Understanding the glucose tolerance of an archaeon β-glucosidase from Thermococcus sp.

Article abstract of DOI:10.1016/j.carres.2019.107835

β-glucosidase hydrolyzes the β-1,4 linkage of cellobiose, a product generated from the action of endoglucanase and cellobiohydrolase on cellulose, and generates glucose. Accumulated glucose during saccharification leads to product inhibition of β-glucosidase, which in turn cause an accumulation of cellobiose and inhibition of other cellulolytic enzymes. Thus, glucose tolerant and active β-glucosidase is required for the efficient saccharification of biomass. O08324 is a glucose tolerant β-glucosidase isolated from archaeon Thermococcus sp. which shows no loss in enzyme specific activity in the presence of up to 4 M glucose and is active at 78 °C. Since O08324 has such high glucose tolerance, knowing the rationale for glucose tolerance will be helpful in engineering glucose tolerant β-glucosidase. In the present study, we designed mutations at eleven sites across the gatekeeper, aglycone, and glycone region. Based on the kinetic studies of O08324 mutants, the gatekeeper residues at positions 160, 166, 167, 168, and the aglycone binding residue 156 were identified to play a role in glucose inhibition. However, only residues at the tunnel entrance, and not all gatekeeper residues contribute to glucose tolerance. This study sheds some light on the unusual glucose tolerance of O08321 archaeal GH1 β-glucosidase.

Full text of DOI:10.1016/j.carres.2019.107835

Journal Pre-proof
Understanding the glucose tolerance of an archaeon β-glucosidase from
Thermococcus sp.
Sushant K. Sinha, K. Prakash Reddy, Supratim Datta
PII:
S0008-6215(19)30421-5
DOI:
https://doi.org/10.1016/j.carres.2019.107835
CAR 107835
Reference:
To appear in: Carbohydrate Research
Received Date: 17 July 2019
Revised Date: 29 September 2019
Accepted Date: 10 October 2019
Please cite this article as: S.K. Sinha, K. Prakash Reddy, S. Datta, Understanding the glucose tolerance
of an archaeon β-glucosidase from Thermococcus sp., Carbohydrate Research (2019), doi: https://
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Understanding the glucose tolerance of an archaeon β-glucosidase from Thermococcus sp.
Sushant K Sinha1, K. Prakash Reddy1, Supratim Datta1,2,3,*
1Protein Engineering Laboratory, Department of Biological Sciences, Indian Institute of Science
Education and Research Kolkata, Mohanpur, West Bengal
2Center for the Advanced Functional Materials, Indian Institute of Science Education and
Research Kolkata, Mohanpur, West Bengal
3Center for the Climate and Environmental Sciences, Indian Institute of Science Education and
Research Kolkata, Mohanpur, West Bengal
*To whom correspondence should be addressed: Supratim Datta, Department of Biological
Sciences, Indian Institute of Science Education and Research, Mohanpur 741246, India, Fax: 91-
33-25873020; supratim@iiserkol.ac.in
1

Abstract
-glucosidase hydrolyzes the β-1,4 linkage of cellobiose, a product generated from the action of
endoglucanase and cellobiohydrolase on cellulose, and generates glucose. Accumulated glucose
during saccharification leads to product inhibition of -glucosidase, which in turn cause an accumulation
β
β
of cellobiose and inhibition of other cellulolytic enzymes. Thus, glucose tolerant and active
is required for the efficient saccharification of biomass. O08324 is a glucose tolerant
β
β
-glucosidase
-glucosidase
isolated from archaeon Thermococcus sp. which shows no loss in enzyme specific activity in the presence
of up to 4 M glucose and is active at 78 °C. Since O08324 has such high glucose tolerance, knowing the
rationale for glucose tolerance will be helpful in engineering glucose tolerant β-glucosidase. In the present
study, we designed mutations at eleven sites across the gatekeeper, aglycone, and glycone region. Based
on the kinetic studies of O08324 mutants, the gatekeeper residues at positions 160, 166, 167, 168, and the
aglycone binding residue 156 were identified to play a role in glucose inhibition. However, only residues
at the tunnel entrance, and not all gatekeeper residues contribute to glucose tolerance. This study sheds
some light on the unusual glucose tolerance of O08321 archaeal GH1 β-glucosidase.
2

