In Silico Biology 10, 0010 (2010); ©2009, Bioinformation Systems e.V.  

A combination of evolutionary trace method, molecular surface accessibility and hydrophobicity analysis to design a high hydrophobicity laccase

Saharuddin Bin Mohamad1*, Ai Ling Ong2, Raja Farhana Khairuddin3 and Adiratna Mat Ripen4

1Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia
2Multimedia University, Jalan Ayer Keroh Lama, 75450 Melaka, Malaysia
3Department of Biology, Faculty of Science and Technology, Universiti Pendidikan Sultan Idris, 35900 Tanjong Malim, Perak
4Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia

* Corresponding author


Edited by E. Wingender; received June 13, 2009; revised October 18, 2009; accepted November 02, 2009; published March 14, 2010


Laccases are industrially attractive enzymes and their applications have expanded to the field of bioremediation. The challenge of today's biotechnology in enzymatic studies is to design enzymes that not only have a higher activity but are also more stable and could fit well with the condition requirements. Laccases are known to oxidize non-natural substrates like polycyclic aromatic hydrocarbons (PAHs). We suppose by increasing the hydrophobicity of laccase, it would increase the chance of the enzyme to meet the hydrophobic substrates in a contamination site, therefore increasing the bioremediation efficacy of PAHs from environment. In this attempt, the applications of evolutionary trace (ET), molecular surface accessibility and hydrophobicity analysis on laccase sequences and laccase's crystal structure (1KYA) are described for optimal design of an enzyme with higher hydrophobicity. Our analysis revealed that Q23A, Q45I, N141A, Q237V, N262L, N301V, N331A, Q360L and Q482A could be promising exchanges to be tested in mutagenesis experiments.

Keywords: laccase, evolutionary trace (ET) analysis, enzymatic bioremediation, hydrophobicity, polycyclic aromatic hydrocarbons (PAHs)


First described by Yoshida in 1883, l>accases (E.C. are now known as members of the ubiquitous blue multi-copper oxidase family. They are widely distributed ranging from fungi, plant to bacteria [Claus, 2004]. Their role in synthesis and/or degradation of the biopolymer lignin is well known. However, their physiological function is still under intensive investigation. Due to their ability to catalyze the oxidation of organic substances such as phenols, aromatic amines and non-phenolic compounds [Riva, 2006], laccases have received much attention in industrial application, especially in biopulping [Wong et al., 2000] and biobleaching [Sigoillot et al., 2004]. Their catalytic properties also evoked particular interest in enzymatic treatment for the removal of toxic xenobiotic from environment such as in bioremediation of wastewater and soil [Novotný et al., 2000].

Polycyclic aromatics hydrocarbons (PAHs) are organic compounds composed of fused aromatic rings. They may contain 4 to 7-member rings, but the most common PAHs are those with 5 or 6-member rings. They are environment contaminant produced as byproducts of organic matter combustion. In a contamination site such as soot, PAHs are usually found as a mixture of more than 2 types of these compounds. Many of them have mutagenic and carcinogenic properties [Groopman and Kensler, 1993]. Due to their hydrophobic nature, they possess low aqueous solubility and preferentially associate with the carbon phases of particles [Sartoros et al., 2005]. They accumulate in soils and sediments and finally contaminate the food chain. The high solid-water distribution ratios are among the physical properties that increase the resistance of PAHs against biodegradation [Zhang et al., 2006].

Laccases from fungi have been reported with the availability to catalyze the oxidation of PAHs [Ryan et al., 2007]. However, improvement of the catalytic properties is a current challenge for an efficient bioremediation of the PAHs. Owing to the complexity of PAHs, we need to design enzymes that could fit as much as possible the bioavailability requirements in order to successfully remediate PAHs from the environment. It is known that the solubility and bioavailability of PAHs decrease with the increase of the PAHs molecular mass. By increasing the hydrophobicity of laccase, it would increase the chance of the enzyme to meet the hydrophobic PAHs molecules. This would enhance the bioremediation efficiency of laccase against the PAHs. However, we need to identify the residues that could be manipulated without affecting the protein folding that could devastate the enzymatic function. Evolutionary trace (ET) analysis at the active site of laccase has been reported in the design of a higher substrate spectrum laccase [Mohamad et al., 2008]. In this report, we analyzed the relationship between ET conservation, molecular surface accessibility and hydrophobicity pattern of laccase in order to identify the suitable residue(s) for site-directed mutagenesis experiments to design laccase with higher hydrophobicity. The results would provide details on potential amino acid residues that could be manipulated without affecting its structure and function.

