2.1 Compactional screening the mutant candidates with better
catalysis efficiency and thermostability
α-L-rhamnosidases, a glycoside hydrolase, exhibit a low sequence
identity at only 20%–30%, but share a similar (α/α)6-barrel catalysis
domain and several β-sandwiches.36-40 Six crystal
structures (PDB: 2OKX, 3W5M, 3CIH, 4XHC, 6GSZ and 6I60) from the GH78
family have been determined so far. By homology modelling with the
crystal structures of α-L-rhamnosidases 2OKX, 3W5M, and 4XHC as
templates, the structure of r-Rha1 was built and used for our research.
In order to find out how the non-active-site residues of
α-L-rhamnosidase contribute to the catalytic efficiency, bioinformatics
analysis was performed. Specifically, docking between r-Rha1 and its
substrate p NPR were performed and the interaction between them
could offer a guide for site-directed mutagenesis. For example, Lu et
al.45 have reported that some amino acids identified
from protein docking play key roles in catalysis. Bernardi has reported
that hydrogen bonds involved amino acids around the catalytic domain
contribute most to the contact between enzyme and substrate, which could
be mutated to adjust enzymatic activity.46 In the
binding mode of r-Rha1 and substrate p NPR (Fig.1A),9 residues
(Trp253, Tyr293,
Thr301, Val302,
Ser303, Ala355,
Ser356, Asp525,
Trp640) of r-Rha1 were located around p NPR
(Fig, 1B). Notably, some of these residues could be significant for
enzyme activity and should not be mutated. According to structure of
bound L-rhamnose from Fujimoto37, which was sandwiched
between two aromatic residues, Trp640 and
Trp747, the corresponding residues in r-Rha1,
Trp253 and Trp640, are such
essential residues and should not be modified in site-directed
mutagenesis. An amino acid sequence alignment of the available
α-L-rhamnosidases from different sources revealed that, r-Rha1 shared a
low similarity with other α-L-rhamnosidases (Figure S1), but the
residues in the catalytic domains are well conserved (Table 1). The
model of r-Rha1 exhibited that the side chains of
Tyr293 and Trp253 were located near
the substrate binding loop which were important in the binding of the
substrate. In the predicted catalytic site, the aromatic rings of
Tyr293 and Trp253 were parallel to
the ring of rhamnose and presumably play roles in fixing the substrate
through the pi-pi stacking interaction, which is consistent with what Xu
et al 47 have showed, i.e. hydrophobic residues were
located around the catalytic pocket. As a consequence,
Trp253, Tyr293 and
Trp640 should be excluded for mutagenesis. Thus, the
rest residues identified from the molecule docking,
Thr301, Val302,
Ser303, Ala355,
Ser356, Asp525, were selected for
mutation to test the effect on its catalytic activity. Particularly, 14
mutations, T301S, T301Q, T301G, V302S, V302A, V302N, S303V, S303G,
A355N, A355G, S356I, S356Y, D525N, D525G were designed, according
results of sequence alignment (Table 1).
To filter out mutants with high thermostability, the thermostability of
14 mutants were evaluated by mutation energy (stable) module of
Discovery studio 2019 at 60, 65, 70, 75, 80 ºC, and the calculation
results were shown in Fig 1C. As a result, seven mutants were found to
be stable at 60, 65, 70, 75, 80ºC. They are D525N, S356Y, D525G, S356I,
A355N, S303V, V302N, with the stability from high to low in sequence.
While the structure of mutant T301Q, T302S, T301S, V302A, S303G, A355G,
T301G were unstable at different temperatures, it means the seven
mutants have lower thermostability (see Table S2 for detailed data).
Therefore, we obtained seven mutants D525N, S356Y, D525G, S356I, A355N,
S303V, V302N through two rounds of screening, which may have both high
enzyme activity and good thermostability, and could be verified
experimentally.