Introduction
-Glucosidase (EC 3.2.1.21) is a glycosyl hydrolase enzyme that cleaves
or similar substituted molecule from the non-reducing end and releases glucose [1]. An essential
application of -glucosidase is in the enzymatic cocktail for the hydrolysis of cellulose polymer to
β
β-1,4 linkage of the cellobiose
β
glucose monomer, which is a precursor for various chemicals and fuels such as bioethanol, fatty acid
esters, and biodiesel. The enzymatic cocktail is a mixture of at least three enzymes, cellobiohydrolase,
endoglucanase, and β-glucosidase. Endoglucanase (EC 3.2.1.4) randomly cleave the β-1,4 glycosidic
linkages of long cellulose polymer into small oligomers, then cellobiohydrolase (EC 3.2.1.91) attack the
oligomers and cleaves off cellobiose, a dimer of glucose, from reducing and non-reducing ends. Finally,
β
-glucosidase (EC 3.2.1.21) acts on cellobiose to generate two molecules of glucose.
High substrate loading during the saccharification process leads to an initial accumulation of high
concentrations of the reaction product glucose, leading to an inhibition of
Product inhibition of -glucosidase by glucose varies between µM to M range [1]. However, there are
only a few naturally occurring glucose tolerant -glucosidase, with glucose tolerance in the molar range.
In recent years, transglycosylation of glucose and allosteric activation of -glucosidase by glucose
binding to allosteric sites have been proposed to explain glucose tolerance and stimulation of
β-glucosidase catalytic activity.
β
β
β
β
-
glucosidase [2-4]. In another study, it has been suggested that glucose relieves substrate inhibition
leading to stimulation of enzymatic activity by glucose [5]. However, to date, the mechanism of
glucose tolerance is unknown. It has been proposed that the amino acid residues located in the aglycone
binding sites are responsible for glucose tolerance [6, 7]. Giuseppe et al. have reported that
with low glucose tolerance generally possess a larger active site tunnel entrance and an electronegative
patch along the tunnel while -gluco,sidase with high glucose tolerance have a narrower tunnel entrance,
smaller tunnel length, and the gatekeeper region at the entrance occupied by neutral residues [6]. It has
also been proposed that glucose tolerant -glucosidase may contain secondary glucose binding sites inside
β-glucosidase
β
β
the tunnel and thus the structure inside the tunnel is critical for glucose tolerance and substrate specificity
[4, 8]. Thus, there is a need to understand the basis of such tolerance in order to engineer glucose
tolerance.
Thermostable, active
saccharification processes at industrial scale. While there are a few reports of engineering of
glucosidase for glucose tolerance from bacterial and fungal sources, in which residues have been
highlighted to be responsible for glucose tolerance and stimulation, no such reports are available for
β-glucosidase which can tolerate high glucose concentration is needed for
β
-
β
-
glucosidase of archaeal origin [2, 9]. Many archaea are extremophiles, and their enzymes are
extremozymes due to their adaptation to the most extreme environments [10]. Archaeal enzymes are
3

industrially more preferable for their robustness at elevated temperature, extremes of pH, and high ionic
strength. Thus, understanding the glucose tolerance of O08324 is essential. O08324 is a small protein
with only 418 residues than the typical GH1
β-glucosidase containing 440-480 residues [11]. Since
O08324 is not transglycosylated in the presence of excess glucose, and no glucose binding sites could be
detected, probing the enzyme for more insights into the basis of glucose tolerance was necessary [12]. In
this study, we report the contributions of residues across eleven sites to glucose tolerance. The rationally
designed mutations span the gatekeeper, aglycone, and glycone binding region of the active site tunnel
and help identify residues that play a role in glucose tolerance.
Material and Methods
Chemicals: Oligonucleotides used for cloning and mutagenesis were synthesized by Xceleris
(Ahmedabad, India). DNA polymerase, restriction endonucleases were purchased from NEB (MA, USA),
dNTPs, and DNA ligase were purchased from Thermo scientific (Mumbai, India). All the substrate and
chemicals purchased from Sigma-Aldrich (St. Louis, MO, USA). Plasmid prep kit and Agarose gel
purification kit were purchased from Qiagen (Hilden, Germany). Centrifugal concentrator 30 kDa cut-off
was purchased from Amicon-Ultra-15, Merck Millipore (Bangalore, India). All other chemicals and
buffers used were of analytical or HPLC grade.
Bacterial strains, culture conditions, and plasmids: The synthetic gene corresponding to the
β-
glucosidase from Thermococcus sp. was constructed (Gene accession number BankIt1899421
BG_Thermo KU867869) as reported previously and expressed in Escherichia coli BL21(DE3) expression
strain (Thermo Fisher Scientific, Waltham, USA) [12]. The cells were centrifuged at 4000 ×g for 10 min
at 4 °C and the pellet stored at -20 °C until purification of the protein.
Mutagenesis and cloning: Mutations in O08324 were created by mutagenic megaprimer based
polymerase chain reaction (PCR) based mutagenesis strategy. Briefly, three primers were used - a
template specific forward and reverse primer and the mutant primer either towards the forward or reverse
direction, depending on the position of the mutation in the gene. The first PCR was run using the mutant
primer and template-specific primer to generate the megaprimer containing the mutation. The megaprimer
was extended during the second PCR by another sequence-specific primer. While the single mutants were
generated from the wild-type DNA, the template containing single mutations were used to generate the
double mutants. [13]. The sequence of mutagenic primers used in this study is listed in Table S1.
Primers were designed using OligoAnalyzer (IDT Technology) and ApE (ApE Plasmid Editor, version
4