Materials and methods


Laccase from Trametes versicolor with Swiss-Prot accession number Q96UT7 was used as seed sequence to search for homologous sequences from the Swiss-Prot database [Altschul et al., 1990]. Thirty-one protein sequences with more than 50% identity were selected by BLASTP [Bairoch et al., 2004] and aligned together with ClustalW [Thompson et al., 1994] using the Gonnet protein weight matrix [Gonnet et al., 1992]. A rooted phylogenetic tree was constructed based on the neighbor-joining algorithm from the multiple sequence alignment and visualized by PhyloDraw [Choi et al., 2000].

Evolutionary trace (ET) analysis

We generated a single partition identity cutoff (PIC) which divided the phylogram into 5 groups. Sequences within different groups were separately aligned, and the resultant aligned groups were compared to derive their consensus residues. Consensus residues from the multiple sequence alignment were classified as neutral, conserved and group-specific. Neutral residues are amino acids that are not conserved whereas conserved residues are conserved in the multiple sequence alignment. Group-specific residues are amino acid residues that are conserved within the group, but they differ from one group to another. The trace residues were then mapped onto the known 3D structures of laccase (1KYA) [Bertrand et al., 2002] obtained from Protein Data Bank (PDB) [Berman et al., 2000] and the mapped structure was visualized by Rasmol [Sayle et al., 1995].

Molecular surface accessibility

The molecular surface accessibility of 1KYA was analyzed by WHATIF web server ( [Vriend, 1990]. The accessible residues were mapped onto 1KYA structure and visualized by Rasmol.

Hydrophobicity analysis

We analyzed the hydrophobicity of 1KYA amino acid residues using 3 different hydrophathy definition developed by Kyte and Doolittle [Kyte and Doolittle, 1982], Rose et al. [Rose et al., 1985] and Lins et al. [Lins et al., 2003]. Residues are classified as hydrophilic and hydropobic in Kyte-Doolittle definition. According to Rose et al., 1985, hydropathy classification, amino acid residues are divided into hydrophilic, hydrophobic and neutral as defined by Biological Magnetic Resonance Data Bank (http:/ [Ulrich et al., 2007]. Lins et al., 2003, defined the residues as hydrophilic, hydrophobic, intermediate and special features. Hydrophobicity status are mapped onto the accessible residues of 1KYA, and visualized by Rasmol.

Results and discussion

A multiple sequence analysis was done to a group of 31 laccases that are more than 50% identical to laccase from Trametes versicolor (Q96UT7) (Supplemental Figure 1). We generated a phylogenetic tree based on the neighbor-joining algorithm from the multiple sequence analysis and visualized by PhyloDraw. The multiple sequence alignment and the phylogram were used as input for ET analysis. ET analysis is a method that extracts evolutionarily important information based on both sequence and structural details [Lichtarge et al., 1996]. Classes or groups were generated by dividing the phylogram with evolutionary time cutoff lines. A single ET partition cutoff was drawn to the phylogram that divided the tree into 5 groups of protein sequences (Fig. 1).

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Figure 1: Evolutionary trace based dendrogram of selected laccases. ET partition cutoff divides the phylogenetic tree into 5 groups.

The members of Group 1 have 55 to 59% identity to Q96UT7. Even though members of Group 2 and Group 3 are about 61 to 66% identical to Q96UT7, however they are not in the same group as they are separated on the nod earlier from the partition cutoff. All members of Group 5 have 72% identity, while members of Group 4 have more than 75% identity to Q96UT7. Sequences from each group were separately aligned and compared to each other to develop a consensus sequence. The consensus sequence from ET analysis was aligned with the seed sequence (Q96UT7) to identify the position of conserved and group-specific residues on the seed sequence (Fig. 2).

