and –OH groups are towed inward by the negative charge of this deprotonated carboxylate group and directed to the histidine ligand (H458).

3.2. Steric Hindrance Due to Bulky Structures

Steric hindrance can also be an activity-determining factor, as bulky substrates may find it difficult to bind at the substrate binding pocket lined by residues with large side chains ^[34][35][58,59]. Tadesse and coworkers investigated the comparative participation of the oxidation–reduction and steric features of presumed substrates in finding their propensity for oxidation in Trametes villosa (E⁰ = +790 mV) and Myceliophthora thermophila (E⁰ = +470 mV). Even though ∆G⁰ between the substrate and T1 Cu site is a rate-determining reaction step, the results of the investigation show that some of the substituted anilines and phenols did not get oxidized by the enzyme even if they had suitable redox potential. A description was made relating the substrate consumption to the maximum dimension of substituted phenols. Hydrophobic residues (F162, L164, and F265) delimit one of the binding pocket’s sides, while other residues (F332, F337, and P391) are part of the opposite wall. Occupied by the two phenylalanine residues (F332 and F265), which are separated by a distance of 10.8 Å forces involved in interactions between substrate and active site residues of the laccase (excluding the hydrogen atom’s van der Waals radii) form the entrance path of the substrate. Another very good example supporting steric hindrance as an activity-determining factor was demonstrated by Galli and co-workers. A site-directed mutagenesis strategy was used in TvL to examine how the substitution of the hydrophobic and bulky phenylalanine residues located at the prime positions at the T1 Cu site with small apolar residues that are less hindered, such as alanine, could probably expedite the access of large substrates while simultaneously preserving the non-polar feature of the active site. Oxidation of thymol by wild-type (WT), F162A, and F332A mutants was found to be similar, whereas F265A was comparatively less efficient. The extent of oxidation of thymol by WT and mutants (F162A and F332A) was anticipated to be similar due to the smaller size of the substrate. It has a width of 7.7 Å, which is smaller than the distance of 10.8 Å between the two phenylalanines, F332 and F265, in TvL, which define the entry to the enzyme’s active site. Hence, the alterations done to enlarge the width of the binding pocket entrance should not affect the activity of the mutants (F162A and F332A). While, for the oxidation of 3,5-di-t-bu-phenol, the efficiency of F332A appeared similar to that of WT, F162A exhibited a higher conversion rate as compared to WT, and F265A again showed a lower conversion rate. Docking simulations rationalized the results by showing that F332 is far away from the substrate and does not interact with it; therefore, the F332A mutation did not result in any change in the reactivity. In another study of conformations of the substrate at the active site by docking, it was shown that the bulky planar ring structure of the residues in the catalytic site of laccase creates steric hindrance due to which the substrate finds it difficult to enter the catalytic site of the enzyme ^[34][58]. However, the conformations of mediators at the catalytic site revealed that mediators can easily enter the catalytic site and interact with residues without any steric repulsion. The relatively small size of the mediator molecules allows them to easily reach the active site residues through the narrow path. The mediator molecules bind in the vicinity of the T1Cu site ^[36][63]. A docking study revealed that, if the distance between amino acids and the mononuclear copper site is less than 25 Å, the percentage of dye decolorization decreases to <20%.

3.3. Orientation of Substrate in Binding Site

The crucial element affecting how quickly the substrate oxidizes is the substrate’s orientation in the laccase’s substrate binding pocket. Various site-directed mutagenesis studies have been conducted, which support the importance of the substrate’s orientation in the binding site. Substitution of V148L has resulted in an increased activity of mutant laccase compared to the parental enzyme. The presence of the aromatic ring of Y208 in the vicinity of the leucine side chain may have changed the loop (204–208) conformation at the substrate binding site containing the conserved D205, which interacts with the binding substrate. Conformational changes may have resulted in a favorable orientation of D205 towards the binding substrate, which has increased the reducing substrate’s oxidation, thus increasing the activity of the mutant ^[37][67]. Mutation of M168G at the putative substrate binding site of a small low-redox-potential laccase from Streptomyces coelicolor resulted in improved k_cat (fourfold increase) and K_m (tenfold lower), which consequently resulted in around a 40-fold improvement in k_cat/K_m over the WT enzyme.

3.4. Hydrophobic Environment of the Enzyme Pocket

The hydrophobic milieu of the substrate-binding pocket also plays a crucial role in determining the activity of the laccase ^[32][52]. The hydrophobic interaction between the xylidine and laccase through the hydrophobic amino acids that delimit the enzyme’s substrate binding pocket is considered the initial stage of the catalytic pathway ^[38][74]. Increased hydrophobic interactions caused by L386W/G417L mutations in Bacillus subtilis CotA-laccase were also reported to be responsible for increased enzyme activity ^[39][75]. Chen et al. also described that hydrophobic forces are essential for the interaction of laccase with lignin/lignin model compounds ^[40][76]. The activity of the double mutant (L386W/G417L) of Bacillus pumilus CotA-laccase for the decolorization of dyes was enhanced due to increased hydrophobic interactions between the redox mediator 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and the catalytic residues ^[41][77]. It was also observed that hydrophobic interactions are crucial in maintaining the favorable orientation of the substrate at the binding site ^[42][43][72,78]. The residues F162, L164, F265, F332, and F337 in TvL offer great hydrophobic binding to the subjected substrate to retain it close to the T1 site. The duration of residence of the substrate’s active pose is exploited, which facilitates electron transfer to T1.

4. Conclusions

Engineering laccases is of great interest because of their broad range of functions, including industrial and environmental applications. Therefore, the number of laccases has been engineered to increase/enhance their activity and selectivity. But engineering the enzymes with rational methods has always been an appealing approach to saving time and effort. The most logical point that is usually considered for the rational engineering of laccase is to increase the redox potential of T1Cu at the active site. However, increasing the redox potential is a difficult task, as knowledge about increasing the redox potential of T1Cu effectively is still in its infancy. However, there are some other determinants at the active site of laccase that control its activity and can be engineered efficiently (Figure 47). The electrostatic environment at the active site of laccase aids in concerted electron/proton transfer during the oxidation of the substrate. Another feature that regulates the activity of laccase is the steric effect, i.e., whether the substrate is able to find its way to the substrate binding pocket or not. Sometimes, the size of the substrate is so bulky that it is not able to reach the substrate binding site and hence affects the activity. This can be addressed by widening the substrate binding pocket of the laccase. Proper orientation of the bound substrate towards the T1Cu is also very important in determining the activity of laccase. If the bound substrate is more buried towards the T1Cu of the laccase, it facilitates electron transfer from the substrate to T1Cu and hence increases the oxidizing ability of the laccase. Last but not least, the hydrophobic environment at the substrate binding site plays an important role in the binding of the substrate with the enzyme, as the affinity of the enzyme for the substrate is an important feature of enzyme catalysis. Therefore, improving binding affinity of enzyme to the substrate will results in an increased activity. Although a number of features of the active site of the laccase that determine its oxidizing ability have been discovered, extensive research is still required to explore to what extent these features are specific to controlling the activity of the laccase. Moreover, these discussed features should be extensively targeted to engineer the laccases rationally.