2.0.49 by M. Wayne Davis). The DNA sequences encoding the mutants were obtained from both strands
by automated DNA sequencing at the IISER Kolkata sequencing facility.
Enzyme activity assays: The pH dependence of O08324 mutants was determined by measuring enzyme
specific activities on p-nitrophenyl-D-glucopyranoside (pNPGlc) in the pH range of 5.0 to 8.0 at 78 °C,
after incubating the enzyme overnight at 4 °C in each buffer. The effect of temperature on enzyme
activity for pNPGlc was measured between 55 to 88 °C while incubating in McIlvaine buffer, pH 6.5.
Based on the initial rate measured, the amount of enzyme to be used, and the assay time was optimized.
To determine the specific activity, the mutants were assayed at the Topt and pHopt of each enzyme using
saturating concentrations of the substrate pNPGlc, as per previously published protocol [12].
Kinetic analysis of O08324 mutants: The kinetic parameters of all the mutants were determined at
concentrations, ranging between 0.5 mM to 70 mM, of substrates pNPGlc as previously reported (2).
GraphPad PRISM version 7.0 (GraphPad Software, La Jolla, CA) was used to calculate all kinetic
constants by a non-linear regression fit of the Michaelis- Menten equation.
Effect of glucose on the kinetics of O08324 mutants: The effect of glucose on the enzyme activity were
determined in the presence of an increasing concentration of glucose (0-3 M), using pNPGlc as substrate
under the optimum assay conditions determined for each mutant and tabulated in Table 2. Briefly, in the
total reaction volume of 100 µL, the enzyme and substrate were incubated in Mcllvaine buffer of pHopt for
each mutant and at the Topt of each mutant for 5 minutes. The assay was stopped by addition of 100 µL
glycine-NaOH buffer (pH 10.8). The specific activity of each mutant in the absence of glucose was
considered to be 100 %, and the % relative specific activity in the presence of glucose was determined.
The kinetic parameters of each mutant were determined for a minimum of three concentrations of glucose
(0.1 M, 0.25 M, and 0.5 M).
Results and discussions:
Homology modeling and structural comparison: The lack of O08324 structure necessitated the
generation of a structural model by the homology modeling tool ModBase, using available
GH1 structures in the PDB database [14]. The O08324 model was selected from the highest sequence
identity match (63 % similarity) to the -glucosidase from the archaea Pyrococcus horikoshii (PDB:
β-glucosidase
β
1VFF) [15]. The model quality of O08324 was within the quality thresholds as per the program.
Additionally, the model was validated by Ramachandran plot analysis using PROCHECK and ProSA
(Figure S2). PROCHECK examines the stereo-chemical quality of a modeled structure using
5