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Figure 2: Sequence alignment of seed laccase protein sequence (Q96UT7) to ET consensus sequence. Underlined residues are the amino acid residues for 1KYA.

We identified 172 ET residues at our cutoff partition with 166 positions of conserved residues and 6 positions are marked as group-specific (Fig. 2 and Tab. 1). Tab. 1 summarized the number of ET residues of the selected laccases in our analysis. ET analysis revealed that our selected laccases are 33.3% evolutionarily conserved. Then we mapped the position of ET residues onto the known 3D structure of laccase (1KYA) (Fig. 3a). We identified that most of the conserved residues are inside the 3D structure (Fig. 3b).

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Figure 3: ET residues mapped onto 1KYA. (a) 1KYA structure rotated on y-axis. (b) Slab function of Rasmol to show that most of the ET residues are in the core of the protein.

Those conserved residues may have an important role in stabilizing the structural stability of laccase. This observation is particularly true for cysteine residues, where all the cysteine residues involved in disulfide bridges are conserved in our analysis (Tab. 1). Consequently, we think that the residues on the surface and not conserved in ET analysis are the possible candidates for our purpose.

Table 1: Summary for amino acid residue composition of 1KYA, ET residues and surface accessible residues.
 Composition in 1KYAET residue    Surface accessible residueET and surface
accessible residues
Ala521019.2 2242.3 0
Arg151066.7 533.3 1
Asn34926.5 2676.5 6
Asp381436.8 2052.6 2
Cys55100 00 0
Gln16637.5 956.3 0
Glu7342.9 342.9 0
Gly402050.0 1947.5 7
His161381.3 425.0 1
Ile29724.1 413.8 0
Leu321340.6 928.1 0
Lys500 5100 0
Met400 125.0 0
Phe301136.7 723.3 2
Pro401537.5 2255.0 2
Ser35617.1 1542.9 1
Thr39615.4 2256.4 0
Trp7571.4 00 0
Tyr151173.3 533.3 1
Val40820.0 40100 0
Total49917233.3***238 23
*The percentage of ET residues of each amino acid residue over the total number of the same amino acid residue in 1KYA.
* *The percentage of surface accessible residues of each amino acid residue over the total number of the same amino acid residue in 1KYA.
***The percentage of total conserved residues over the total amino acid residues in 1KYA.

We ran the structure to WHATIF web server to calculate the molecular accessibility surface of 1KYA. We identified 238 residues that were considered as surface accessible (Tab. 1) and mapped them onto 1KYA and visualized by Rasmol (Fig. 4). Supporting the result of Fig. 1d, only 23 out of 238 surface accessible residues are conserved in ET analysis (Tab. 1).

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Figure 4: Accessible molecular surface residues (cyan) mapped onto 1KYA.

Then, we identified the hydrophobicity of the surface accessible residues on the surface of 1KYA. Numerous hydropathy scales have been developed to quantify the relative hydrophobicity of amino acids. the Kyte-Doolittle scale is widely applied for delineating the hydrophobic character of a protein. The scale was derived from the physicochemical properties of amino acid side chains. On the other hand, the Rose scale was constructed by examining proteins with known 3D structures. The hydrophobic character was defined as the tendency for a residue to be found inside of a protein rather than on its surface. The accessible molecular surface residues identified were then applied to hydrophobicity analysis. Hydrophobic distribution of the molecular surface residues according to Kyte-Doolittle (Fig. 5a) and Rose (Fig. 5b) definition was mapped onto 1KYA.

