyme dramatically, the activity of the mutant
D
o
48 h (Fig. 5C). The results indicated that the representing less than 1% of the wild-type activity w
n
individual modules exhibit decreased stability in on pNPX (0.89%). lo
a
d
solution but upon re-association form a stable Re-association of GH43 and mutated Module- ed
complex. A(F518A). In order to further examine the role of fro
m
the Phe518 residue in the catalysis, Module-A was h
In order to gain insight into the molecular ttp
basis of re-association of the GH43 and Module-A, replaced with alanine in a manner similar to that of ://w
the wild-type enzyme. As described for the wild- w
we employed the Protein Interaction Calculator w
(PIC) server (34) to analyze the known 3D type modules, non-covalent association of GH43 .jbc
and the mutated Module-A(F518A) was .o
structure of the family-GH43 XynB3 β-xylosidase rg
performed. A strong interaction between the two b/
from G. stearothermophilus. Using this server, the y
modules was observed (data not shown), g
various interactions involved in the interface ue
between the catalytic and the C-terminal Module- suggesting that Phe518 does not play a dominant st o
role in the strong association between the two n A
A were determined, and the homologous p
icdoennsteirfvieedd. r eTsiadbulees i2n Tsh. ofuwssc at hXey l4m3aAjo rw errees itdhueens , mmoodduulleess., βN-xeyvleorsthidealesses ,a cutipvoitny wasasso cnioatt iorenc oovfe rtehde, ril 5, 20
suggesting that the role of this particular amino 1
predicted to be involved in the intramodular 9
acid is directly related to catalysis per se.
interface. The data suggest strong reciprocal

binding interaction between the two modules of T.
fusca Xyl43A and provide an explanation for their DISCUSSION
observed re-association.
A number of GH43 family members (as well
Production of mutated Xyl43A(F518A).
as GH3, GH51 and GH54 families) have been
According to the crystal structure of the family
reported to exhibit simultaneously both β-
GH43 β-xylosidase (XynB3) from G.
xylosidase and α-L-arabinofuranosidase activities,
stearothermophilus T-6 (32), the active site of the
probably due to the active site geometry, that does
enzyme possesses a pocket topology, mainly
not allow the enzyme to distinguish between the
constructed from β-propeller domain residues. In
two saccharides. Indeed, both activities seem to
addition, a conserved phenylalanine residue
occur in the same active site (35,36). The
(Phe518 of T. fusca Xyl43A, homologous to
Thermobifida fusca Xyl43A, characterized here,
Phe506 of G. stearothermophilus XynB3) is
exhibited primarily β-xylosidase activity and only
located on a loop of Module-A in the latter
nominal levels of α-L-arabinofuranosidase
structure. The loop serves to close one side of a
activity.
cleft, formed by the catalytic module, and inserts
8