Ramachandran plot while ProSA gives the details of the fold quality of the modeled protein. Figure 1
shows the three-dimensional representation of the O08324 model structure containing the typical
(
α
/
β
)8 TIM barrel fold found in GH1
as catalytic residues located deep in the active site tunnel. Since the glucose dependence of the
glucosidase from Pyrococcus horikoshii is not known, we compared the O08324 structure with the
structure of glucose tolerant -glucosidase from Halothermothrix orenii (IC50,Glc=2.6 M) and a glucose-
sensitive -glucosidase from Paenibacillus polymyxa (IC50,Glc= 300 mM) [16, 17]. The structural
β-glucosidase, with the glutamic acid at fourth and seventh β-strand
β
-
β
β
comparisons reveal significant changes within the surface-exposed loops (Figure 2). O08324 is smaller
by around 30 residues compared to either of the above mentioned two proteins. The structural integrity
and folding in O08324 are possibly enabled through the shorter loops (Figure 2). In proteins, loops are an
essential structural feature and highly flexible even in stable proteins. The sequence and structural
variability of loops among homologous proteins could lead to differences in the protein folding, stability,
and biological function [18]. It has been recently reported that glucose affects the flexibility of
β-
glucosidase and is responsible for the widening of the active site tunnel, leading to glucose accumulation
and inhibition [19]. Thus, the smaller loops of O08324 may help resist the glucose-induced
conformational changes, resulting in high glucose tolerance.
The basis of mutant selection: There are only a few archaeal
(Table 1), and very little is known about glucose tolerance of
Understanding the rationale behind the glucose tolerance of O08324 is essential as it is first such kind of
study with an archaeal -glucosidase, which, unlike other previously archaeal -glucosidase, has very
β
-glucosidase characterized thus far
β
-glucosidase from archaea.
β
β
high glucose tolerance. To understand the molecular basis of high glucose tolerance of O08324 and
identify residues that could be responsible for imparting glucose tolerance and/ or stimulation, we
compared its sequence with GH1
β
-glucosidase of known glucose tolerance and also with archaeal
β-
glucosidase reported in the literature (Figure 3, Figure S1). The active site tunnel of GH1
β
-glucosidase
is typically divided into three regions- glycone binding (subsite -1), aglycone binding (subsite +1) and
gatekeeper region (Figure S3) [13]. In general, the gatekeeper and aglycone residues are mostly non-
conserved, while the glycone-binding pocket residues are conserved across the GH1 β-glucosidase [20].
In particular, residues in proximity or inside the active site tunnel were chosen towards a rational
designing of mutants with increased sensitivity to glucose and lower glucose tolerance.
Glycone binding site: In O08324, the glycone binding residues at position 110 is not conserved, with a
Phe instead of a Trp, so we chose to mutate the Phe to a Trp at this position to observe the effect of this
substitution (Figure 4). In the catalytic motif “VTENGI” both Val318 and Ile323, were replaced by Lys
and Ala respectively. Position 318 is non-conserved (Figure 4) and occupied by non-polar residues, Ile, or
6

Val. To observe the role of the non-polar residue at this site, Val was substituted by a polar Lys. Ile323
was replaced by Ala to understand the effect of volume of the side chain.
Aglycone binding site: At the aglycone binding site, residue 156 is near to the catalytic residue E153, and
is alternately occupied by Val or Cys across GH1
substituted by a polar Cys.
β-glucosidase. The non-polar V156 was therefore
Gatekeeper residues: Gatekeeper residues A160, T166, W167, P168, P208, F230, S233 were mutated to
change hydrophobicity and size at the tunnel entrance. The residue A160 was substituted by polar Glu
and nonpolar Leu. T166 was replaced with more polar Tyr, which is present in Humicola grisea
β-
glucosidase (21), glucose-sensitive and glucose-stimulated -glucosidase (Figure 3). The residue W167
β
was substituted by His and Lys, as this position is occupied by His in Td2f2, Thermoanaerobacterium
saccharolyticum, Agrobacterium tumefaciens, Halothermothrix orenii and Paenibacillus polymyxa, and
by Lys to make this position highly hydrophilic (Figure 3). Similarly, P168 was substituted by Ala as
present in most of the glucose tolerant
β
-glucosidase and by Lys to increase hydrophilicity. P208 was
-glucosidase like Td2f2, Halothermothrix orenii,
substituted to Thr, as most of the glucose-stimulated
β
and Thermoanaerobacterium saccharolyticum has a Thr at that position. The residue F230 was replaced
with smaller side-chain Ala to decrease the sterics. The residue S233 was replaced with the Trp as most of
the β-glucosidase have it at a similar position. Thus, we have generated mutants in different regions of the
active site tunnel, taking into account properties like residue volume and charge to understand how these
affect glucose tolerance (Table S2).
Biochemical characterization of the O08324 mutants: The optimum pH (pHopt) and temperature (Topt)
were determined for each mutant using pNPGlc as the substrate (Table 2). The optimum temperature of
mutants V156C and A160L were increased by 2 °C and 4 °C respectively as compared to the wild type
(78 °C), while the Topt of rest of the mutants remained similar to the wild-type. In mutant A160L, the
increase in thermal stability could be due to a bulkier Leu side chain instead of the smaller Ala, making
the structure rigid and stable without affecting major electrostatic interactions. The optimum pH (pHopt) of
all mutants was 6.5 except W167K, which increased to 7.0. The mutant kinetics in the absence and
presence of glucose was measured at their respective optimum temperature and pH. The small changes in
optimum pH and temperature among mutants could arise due to its location and changes in interaction
with the neighboring residues and solvent. Though we did not observe much differences in the
thermodynamic properties, Topt and melting temperature, Tm (data not shown) of the mutants, there are
significant differences in the kinetic properties.
7

