Redox Potential Features of Laccase

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This entry is adapted from the peer-reviewed paper 10.3390/molecules28176209

Laccase, one of the metalloproteins, belongs to the multicopper oxidase family. It oxidizes a wide range of substrates and generates water as a sole by-product. The engineering of laccase is important to broaden their industrial and environmental applications. The general assumption is that the low redox potential of laccases is the principal obstacle, as evidenced by their low activity towards certain substrates. Therefore, the primary goal of engineering laccases is to improve their oxidation capability, thereby increasing their redox potential. Even though some of the determinants of laccase are known, it is still not entirely clear how to enhance its redox potential. However, the laccase active site has additional characteristics that regulate the enzymes’ activity and specificity. These include the electrostatic and hydrophobic environment of the substrate binding pocket, the steric effect at the substrate binding site, and the orientation of the binding substrate with respect to the T1 site of the laccase.

laccases rational engineering electrostatic environment hydrophobic environment

1. Introduction

Laccases (EC 1.10.3.2, benzenediol:oxygen oxidoreductases) are metalloenzymes that are members of the multicopper oxidase family and are characteristically extracellular monomeric glycoproteins ^[1]^[2]^[3]. Their catalytic function is to oxidize a wide array of phenolic and non-phenolic compounds. This catalytic reaction uses oxygen as an acceptor of electrons, and the only by-product is water ^[4]^[5]^[6]. These enzymes express a broad spectrum of redox potential vs. NHE (normal hydrogen electrode) from +430 to +800 mV, irrespective of their very alike structures ^[1]^[6]^[7]^[8]. The utilization of oxygen as a final acceptor of electrons without involving expensive co-factors, the generation of water as a lone secondary product, and the promiscuity of these enzymes for a broad variety of organic compounds make them an ideal candidate as biocatalysts for diverse technological purposes ^[9]^[10]^[11]. These purposes include applications in the food industry, chemical synthesis, the paper and pulp industry, and bioremediation ^[12]^[13]^[14]^[15]^[16]^[17].

2. An Overview of the Laccase Structure

2.1. Overall Structure of the Laccase

Laccases exhibit a peculiar fold that consists of three cupredoxin domains, and their active site consists of four copper atoms. These atoms are categorized as type 1 copper (T1Cu) in the T1 site (mononuclear copper center) and a cluster of type 2 and type 3 coppers (T2Cu, T3Cu, and T3’Cu) in the T2/T3 site (trinuclear copper center) as shown in the Figure 1. The distinctive absorbance of the T1 copper site has been found somewhere around 610 nm. This site is responsible for the blue color of the enzyme. The T2 copper cannot be identified by spectrophotometry; however, it generates a distinctive EPR signal ^[2]^[18]^[19]. The absorbance peak can be seen at 330 nm, which is due to the diamagnetic nature of the T3 copper site ^[20]. Their optical and electro paramagnetic resonance characteristics serve as the main criteria for this classification ^[2]^[21]^[22]. For some laccases, it has been reported that T1 is the primary site where electrons are accepted from reduced substrates. In addition, the catalytic efficiency (k_cat/K_m) is influenced by the redox potential of the T1 copper site ^[2]^[23]^[24]. Mononuclear and trinuclear copper centers are localized in the third domain and in between the first and third domains, respectively (Figure 1) ^[25]^[26]. The substrate binding pocket is located near the mononuclear copper center and is constituted by the residues of the second and third domains ^[27]^[28].

Figure 1. Crystal structure of fungal laccase representing the mononuclear site (T1Cu) and the trinuclear site (TNC).

2.2. T1 Site

The T1Cu atom is coordinated by the side chains of one cysteine residue and two histidine residues. The Cu atom and the atoms taking part in its coordination fall in the same plane, while on both sides, side chains of the axial hydrophobic residues are present ^[29] (Figure 2).

Figure 2. Detailed representation of T1 site.

2.3. T2/T3 Site

The T2/T3 site consists of three Cu atoms (T2Cu, T3Cu, and T3’Cu) and eight coordinating histidine residues. The T2Cu atom is coordinated by two histidine residues, and it can be additionally linked by one or two oxygen ligands. This results in the development of a square-planar structure. The T3Cu and T3’Cu atoms are coordinated by six histidine residues. These are coordinated with each other through the oxygen as a ligand shown in Figure 3 ^[25]^[30].

Figure 3. Detailed representation of T2/T3 site.

3. Characteristics That Determine Activity Other Than the Redox Potential

3.1. Electrostatic Environment of the Enzyme Pocket

It was first shown by Xu and co-workers that the characteristics of the enzyme pocket determine substrate binding and electron transfer. During the process of substrate oxidation, the presence of an electrostatic environment within the active site of laccase is beneficial to the coordinated transfer of electrons and protons. A significant drop in k_cat and increase in K_m but no change in the redox potential of T1Cu was observed in Rhizoctonia solani (E⁰ = +710 mV) and Myceliophthora thermophila (E⁰ = +470 mV) laccase substrate binding pockets with a triple mutation of a tripeptide (LEA and VSG, respectively). The observed changes in substrate docking were attributed to steric and electrostatic hindrances introduced upon mutation ^[31]. Later on, it was also documented that the substrate forms a hydrogen bond with aspartate (or glutamate) residues placed at the base of the binding pocket, which is completely conserved in fungal laccases ^[32]. In fact, it was observed in a study that the D205R mutation in POXA1b resulted in a significant waning of catalytic features along with a reduction in its stability. It was revealed through molecular dynamics simulations that the structure of mutated POXA1b is perturbed by the R205 mutation in a greatly conserved region. This leads to a large reorganization of the structure, therefore decreasing both enzymes’ activity and stability ^[33]. The carboxylate group (D206) is deprotonated at physiological pH (pKa 3.9). Therefore, substrates carrying –NH₂ 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]. 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]. 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]. 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]. 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]. 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]. Increased hydrophobic interactions caused by L386W/G417L mutations in Bacillus subtilis CotA-laccase were also reported to be responsible for increased enzyme activity ^[39]. Chen et al. also described that hydrophobic forces are essential for the interaction of laccase with lignin/lignin model compounds ^[40]. 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]. It was also observed that hydrophobic interactions are crucial in maintaining the favorable orientation of the substrate at the binding site ^[42]^[43]. 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 4). 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.

Figure 4. Demonstration of activity-determining features of laccase.

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