The xylanase activity of XynA
To
explore the catalytic properties of XynA, we first investigated the
optimal enzyme concentration and the xylanase activity of XynA. Using
0.5% (w/v) beechwood xylan (BWX) as substrate in 100 mM sodium citrate
buffer, pH6.5 at 65°C for10 minutes, as the concentration of XynA
protein increased, the percentage of degraded sugar increased until it
reached a plateau (Figure 4A) . Based on this reaction curve,
500 nM is regarded as the optimal concentration for XynA.
The
activity of GH10 family enzymes requires two conserved catalytic
residues glutamate, and the mutation of glutamate will completely
inactivate the protein (Derewenda et al., 1994). To verify the effect of
these two glutamic acids on XynA activity, we double mutated Glu182 and
Glu280 to alanine and produced the mutant XynA E182A/E280A protein.
The
xylanase activity of the mutants (XynA E182A/E280A) was determined using
the DNS method. The results displayed that WT had the highest activity
at a concentration of 500 nM, while the E182A/E280A mutant is inactive,
even at an extremely high concentration (1 mM) (Figure 4B) .
To
analyze the binding mode and interaction patterns of XynA and its
substrates, we have tried hard to prepare the crystals of XynA WT,
E182A/E280A or E182Q/E280Q with cellobiose, cellohexaose, or xylohexaose
complexes, respectively. However, all trials failed, only the apo
crystals of WT and mutant XynA were obtained. This may be explained by
the weak and difficult detection features for the GH10 family proteins
and their substrates according to previous studies (Baumann et al.,
2011).
However,
through Surface Plasmon Resonance (SPR) assay, we detected the binding
affinity of BWX to both the WT and E182A/E280A mutant XynA(Figure 4C) .
The
binding constants indicated that the binding ability of the WT to BWX
was stronger than that of the E182A/E280A mutant. We found that WT does
not bind to oligosaccharides by SPR assay, which is consistent with the
fact that it is difficult for oligosaccharides to enter the crystal
structure (Figure 4C) .
The protein thermal shift assay indicates that XynA is much
thermostable, as evidenced by the Tm value 73.53 °C. Moreover, the Tm
value 75.5 °C of XynA bound with BWX is higher than that of the apo
protein, indicating that substrate binding could stabilize XynA(Figure 4D) . Similarly, the Tm value of the mutant before and
after the BWX binding also increased, indicating that the substrate
binding also affects the stability of the mutant.
Crystal
structure can provide direct hints to study the protein features and
properties. To elucidate how E182/E280 affect the enzyme activity of
XynA, we determined the crystal structure of XynA E182A/E280A at 2.9 Å.
The overall architecture of XynA E182A/E280A is highly similar to the WT
XynA, yielding a Cα RMSD of 0.218 Å. A major difference observed between
the
WT
and mutant is an angle swing in the N- and the C-terminus.
We
found that the previously established hydrogen bonds between Glu182 and
Trp137, Asn181 and Gln250 as well as Glu280 and with Asn217 and His252
disappeared in the
E182A/E280A
mutant (Figure 4E, 4F) .
Molecular
docking of XynA with xylohexaose and cellohexaose and the mutant
activity confirmation
Since
the cocrystal structure of XynA complexed with substrates is
unsuccessful, to investigate the catalytic mechanism of XynA, we
performed molecular docking studies. The sugar chains of xylan and
cellulose are too long to suitable for molecular docking, therefore,
xylohexaose and cellohexaose are substrates for docking. Viewing the
putative binding mode, these two substrates situated in the center of
the mouth of the XynA ”mug” structure (Figure 5A, 5B) .
Xylohexaose formed ten hydrogen bonds with surrounding residues in the
active pocket (mug mouth), including Asp141, Asn143, Glu96, Lys100,
Trp314, Asn220 and Ser223. Cellohexaose formed eleven hydrogen bond
interactions with surrounding residues, such as Asp74, Glu96, Glu98,
Try102, Glu182, Gln250, His252 and Asn220.
Among
the residues in putative substrate binding pocket, only five residues
are varied between bifunctional enzymes XynA and CbXyn10C, namely 140,
216, 223, 257 and 288, indicating conservation in sequence and structure
(Figure 5F ). The centric Glu182 and Glu280 are surrounded by
Glu96, Asn97, Lys100, Tyr216 and Asn217, forming a pocket embracing the
sugar chains of substrate. Based on molecular docking results, residues
Gln250 and His252 are speculated with relation to the bifunctionality of
XynA.
According
to the results, Asp74, Glu96, Gln250, His252 and Asn220 formed hydrogen
bond with cellohexaose.