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Figure 5: Hydrophobicity analysis mapped onto 1KYA. (a) Kyte-Doolittle definition of hydrophobicity. Hydrophobic residues are colored blue and hydrophilic residues are colored red. (b) Rose et al. definition of hydrophobicity. Hydrophobic residues (blue), hydrophilic residues (red) and neutral residues (green). (c) Lins et al. definition of hydrophobicity. Hydrophobic residues (blue), hydrophilic residues (red), intermediate residues (green) and special residues (yellow).

It seems that not much difference can be found in the hydrophobic distribution between both hydrophobicity scales. We hardly identified any hydrophobic clustering on the surface of 1KYA. We assume that the hydrophilic distribution in 1KYA is scattered all around the molecule surface to ensure that the protein is soluble in water phase. We suppose those hydrophilic molecules that are not conserved in ET analysis are the possible residues to be manipulated in order to produce a higher hydrophobicity laccase for bioremediation. We tested the hydrophobicity analysis with a different hydropathy definition as reported by Lins et al, 2003. Lins et al. analyzed the relationship between amino acids hydrophobic and hydrophilic accessible surface in extended state and in folded protein. Based on the relationship, they classified 3 groups of amino acids. Ile, Leu, Val, Phe, Met, Ala and Gly are grouped as hydrophobic residues, while Asp, Asn, Glu, Gln and Arg are grouped as hydrophilic residues. The third group that holds an intermediate property was also identified, which consists of His, Tyr, Trp, Ser and Thr. However, Pro and Lys were reported to show a distinct property and were not classified into any group. There were no much changes found in the hydrophobic and hydrophilic distribution of Lins definition (Fig. 5c), compared to the hydrophobicity definition of Kyte-Doolittle and of Rose.

The residue of choice for the design of high hydrophobicity laccase should be the one that was not conserved in ET analysis, located on the surface of the 3D structure and a hydrophilic residue. The hydrophobicity analysis showed that we are able to choose Asp, Glu, Arg, Asn and Gln as candidate for mutagenesis experiments. However, we would consider avoiding residues of Asp, Glu and Arg because modifying them would alter the pI of the protein. We advert Asn residues that may be glycosylated since laccase is known as a highly glycosylated protein. Considering ET, molecular surface accessible, hydrophobicity analysis and the factors mentioned above, we concluded that Asn at the position 141, 262, 301, and 331 and Gln 23, 45, 237, 360 and 482 as possible candidates for the purpose (Tab. 2). All the predicted residues were applied to CUPSAT server ( [Parthiban et al., 2006] to analyze the protein stability changes upon a single point mutation. The residues listed in Tab. 2 are the residues that were predicted to support stabilization of the protein structure at favorable angle torsion. All the predicted site mutations on 1KYA with their correspondence substitute amino acids were analyzed by ERIS, a protein stability prediction server ( [Yin et al., 2007]. The analysis result showed that the mutations at amino acid position (Q23A, Q45I, N141A, Q237V, N262L, N301V, N331A, Q360L and Q482A) are stabilizing mutations with the ΔΔG = −49.18 kcal/mol.

Table 2: Potential residues for mutagenesis experiments.
Residue no.Wild-typeMutation Predicted DDG (kcal/mol)

PAHs are unique contaminants in the environment and their low solubility is among the factors that reduce their bioavailability for remediation, thus promote their accumulation in the environment. While laccase is applicable for PAHs enzymatic bioremediation, the design of laccase to optimize PAH enzymatic bioremediation is a challenge for today's biotechnology. Not all residues in a protein are carrying the same weight for a protein to function properly. Mutation in some residues may cause the protein to lose its function. In some case, residues which are known as specificity-determining residues, mutation will cause the protein to have a modified function. Another important group of residues are the one that responsible for the protein's structure stability. Mutation in such residues may jeopardous the whole protein function. Therefore, identifying which residue to be manipulated is a crucial step in designing a protein for specific purpose.

In conclusion, in this report, we combined ET method, hydrophobicity studies and accessible surface analysis to identify the suitable residues to be manipulated, in order to design laccase with higher hydrophobicity. This analysis provides basic knowledge for designing high hydrophobicity laccase which can be exploited for PAHs enzymatic bioremediation, as well as in industrial usage in future.