stability (from 1.0 to 10.5) by creating a saturated mutation in amino acid residues S3
and D35 in the recombinant enzyme obtained. They also performed structural
analysis of TlXyn11B and its mutants, S3F and D35I, by homology modeling and
MD simulations. As a result, they determined that the overall structural rigidity
(improved thermostability) was increased in S3F and D35I double mutants
(S3F/D35I).
In conclusion, molecular docking analysis (including molecular dynamic simulations) and 3D enzyme/protein modeling tools offer unprecedented advantages that
no pure experimental approach can provide in studies such as enzyme-substrate
affinity or enzymatic yield increment through random/site-directed mutagenesis in
biotechnology. In this context, the superiority of in silico design versus experimental
approaches can be summarized as follows:
1. Possible noncovalent (non-bonded) interactions (hydrogen, electrostatic, hydrophobic, halogen and others) occur during enzymatic catalysis between the ligand
and the receptor cannot be experimentally demonstrated at the atomic level.
2. Neither all enzymes are experimentally isolated nor their crystallographic structures are determined. In addition, it is almost impossible to produce an enzyme
from the scratch which is functional in the cell. Therefore, the catalytic properties
or affinity of each enzyme to its substrate may not be studied experimentally.
3. Due to the probabilistic nature of mutagenesis, a lot of experiments may be
required to increase enzymatic yield through amino acid substitutions.
Therefore, when molecular docking, molecular dynamics, and 3D molecular
modeling methods are used in conjunction with special visualization software,
they essentially provide an invaluable atomistic perspective in protein-ligand interactions, and molecular dynamic simulations add the time scale into these interactions, providing us with valuable information about the stability of protein-ligand
complexes.
However, as these three different approaches mentioned above have reached a
very professional level today, and although the software produced or web servers
designed for this purpose are relatively easy to use, computer-assisted protein-ligand
modeling and the interpretation of the interactions between protein-ligand complexes still require a solid background.
Finally, with a small-scale molecular docking analysis that we performed for this
review, we have demonstrated how useful molecular docking applications are in
atomic-level demonstration of enzyme-substrate interactions in biofuel production.
In these examples, the best poses from docking analyses of xylanase (10B from
Thermotoga petrophila RKU-1, PDB ID: 3NJ3) enzyme and its substrate xylobiose
are demonstrated (Santos et al. 2010). The examples clearly explain the interaction
mode and binding energy of both wild-type and mutated xylanase enzyme with its
corresponding substrate, xylobiose. 87.5% of the amino acids of xylanase in the
substrate binding region consist of polar amino acids. Therefore, for trial purposes,
all amino acids in this region have been replaced by hydrophobic alanine (except
tryptophan) to show how amino acid substitution events in the binding site will
affect the binding affinity of the ligand and to show how useful molecular docking
analysis is in site-directed mutations.
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T. Karaytuğ et al.
and D35 in the recombinant enzyme obtained. They also performed structural
analysis of TlXyn11B and its mutants, S3F and D35I, by homology modeling and
MD simulations. As a result, they determined that the overall structural rigidity
(improved thermostability) was increased in S3F and D35I double mutants
(S3F/D35I).
In conclusion, molecular docking analysis (including molecular dynamic simulations) and 3D enzyme/protein modeling tools offer unprecedented advantages that
no pure experimental approach can provide in studies such as enzyme-substrate
affinity or enzymatic yield increment through random/site-directed mutagenesis in
biotechnology. In this context, the superiority of in silico design versus experimental
approaches can be summarized as follows:
1. Possible noncovalent (non-bonded) interactions (hydrogen, electrostatic, hydrophobic, halogen and others) occur during enzymatic catalysis between the ligand
and the receptor cannot be experimentally demonstrated at the atomic level.
2. Neither all enzymes are experimentally isolated nor their crystallographic structures are determined. In addition, it is almost impossible to produce an enzyme
from the scratch which is functional in the cell. Therefore, the catalytic properties
or affinity of each enzyme to its substrate may not be studied experimentally.
3. Due to the probabilistic nature of mutagenesis, a lot of experiments may be
required to increase enzymatic yield through amino acid substitutions.
Therefore, when molecular docking, molecular dynamics, and 3D molecular
modeling methods are used in conjunction with special visualization software,
they essentially provide an invaluable atomistic perspective in protein-ligand interactions, and molecular dynamic simulations add the time scale into these interactions, providing us with valuable information about the stability of protein-ligand
complexes.
However, as these three different approaches mentioned above have reached a
very professional level today, and although the software produced or web servers
designed for this purpose are relatively easy to use, computer-assisted protein-ligand
modeling and the interpretation of the interactions between protein-ligand complexes still require a solid background.
Finally, with a small-scale molecular docking analysis that we performed for this
review, we have demonstrated how useful molecular docking applications are in
atomic-level demonstration of enzyme-substrate interactions in biofuel production.
In these examples, the best poses from docking analyses of xylanase (10B from
Thermotoga petrophila RKU-1, PDB ID: 3NJ3) enzyme and its substrate xylobiose
are demonstrated (Santos et al. 2010). The examples clearly explain the interaction
mode and binding energy of both wild-type and mutated xylanase enzyme with its
corresponding substrate, xylobiose. 87.5% of the amino acids of xylanase in the
substrate binding region consist of polar amino acids. Therefore, for trial purposes,
all amino acids in this region have been replaced by hydrophobic alanine (except
tryptophan) to show how amino acid substitution events in the binding site will
affect the binding affinity of the ligand and to show how useful molecular docking
analysis is in site-directed mutations.
288
T. Karaytuğ et al.