Based
on the classification of residues, we designed mutants D74A, E96A,
Q250A, Q250E, H252A and N220E. In light of the residues of CbXyn10C
interacting with the substrate, Val216 and Ala254 are potential residues
that could interact with the substrate in the XynA
sequence
(Chu et al., 2017).
We
designed mutants N97E, V216E and A254E.
We used WAX, BWX and barley
β-glucan as substrates, and the activities of these mutants relative to
that of the XynA (abbreviated as WT in Figure 5 ) was determined
by the 3,5-dinitrosalicylic acid (DNS) method (Figure 5C, 5D,
5E) . We found that the four mutants
(E182A/E280A, E182Q/E280Q, Q250A, and H252A) were inactive towards three
substrates. The mutants E96A and N97E lost their activity towards BWX
and were unable to degrade WAX and
barley β-glucan by more than 50%.
The mutants V216E, N220E, and A254E
showed significantly reduced degradation activity on WAX, BWX, and
barley β-glucan. The mutant Q250E
showed reduced degradation activity on WAX and BWX and no activity
against barley β-glucan. The mutant D74A showed significantly weaker
degradation activities against WAX and barley β-glucan.
E182A/E280A
and E182Q/E280Q were completely inactive against these three substrates,
further confirming that Glu182 and Glu280 were the key catalytic residue
residues.
Discussion
The
most important features of XynA are its thermal stability,
bifunctionality and low homology. It has been reported that its
half-lives at 65 °C and 70 °C are 12 hours and 1.5 hours, respectively
(Wang et al., 2019). We can take advantage of the thermal stability of
this protein. We tried to purify the protein by heating it in a water
bath at 65 °C for 1 hour, which may affect the structure of the protein,
so we also used an anion-exchange column QHP for purification. During
the crystallization process, we found that the protein purified by the
QHP column crystallized quickly, and the crystal morphology was regular,
while the protein purified by heating crystallized slowly, and the
crystal morphology was irregular. These two kinds of crystallization
indicate that the high temperature influences the conformation and
stability of the protein, resulting in differences in the
crystallization.
In
addition to the typical (α/β)8 TIM-barrel fold GH10
domain structure, XynA has N-terminal and C-terminal. By homology
analysis of the overall structure of XynA, the homology of the ”mug
handle” formed by the N-terminal and C-terminal parts of XynA is low,
the β-sheet conservatism inside the ”mug” is high, and the outer part of
the α-helix outside the “mug” is not conserved, while the inner part
of the α-helix is very conserved (Figure 5F).It
has been reported that the N-terminus and C-terminus might affect the
thermal stability, activity and substrate specificity of the protein
(Mahanta et al., 2015; Song, Tsang, & Sylvestre, 2015; Wang et al.,
2019; Zheng et al., 2016). By truncating the N-terminal α helix (1-36),
we proved that the N-terminal did not affect the thermal stability of
XynA, and truncating the N-terminal alpha helix enhanced the activity of
XynA (Figure 2B, 2C) . In the structure, we see that the
N-terminal alpha helix (1-36) interacts with the GH10 domain by hydrogen
bonding (Figure 2D) . This hydrogen bond restricts the (α/β)8
TIM-barrel fold and cannot swing better to accommodate the substrate and
promote its hydrolysis. We tried many C-terminal truncations, but none
of them were soluble. This proves that the presence of the C-terminus is
essential for protein expression. Our analysis showed that the inside of
the ”mug” is bound to the substrate and degrades the substrate, so the
residues are more conserved.
We found and verified two key residues Glu182 and Glu280 by structural
analysis of XynA. In the catalytic process, Glu182 and Glu280 are
catalytic nucleophiles/bases and catalytic proton donors, respectively
(Withers et al., 1986). Comparing the properties of WT with those of the
double mutant showed the activities, structures and stabilities were all
different, proving the importance of Glu182 and Glu280. Glu182 and
Glu280 formed hydrogen bonds with surrounding residues could “lock”
the substrate to be catalyzed after entering the active pocket. The
hydrogen bonds formed by the side chains of Glu182 and Glu280 with
surrounding residues account for the majority interaction. Since the
residues 182 and 280 are located in the loop area, the reduced
interaction also makes this part more flexible, thereby reducing the
binding affinity to substrate. This is in consistent with the SPR
result. At the same time, WT has the ability to hydrolyze the substrate,
while the E182A/E280A has no hydrolysis ability, which also shows that
the mutation affects the hydrolysis activity by affecting the binding of
the substrate.
We
attempted to determine the cocrystal structure of the proteins (WT,
E182A/E280A, and E182Q/E280Q) with substrates xylohexaose and
cellohexaose to reveal the specific catalytic mechanism. However,
neither the incubation of the protein with the substrate nor the
immersion of the crystal in the substrate successfully afforded
cocrystals.