The specific activity of F110W, V156C, W167H, P208T, and I323A under their optimum assay
conditions decreased by fifty percent as compared to the wild type (Table 2) while for the rest of the
mutants (A160E, E167K, V156C, and T166Y) it is similar or more than the wild-type. Two mutants,
F110K and V318K, do not show any activity. As F110 is a glycone-binding residue and V318 is very
close to the catalytic residue E320, mutation to a charged polar residue Lys could lead to disruption of the
hydrogen bonding network essential for the substrate positioning and catalysis. Table 3 lists the kinetic
parameters of the mutants.
Effect of glucose on O08324 mutants: The mutants were assayed in the presence of a varying
concentration of glucose and the inhibition quantitated either by a decrease in specific activity or an
increase in Km with increasing concentration of glucose. The mutant F110W show glucose tolerance and
50 percent stimulation of specific activity in the presence of 2-3 M glucose (Figure 5). This site is
essential for glycone binding and has been previously shown to play a crucial role in glucose tolerance in
B8CYA8 and H0HC94 [21, 22].
V156 is an aglycone binding site and forms a small hydrophobic patch near the catalytic domain.
Kinetic assay of V156C showed an increase in Km with increasing glucose concentration, without any
change in kcat and was inhibited by glucose. This inhibition might be due to the substitution of polar Cys
residue which potentially disturbs the hydrophobic patch near the catalytic site. The polar Cys residue can
interact strongly with glucose and thus increase the competition with a substrate for the catalytic site,
leading to high Km in the presence of glucose.
Gatekeeper residues may help maintain the dynamics of substrate and product inside the active
site tunnel and can play an essential role in the glucose tolerance of β-glucosidase. Among the mutations
at the gatekeeper region, hydrophilic substitutions like in A160E, T166Y, W167K W167H, and P168K
show an increase in inhibition with increasing concentration of glucose. The percent specific activity of
A160E, W167K and W167H decreased by 25 %, 35 %, and 20 % respectively (Figure 5), while the
increase in Km is almost 2-fold in the presence of 0.5 M glucose as compared to the absence of glucose
(Table 3). The mutants, T166Y and P168K, also show an increase in Km with an increase in glucose
concentration, though there is no decrease in a specific activity. The S233W mutant shows an increase in
Km and kcat in the presence of glucose, indicating both stimulation and inhibition in the presence of
glucose. The A160L mutant does not show any inhibition in the presence of up to 3 M glucose. In I323A,
the smaller side chain in Ala decreases the steric interactions at this site and facilitates more glucose entry
inside the tunnel leading to glucose inhibition. In A367K, there is no effect on glucose tolerance or
8

change in Km, probably because it is situated outside the tunnel where the change in charge does not seem
to play any role in modulation of enzyme activity by glucose.
P208T shows a 75 % stimulation in specific activity in the presence of a higher concentration of
glucose. At this particular site, Thr has a role in glucose-induced stimulation in specific activity as
previously reported in Td2f2, Halothermothrix orenii, and Thermoanaerobacterium saccharolyticum, all
of which show similar glucose-induced stimulation [3, 12, 23]. Most of the glucose tolerant bacterial β-
glucosidase also show a glucose-induced stimulation. At low concentrations of glucose, typically the
Km and kcat increases, while at higher glucose concentrations Km increases along with a decrease in kcat
resulting in the decrease of kcat/Km [2-4, 8]. In contrast, in wild-type O08324, the kcat/Km remains
unchanged in the presence of all concentrations of glucose.
Among the archaeal
saccharovorans (Ki, Glc 158), spring metagenome (Ki, Glc=150 mM) and, Pyrococcus furiosus (Ki, Glc=300
mM), the glucose tolerance is low [24-27]. Alignment with such reported glucose tolerant archaeal
β-glucosidase, like in Sulfolobus solfataricus (Ki, Glc= 96 mM), Acidilobus
β
-
glucosidase reveals structural difference mainly arising due to the shorter length of O08324 and is
evident from the gaps in the alignment (Figure S1). The gatekeeper residues are non-conserved and
have less similarity with O08324 than amongst the rest. For example in other archaeal β-glucosidase,
the gatekeeper residue A160 of O08324 is occupied by Ile, Leu, and Gln, T166 is occupied by Asn
and Gly, W167 is by Phe, P208 is by Ser and Ala, and S233 is by Ala (Figure S1). Such variability is
also observed across gatekeeper residues in previously reported bacterial GH1
All previous studies on the mechanism of glucose tolerance and stimulation were on the
bacterial and fungal GH1 -glucosidase [1]. Here we investigated the role of the active site tunnel of
β-glucosidase.
β
O08324 towards understanding the unusually high glucose tolerance of this enzyme. The mutational
study and changes in enzyme activity and kinetic parameters with increasing glucose concentration reveal
a trade-off between hydrophilicity at gatekeeper positions A160, T166, W167 and P168, and aglycone
residue 156 and glucose tolerance. The hydrophilic changes in the vicinity of the active site tunnel
entrance increase the glucose-induced inhibition and facilitate the entry of more glucose and depletion of
the water molecule, which functions as a proton donor, at the substrate-binding site in the active site
tunnel. The other gatekeeper residues at P208, F230, S233, A367, however, did not show much effect on
glucose tolerance probably because not all gatekeeper residues are the same. Residues far from the
catalytic site and situated at the different ??-sheets than the one at which catalytic residues may have no
role to play in any change in conformation of the active site region. The study shows the importance of
the gatekeeper residue and the active site tunnel in glucose tolerance and stimulation.
.
9