In former research (37), Asp and Glu residues the active site in G. stearothermophilus T-6
of family GH43 enzymes have been directly arabinanase (AbnB) resembles a groove with a
implicated in substrate hydrolysis, in line with the narrow bridge formed by Phe104 (conserved in
classical general acid (proton donor) and family GH43 arabinanases) and Cys222. A
nucleophile/base that characterize enzymatic neighboring cysteine (Cys221) bridges with the
hydrolysis of the glycosidic bond (38). Multiple- Cys222 and covers the cleft formed in the active
sequence alignment of glycoside hydrolase (GH) site. The Phe and both Cys residues are absent
families 32, 43, 62, and 68 revealed three from β-xylosidases in general, and T. fusca β-
conserved blocks, each containing an acidic xylosidase in particular. Moreover according to
residue at an equivalent position in all of the Brux, et al. (32), in the β-xylosidases, an extended
enzymes; site-directed mutagenesis studies have arabinopyranose ligand would clash with the
shown that Asp/Glu (block I), Asp (block II), and hydrophobic area generated by conserved
Glu (block III) are essential for catalytic activity of xylosidase residues, i.e., Trp74, Phe127, Phe32,
GH43. This indicates direct involvement of the and Phe506 of G. stearothermophilus XynB3. In
conserved residues in substrate binding and the case of galactopyranose, there is also a steric
hydrolysis, and substitutions of these specific clash caused by the additional hydroxymethyl
residues inactivate the enzyme or considerably group, which encounters Phe506 (1.4 Å). These
reduce its activity. These three strictly conserved criteria explain the inability of the T. fusca D
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key acidic residues are located in a funnel-shaped catalytic GH43 module to degrade arabinan, and w
n
active site that comprises two subsites with a underscore the sophisticated divergence of the loa
d
single route for access by ligands in a β-propeller conserved GH43 arabinanases and β-xylosidases. ed
architecture, and the three residues operate with Physical separation and reassociation of the from
the canonical reaction mechanism of inversion of two modular components of Xyl43A (GH43 and http
anomeric configuration (32,39). More recently, the Module-A) enabled us to further analyze the ://w
crystal structures of Cellvibrio japonicus enzymatic and binding properties of this enzyme. w
w
arabinanase 43A and Geobacillus Separating the two modules led to a complete loss .jb
c
stearothermophilus β-xylosidase further in enzymatic activity of the N-terminal module .org
demonstrated the role of these three carboxylates GH43 but did not affect its ability to bind xylan, b/
y
in catalytic activity (19,31). Similar blocks and suggesting that the active site would thus need the gu
e
essential residues were found in T. fusca Xyl43A combined conformation of the complete protein in st o
by sequence comparison: block I Y29PDPS33, order to degrade its substrate correctly. Indeed, a n A
p
block II G142FDPS146 and block III V201TEAPH206. strong non-covalent interaction occurs between the ril 5
In the GH43 family, several modular two modules of T. fusca Xyl43A. This physical , 2
0
architectures are observed (40). One group of interaction led to substantial recovery of β- 19
GH43 enzymes is composed only of a single xylosidase activity (71.8%), demonstrating the role
GH43 module and lacks the C-terminal module of the Module-A in enzymatic degradation of its
(Module-A). These enzymes belong to the substrate. Based on the solved structure of G.
arabinanase class (20-22,41). Another group of stearothermophilus (32,43), several residues play
family GH43 enzymes comprising both modules is an essential role in formation of the strong
defined as either β-xylosidases or α-L- interface between the two modules of the enzyme
arabinofuranosidases (32,42). One possibility is (Table 2).
that the C-terminal module in family GH43 would The re-association of GH43 and Module-A in
be responsible for switching substrate specificity T. fusca Xyl43A provided a key functional residue
from arabinan to xylobiose. However, in the for β-xylosidase activity. The phenylalanine
present work, the T. fusca GH43 module alone was located at position 518 of Module-A was thus
unable to degrade arabinan and no binding ability demonstrated to be inserted in a critical position
of either of the two Xyl43A modules towards and essential for Xyl43A enzymatic activity. The
arabinan was observed, thus suggesting that the F518A replacement, either in the intact enzyme
difference in specificities is a function of the Xyl43A or in Module-A, resulted in the near-
topography and composition of the active site. complete loss of enzymatic activity. According to
According to Alhassid, et al. (22), the topology of
9

Brux et al. (32), this conserved phenylalanine unclear, since it is phylogenetically unconnected
emerges from a loop originated from the β- with any known protein in the databases other than
sandwich domain and contributes to substrate those associated with the family-GH43 enzymes.
binding by a stacking interaction with the xylose Perhaps an ancestral form of Module-A was
unit at the -1 subsite. Interestingly, the amino-acid originally a CBM, since it possesses reminiscent
replacement does not appear to affect the overall characteristics of a CBM (e.g., β-sandwich
folding of the enzyme according to the re- structure); its binding role would presumably have
association of the separated GH43 and mutated been lost during evolvement of its singular

Module-A. catalytic contribution to the GH43 xylosidases.
In a recent article (44), physical re-association Moreover, since Xyl43A does not contain any
of a family-GH9 endoglucanase catalytic module signal peptide, the enzyme appears to remain in
and its ancillary family-3c CBM was also the intracellular milieu in T. fusca and would not
demonstrated with attendant regain of enzyme require a CBM to target the enzyme to its
activity. The latter study, together with the current substrate. Indeed, xylobiose is small enough to
work, promotes the original description of the enter the cell, presumably via an appropriate
combined S-protein and S-peptide to generate an transport apparatus, and can be degraded internally
active form of ribonuclease A (45). In the current into xylose units, so a CBM would be irrelevant
studies on the glycoside hydrolases, however, the for the enzyme. Consequently, as representative of D
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two interacting modules are clearly of distinct the GH43 β-xylosidases, the two linked modules w
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evolutionary origin. of T. fusca Xyl43A should thus be considered as loa
d
Indeed, the two separate modules of Xyl43A an intact, synchronized functional unit, rather than ed
fold on their own, suggesting that originally they a fortuitous pair of independent modules. fro
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may have had distinct functions. The evolutionary h
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source of the C-terminal Module-A, however, is ://w
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