From
the SPR results, we also observed that XynA does not bind to xylohexaose
and cellohexaose. The substrate cannot be immobilized in the protein, so
it is difficult to obtain a cocrystal structure.
We
obtained the simulated structures of XynA and xylohexaose and of XynA
and cellohexaose by molecular docking. The glucose unit has one more
-CH2OH group than the xylose unit in its molecular
structure. Therefore, cellohexaose requires more space than xylohexaose
to accommodate the sugar chains.
It
is found that the XynA active pocket can hold xylohexaose and
cellohexaose. This shows that XynA can accommodate sugar chains of
different substituents and thus exhibit a broad substrate scope.
Compared with the residues that
formed hydrogen bonds with xylohexaose and cellohexaose, Asp74, Glu182,
Gln250 and His252 are unique residues that form hydrogen bonds between
XynA and cellohexaose.
According
to the molecular docking, Glu96, Lys100, Gln250 and His252 are
particularly conserved in the residue sequences of the GH10 family and
interact with substrates in CbXyn10C and SlXyn10A.
We
find that four mutants, E182A/E280A, E182Q/E280Q, Q250A, and H252A are
all inactive towards three substrates. We are not surprised that Glu182
and Glu280 are required for activity, as our results show that they are
key catalytic residues.
After
being replaced with alanine, the activities for the three substrates
completely disappeared, which proves that the two residues His252 and
Gln250 also related to catalysis. Our analysis reveals the conservation
of residue His252 and Gln250 in the GH10 family and the uniformity of
its interactions with substrates (Figure 3 ).
The mutant
H252A
and Q250A only reduce the activity of the CbXyn10C protein (Chu et al.,
2017). However, H252A and Q250A of XynA lose its activity towards the
three substrates, proving that His252 and Gln250 are important residues
in XynA. For most of the xylanases in the GH10 family, the catalytic
residues are Glu residue (Chu et al., 2017). But we find that in
addition to Glu182 and Glu280 as catalytic residues, XynA also has
the
two residues His252 and Gln250.
From
the crystal structure, it is found that Glu280 is located in the loop
region (more flexible). The side chain residues of His252 form hydrogen
bonds with Glu280 side chains, and the main chain residues of Gln250
form hydrogen bonds with Glu280 main chain residues. The hydrogen
bonding interaction makes the structure of Glu280 more stable, so that
the side chain is closer to the substrate, thereby playing the role of
electron transfer. The mutation of His252 prevents Glu280 from getting
close to the substrate, making it difficult to carry out electron
transfer and making the protein inactive (Figure 6) . Glu182 is
also located in the loop area, which is easier to swing and difficult to
bind to the substrate. The side chain residues of Gln250 and Glu182 side
chain residues form hydrogen bonds, which make the structure of Glu182
more stable and play the role of electron transfer (Figure 6) .
The mutation of Gln250 prevents Glu182 from getting close to the
substrate, making it difficult to perform electron transfer. Therefore,
we believe that a hydrogen bond network is formed around Gln250 to
mediate the effects of Glu182 and
Glu280.
The
mutants D74A, E96A, N97E, N220E, V216E and A254E all have reduced
activity to various extents, proving that these sites all contribute to
the activity of the substrate.
We
indicate that the residues that interact with Glu182 including Trp137,
Asn181, Asn217 and Gln250 and the residues that interact with Glu280
including
His133, Asn217, Gln250 and His252 are very conserved in CbXyn10C, and
these residues interact with the substrate, fully reflecting the unity
of the GH10 family.
Finally,
we propose the catalytic mechanism of XynA.
In
the first glycosylation step, E280 acts as a nucleophile (since Glu280
has less interaction with surrounding residues than Glu182, Glu280 is
more flexible to generate electron transfer first), attacking the
anomeric center to displace glycosides and form glycosylase
intermediates. At the same time, Glu182 acts as an acid catalyst and
protonates the glycoside oxygen when the bond is broken. In the second
deglycosylation step, the glycosylase is hydrolyzed by water, and Glu182
acts as a base catalyst, deprotonating the water molecules when it
attacks. As the catalysis progresses, Gln250 and His252 interact with
the catalytic residue to bring the catalytic residue close to the
substrate. At the same time, Gln250 and His252 interact with the
substrate to affect the catalysis (Figure 6) .
XynA is a heat-stable and acid-stable xylanase in the GH10 family, and
Celluclast 1.5L can synergistically hydrolyze pretreated corn stover.
These properties indicate that it may be effective in food, animal feed
and biofuel production. Analysis of the XynA crystal structure can
provide a basis for its transformation, making it more suitable for
commercial applications such as biofuel production.