Conclusions
Through a combination of structural analysis, protein engineering, and kinetic study of the mutants, our
work shows the differences between O08324 and other archaeal and bacterial β-glucosidase. The non-
conserved residues, particularly at the entrance to the active site tunnel play an essential role in glucose
tolerance. Such studies towards understanding glucose tolerance are crucial towards improving the
glucose tolerance of β-glucosidase and a more efficient cellulase cocktail.
Conflict of interest
The authors declare that they have no conflicts of interest with the contents of this article.
Acknowledgments
We thank Mr. Shibashis Das for his initial help in cloning.
Author contributions
SD, SKS, designed the study. SKS, KPR performed the study. SD, SKS, KPR analyzed the data. SD and
SKS wrote the paper.
Footnotes
This work was supported in part by the Science & Engineering Research Board (SERB), Government of
India, EMR/2016/003705 (S.D.) and Academic Research Fund (IISER Kolkata). SKS is supported by
Senior Research Fellowship from CSIR, Govt. of India and K. Prakash Reddy is supported by an Institute
Fellowship from IISER Kolkata
Abbreviations: pNPGlc, p-nitrophenyl-β-D-glucopyranoside; Glc, glucose;
10

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13

Table 1: List of biochemical properties of characterized archaeal β-glucosidase as reported in
GH
Family
GH1
Topt
(°C)
78
Specific activity
(U/mg)
Ki,Glc
(mM)
ND*
Organism
pHopt
References
Thermococcus sp.
6.5
6.5
6.0
5
208
[12]
[24]
[25]
[23]
[26]
[27]
[28]
[29]
[30]
Sulfolobus solfataricus
Acidilobus saccharovorans
Sulfolobus shibatae
Spring metagenome
Pyrococcus furiosus
Thermofilum pendens
Pyrococcus kodakaraensis
Pyrococcus horikoshii
literature.
GH1
GH1
GH1
GH1
GH1
GH3
GH1
GH1
75
93
95
90
95
90
100
90
116
246ꢀ
96
158
NR
150
300
NR
NR
NR
NR
6.5
5.5
3.5
6.5
6
3195
19.8
910.1
kcat =1650 min-1
kcat =194 min-1
ND*: Not detected up to 4 M of glucose
NR: Not reported
14

Table 2. Biochemical properties- pH optimum (pHopt , Temperature optimum (T (˚C), specific
)
)
opt
activity on p-nitrophenyl β-glycoside (pNPGlc) of wild type, and mutants. The assays were as detailed
in the materials and methods, and specific activity is reported in µmol/min/mg. All measurements
were done at least in triplicate.
Specific activity
(µmol/min/mg)
S. No.
Mutants
pHopt
6.5
not active
6.5
Topt ( )
78
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Wild type
F110K
V156C
A160L
A160E
W167K
F230A
A367K
I323A
208
80
82
78
78
78
78
78
78
78
78
78
57
220
173
24
6.5
6.5
7.0
6.5
6.5
6.5
6.5
6.5
22
160
6.0
124
153
114
69
P208T
P168A
P168K
S233W
V318K
W167H
T166Y
F110W
6.5
6.5
not active
6.5
78
78
78
20
26
24
6.5
6.5
15

Table 3. Kinetic parameters of O08324 mutants on the substrate pNPGlc in the presence of different
0 Glc
0.1 M Glc
0.25 M Glc
0.5 M Glc
concentrations of glucose. Kinetic parameters were determined in optimized reaction condition for
each mutant as described in Table 2. In a reaction mixture of 100 µL, the final glucose concentrations
of 0.1 M, 0.25 M, 0.5 M were used. Experiments were done in triplicate and repeated at least thrice.
Errors shown here are standard deviations of independent reactions. Already published data are shown
in italics [12].
16

0 Glc
0.1 M Glc
0.25 M Glc
0.5 M Glc
K
7.6 ± 1.1
8.2 ± 0.6
10.0 ± 1.3
9.3 ± 1.0
Kmm
3.7 ± 0.1
4.7 ± 0.7
3.2 ± 0.2
2.9 ± 0.3
Wild- type
kcat
190.5 ± 11.7
2.75 ± 0.0
25.06 ± 2.3
0.75 ± 0.0
189.3 ± 10.9
3.2 ± 0.1
195.9 ± 8.9
3.3 ± 0.1
200.5 ± 8.9
2.99 ± 0.1
21.60 ± 1.4
1.1 ± 0.1
F230A
kcat
kcat/K
23.01 ± 2.1
0.68 ± 0.1
19.59 ± 1.8
1.0 ± 0.1
kcat/Kmm
Km
kcat
11.11 ± 0.98
22.92 ± 0.57
7.20 ± 0.92
17.49 ± 0.41
8.82 ± 1.7
13.77 ± 0.4
8.47 ± 0.71
18.20 ± 0.21
S233W
F110W
Km
7.62 ± 0.48
9.84 ± 0.66
15.82 ± 1.16
12.70 ± 0.82
kcat/Km
2.07 ± 0.14
2.45 ± 0.26
1.60 ± 0.27
2.16 ± 0.15
Km
kcat
kcat/Km
3.48 ± 0.17
48.35 ± 0.89
13.92 ± 0.69
5.64 ± 0.05
48.58 ± 0.05
8.62 ± 0.07
5.49 ± 0.09
44.65 ± 0.62
8.14 ± 0.02
9.78 ± 0.75
47.63 ± 0.65
4.89 ± 0.32
V156C
A160E
A160L
T166Y
W167H
W167K
P168A
P168K
Km
Kcat
kcat/Km
12.36 ± 1.09
252.96 ± 9.1
20.52 ± 1.03
12.11 ± 1.07
263.55 ± 14.15
21.81 ± 0.86
20.03 ± 0.62
267.40 ± 5.95
13.35 ± 0.19
27.47 ± 0.45
311.70 ± 3.37
11.35 ± 0.11
Km
kcat
kcat/Km
7.6 ± 0.1
339.4 ± 26.9
45.0 ± 2.6
7.6 ± 1.0
424 ± 33.6
56.2 ± 3.3
5.5 ± 0.6
332.5 ± 7.7
60.5 ± 5.8
7.0 ± 0.8
316.2 ± 15.7
45.5 ± 2.8
Km
kcat
kcat/Km
7.48 ± 0.4
192.60 ± 2.47
25.80 ± 1.16
10.29 ± 1.3
202.98 ± 11.9
19.84 ± 1.4
11.82 ± 0.67
216.62 ± 2.31
18.35 ± 0.83
10.83 ± 1.03
195.72 ± 5.61
18.15 ± 1.29
Km
kcat
kcat/Km
9.20 ± 0.92
70.59 ± 2.14
7.71 ± 0.58
13.74 ± 1.19
79.62 ± 3.59
5.81 ± 0.25
19.12 ± 1.44
99.75 ± 6.49
5.22 ±0.08
16.30 ± 0.92
95.99 ± 2.38
5.90 ± 0.23
Km
kcat
kcat/Km
12.4 ± 0.9
78.5 ±1.8
6.3 ± 0.4
17.5 ± 1.8
74.9 ± 7.7
4.3 ± 0.1
22.7 ± 2.2
92.3 ± 7.0
4.1 ± 0.3
34.7 ± 4.1
100.4 ± 7.2
2.9 ± 0.2
Km
kcat
kcat/Km
13.50 ± 0.66
164.07 ± 3.76
12.17 ± 0.44
9.69 ± 0.17
159.16 ± 0.16
16.43 ± 0.28
10.02 ± 0.22
159.36 ± 0.9
15.92 ± 0.29
8.49 ± 0.49
166.71 ± 3.42
19.67 ± 0.8
Km
kcat
kcat/Km
7.53 ± 0.35
140.29 ± 6.0
18.64 ± 0.49
13.08 ± 0.6
161.22 ± 4.77
12.34 ± 0.23
13.48 ± 0.68
150.41 ± 1.81
11.17 ± 0.43
14.48 ± 0.56
157.77 ± 3.35
10.90 ± 0.19
Km
kcat
kcat/Km
15.59 ± 1.8
33.77 ± 1.58
2.18 ± 0.27
23.20 ± 2.3
38.73 ± 2.34
1.67 ± 0.06
12.77 ± 2.59
29.46 ± 1.46
2.36 ± 0.38
14.36 ± 0.88
33.58 ± 1.75
2.34 ± 0.19
P208T
17

kcat
kcat/Km
80.35 ± 1.95
10.57 ± 0.46
91.50 ± 2.29
9.32 ± 0.42
111.04 ± 2.65
7.04 ± 0.37
109.18 ± 3.41
8.61 ± 0.29
Km
kcat
kcat/Km
3.86 ± 0.5
39.95 ± 1.67
10.44 ± 1.04
8.76 ± 0.95
46.23 ± 1.43
5.31 ± 0.51
7.17 ± 1.28
35.65 ± 1.06
5.07 ± 0.85
8.73 ± 0.23
36.22 ± 0.43
4.15 ± 0.16
I323A
Km
kcat
kcat/Km
6.58 ± 0.22
141.66 ± 1.77
21.53 ± 0.59
6.87 ± 0.87
135.15 ± 5.06
19.83 ± 1.93
5.55 ± 0.23
132.18 ± 2.21
23.82 ± 0.88
5.98 ± 0.57
131.04 ± 5.18
22.00 ± 1.21
A367K
Figure 1. Homology model of O08324. The protein sequence of O08324 was uploaded to ModBase
servers (http://modbase.compbio.ucsf.edu) and after processing model showing the highest sequence
similarity with query was taken. The model was built upon ??-glycosidase from Pyrococcus
horikoshii (PDB code: 1VFF:A) with 63% similarity. The two catalytic residues E153 and E320 are
highlighted in the active site tunnel.
18

Figure 2. The structural comparison of O08324 (model structure, tan) with two β-glucosidases, a)
2O9P (Paenibacillus polymyxa, low glucose tolerance), sky blue and b) 4PTX (Halothermothrix
orenii, high glucose tolerant and glucose-stimulated (plum) [22]. The differences in the loops in both
the cases are highlighted with the dotted circles. The figures were made using Chimera [31].
19

Figure 3. Multiple sequence alignment of O08324 with previously reported glucose tolerant and
glucose stimulated β-glucosidase in the GH1 family, using Clustal Omega [32]. The sequences were
from O08324:Thermococcus sp. (100 % specific activity up to 4 M glucose) [12], A0A0F7KKB7:
bacterial metagenome (IC50,Glc = 3.5 M) [33], Td2f2: bacterium metagenome (IC50,Glc > 1 M) [34],
G8YZD7: Fervidobacterium islandicum (Ki,Glc = 211 mM) [35], I3VS74: Thermoanaerobacterium
saccharolyticum (IC50,Glc = 600 mM) [36], O93785 Hypocrea jecorina IC50,glc = 300 mM [37],
20

H0HC94 Agrobacterium tumefaciens 5A Ki,Glc = 686 mM [21], B8CYA8: Halothermothrix orenii,
O93784: Humicola grisea , IC50,Glc > 500 mM [38], P22505: Paenibacillus polymyxa (IC50,Glc = 300
mM). The mutation sites are highlighted with light blue color.
Figure 4. Cross section of O08324 active site tunnel surface representation with the catalytic residues
(E166 and E354) and selected mutant residues at the gate and inside the active side tunnel. The
residues are depicted as sticks.
21

Figure 5. Percentage specific activity of the O08324 mutants in the presence of the different
concentrations of glucose (0-3 M) on the substrate pNPGlc under optimum assay conditions for each
mutant as reported in Table 2. The specific activity of each mutant in the absence of exogenously
added glucose was considered 100 %, and relative to it, activities with glucose determined. The
glucose- induced changes in percent specific activity of each mutant with respect to their zero-glucose
22

activity is ploted as follows. a) mutants A160E (red filled circle, W167H (blue filled triangle),
W167K (green filled inverted triangle) which show inhibition in the presence of the glucose,
compared to Wild-type (black filled square) b) mutants (F110W (red filled circle), P208T (green filled
inverted triangle), F230A (purple filled rotated square) which show glucose stimulation compared to
Wild-type (black filled square). All measurements were done at least in triplicate, and error bars
indicate standard deviation.
23

Figure 1

Highlights:
•
•
•
•
Accumulated glucose during saccharification causes β-glucosidase product inhibition
O08324 from Thermococcus sp. shows no loss in specific activity up to 4M glucose
Hydrophobicity and steric interactions at tunnel entrance regulate glucose tolerance
All gatekeeper residues at tunnel entrance do not contribute to glucose tolerance

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: