Abstract
In this research, 63 complete amino acid sequences of laccase enzymes from fungi (40), bacteria (5), insects (8), and plants (10) were used for a phylogenetic analysis. A common ancestor that diverged in the laccases in prokaryote and eukaryote was observed. Additionally, it was determined that there was a difference in the type of amino acids bound to the histidines linked to the copper atoms of the active site, and that a fungal laccase of approximately 279 Ma was an ancestor of the laccases in the basidiomycetes considered in this study. In contrast, Lacc 6 of Pleurotus ostreatus was used as a template to generate mutants. Through modeling, the changes in the secondary and tertiary structures of this enzyme were observed with the substitution of amino acids adjacent to histidines in the conserved region of the active site, via the presence of amino acids in a similar place in the laccases of bacteria.
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In Silico Generation of Laccase Mutants from Lacc 6 of Pleurotus ostreatus and Bacterial Enzymes
Rubén Díaz,a Mercado-Flores Yuridia,b Gerardo Díaz-Godínez,a Leonardo David Herrera-Zúñiga,c Jorge Álvarez-Cervantes,b and Miguel Angel Anducho-Reyes b,*
In this research, 63 complete amino acid sequences of laccase enzymes from fungi (40), bacteria (5), insects (8), and plants (10) were used for a phylogenetic analysis. A common ancestor that diverged in the laccases in prokaryote and eukaryote was observed. Additionally, it was determined that there was a difference in the type of amino acids bound to the histidines linked to the copper atoms of the active site, and that a fungal laccase of approximately 279 Ma was an ancestor of the laccases in the basidiomycetes considered in this study. In contrast, Lacc 6 of Pleurotus ostreatus was used as a template to generate mutants. Through modeling, the changes in the secondary and tertiary structures of this enzyme were observed with the substitution of amino acids adjacent to histidines in the conserved region of the active site, via the presence of amino acids in a similar place in the laccases of bacteria.
Keywords: Laccase; Phylogenetic analysis; Mutants; Pleurotus ostreatus
Contact Information: a: Laboratory of Biotechnology, Research Center for Biological Sciences, Autonomous University of Tlaxcala, Tlaxcala, Mexico; b: Polytechnic University of Pachuca, Zempoala, Hidalgo, Mexico; c: Division of Environmental Engineering Technology of Higher Studies of East Mexico State, Mexico State, Mexico; *Corresponding author: anducho@upp.edu.mx
INTRODUCTION
The laccases (EC 1.10.3.2) belong to the Multi-Copper Oxidase (MCO) family of enzymes that are produced by different organisms and have gained great importance since the twentieth century in different industries such as paper, textile, and cosmetics to name a few. The enzymes have great potential in degradation applications of toxic compounds and xenobiotics, such as polyphenols and aromatic and non-aromatic amines. The laccases are glycoproteins that catalyze the oxidation of phenolic compounds through the transfer of an electron with the final reduction of oxygen to water (Claus 2004). Laccases have a molecular weight range between 25 to 80 kDa (Díaz et al. 2013), the isoelectric point (pI) of these enzymes is between 2.5 to 6.5, the optimal pH values are between 2.0 and 7.0 (Kirk-Kent and Cullen 1998), and the optimal glycosylation level is between 10% and 25% (Mayer and Staples 2002; Morosova et al. 2007). The first study reported on this type of enzyme was from the Japanese lacquer tree Rhus vernicifera (Yoshida 1883), and a few years later, the existence of laccases in fungi was reported (Bertrand 1896; Laborde 1896). This enzyme type has been found and studied in bacteria, fungi, plants, and insects. More than 100 different proteins have been identified (Shuresh-Kumar et al. 2003).
Laccase’s physiological function in plants is in the lignification processes; while in fungi, its function is in the morphogenesis process (formation of spores, pigments of fruiting bodies), pathogenesis, virulence, and the degradation of lignin. Likewise, it has also been reported that in some insects the analyzed enzyme acts in the formation of cuticles in the process of sclerotization (Kramer et al. 2001), and in bacteria it has the function of monomer cross-linking, polymer degradation, and the breakdown of aromatic rings (Sharma et al. 2007). In contrast, bioinformatic studies of these enzymes have been performed, analyzing the alignment between amino acid sequences in different bacterial laccases as reported by Sharma et al. (2007), in which the presence of conserved regions between the amino acid sequences was observed. Suresh-Kumar et al. (2003) reported the presence of conserved regions in laccases from fungi and plants related to the Cu1 or Cu2 in copper sites. Diez-Fernandez (2015) carried out a bioinformatic analysis to determine the similarities between prokaryotic and eukaryotic laccase genes, observing that there is a phylogenetic relationship between different kingdoms. Hoegger et al. (2006) performed a phylogenetic analysis that supports the classification of multi-copper oxidase enzymes, dependent on their physico-chemical characteristics and the substrates that they are able to degrade.
However, there is no information on the variations of the structure of the laccase and specifically of its active site, due to a series of natural mutations that were carried out over millions of years and that allowed it to have its current activity. Therefore, in this study silico laccase mutants were generated by substituting amino acids adjacent to the active site of Lacc 6 for those present in laccases of bacteria that tolerate high temperatures and low humidity. In addition, the changes in the secondary and tertiary structure of the models of mutants were observed.
EXPERIMENTAL
Materials
Selection of complete sequences of known laccase enzymes
The search for complete amino acid sequences of laccase enzymes from bacteria, fungi, insects, and plants was performed using the public databases of NCBI and UniProt.
Methods
Analysis of the sequences
To determine the family of each enzyme through the obtained sequences, the conserved regions of the active sites associated with copper atoms contained in the laccases were searched, using the Hidden Markov Models (HMMs) corresponding to an algorithm of the Pfam program (version 31.0, European Molecular Biology Laboratory and European Bioinformatics Institute (EMBL-EBI), Hinxton, Cambridge, England). This program compares the similarity of the protein domains to define the family or families to which it belongs (Finn et al. 2014, 2016). From this analysis, the regions where the copper atoms are located and the amino acids in the vicinity of the active site were identified.
Once the sequences with conserved regions of each protein were selected, multiple alignments were performed using the MAFFT program (version 7.0, Immunology Frontier Research Center, Osaka, Japan) (Katoh et al. 2005, 2013). This program was based on the fast Fourier transform (FFT), which correlates the physicochemical properties of the amino acids of each protein and improves the alignment; after which a manual editing was performed based on the histidine residues present in the active site of the multicopper oxidases. These alignments were used for phylogenetic analysis.
Phylogenetic analysis
Phylogenetic analysis was performed with the BEAST program (held by the Centre for Computational Evolution and Department of Computer Science, University of Auckland, v1.8.4, Auckland, New Zealand) (Drummond and Rambaut 2007; Drummond et al. 2012), which was built based on the model reported by Whelan and Goldman (2001); the algorithm estimates empirical amino acid replacement matrices from large databases of aligned protein sequence families. A maximum likelihood procedure is used, and the evolutionary relationships within the families are recorded through the use of phylogenies for each family according to the matrix of Whelan and Goldman (WAG-matrix), as well as, a model of gamma heterogeneity sites and invariant sites with a number of Gamma 4 categories. Later, to estimate the divergence times between the sequences, a Lognormal model with a relaxed clock (Uncorrelated) was used (Drummond et al. 2006). Ten million generations were run using a Markov Chain Monte Carlo algorithm (MCMC). For the second phylogenetic analysis, where the approximate age of onset of fungal laccase enzymes was determined, the estimated time of 290 Ma for the taxonomic group of Agaricomycetes was used (Floudas et al. 2012). Ten million generations were executed using the MCMC algorithm.
The consensus of the phylogenetic tree was realized with the program TreeAnnotator (v1.8.4 supported by the Department of Computer Science, University of Auckland, Auckland, New Zealand), using the parameters preset in the program (Bouckaert et al. 2014; Álvarez-Cervantes et al. 2016). Later, the consensus of the phylogenetic tree was visualized with the program FigTree (v1.4.3 8, Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, Scotland), and finally the image of the tree was edited with the program Inkscape Project (Free Software Foundation, v0.91, Boston, MA, USA).
Protein modeling
Based on the results obtained from the phylogenetic analysis, the sequences corresponding to the bacteria laccases were selected as a model. Due to the geographical region where they were isolated, extreme temperature conditions could have led to the evolution of bacteria causing mutations that resulted in stable and functional laccases under these conditions. In contrast, laccase enzymes called Lacc 6 from Pleurotus ostreatus PC15 with access number KDQ26322.1 was proposed as a mold, because this enzyme showed greater expression and stability to the different pH levels of development of the producer organism (Díaz et al. 2013). The modeling of laccases from bacteria and Lacc 6 was performed using the Swiss-Model program accessible in The Swiss Institute of Bioinformatics (University of Basel Klingelbergstrasse, v8.05, Basel, Switzerland) to observe possible differences in the trinuclear center and their structures, with respect to the alpha helices and beta sheets domains (Biasini et al. 2014; Waterhouse et al. 2018). The obtained models were visualized with the program Python Molecular Viewer (PMV) available in The Molecular Graphics Laboratory (The Scripps Research Institute, v1.4.5, La Jolla, CA, USA) (Sanner 1999; Dallakyan 2010).
Generating mutant
The Lacc 6 enzyme was used as a model. The design of mutants was focused on the active site and the histidine channel where the electron transfer was performed. The bacterial laccase sequences were taken, observing the type and physicochemical characteristics of the adjacent amino acids to the channel of histidines that are united to the atoms of copper, to observe hypothetical changes that have occurred during the past millions of years. The bacterial laccase sequences were of bacteria that grow in environments of high temperature and low humidity.
Structural comparison
For the structural comparison, 46 laccases were studied and those enzymes are of the group that can found as First citation of Laccase on PDB, and are from different kingdoms. It was determined that the following items are included: 37 laccases with structures of 3 domains of Cu-oxidase (L-3MCO) (Zhukhlistova et al. 2008) and 9 with structures of 2 domains of Cu-oxidase (L-2MCO) (Komori et al. 2009; Skálová et al. 2009). A structural study of the domains was also carried out with the Pfam database (Finn et al. 2016). The crystallographic structure of the 46 laccases was superimposed on the crystallographic structure of the enzyme mgLAC L-2MCO (Lawton et al. 2009) (PDB ID: 4E9X), due to its conjecture of “missing link” between the families of oxidoreductases from two to three domains (Komori et al. 2009). The above was done through the Secondary Structure Matching algorithm (SSMA) using the Chimera software and Root Mead Squarer Deviation (RMSD) (Goddard et al. 2017). To compare the architecture of domains through evolution, a phylogenetic tree containing 256 non-redundant laccase structures was performed through the server Simple Modular Architecture Research Tool (SMART) and InterProScan (Letunic et al. 2015).
RESULTS AND DISCUSSION
Selection of Laccase Sequences
Based on the results of the BLAST analysis, 63 protein sequences corresponding to laccase enzymes from different organisms were obtained; 40 of basidiomycete fungi, five of bacteria, 10 of plants, and eight of insects, all above sequences showed regions conserved in the amino acids binding to the copper atoms. The name of the organisms and their laccase access number are described below.
For basidiomycetes, one of Phanerochaete chrysosporium (AAO42609.1), six of Trametes versicolor FP-101664 SS1 (EIW62366.1, EIW63935.1, EIW64151.1, EIW60660.1, EIW58134.1, and EIW58136.1), 12 of Pleurotus ostreatus (AAR21094.1, BAA85185.1, CAA84357.1, CAR48257.1, CAC69853.1, KDQ29908.1, KDQ26322.1, KDQ24506.1, KDQ26265.1, KDQ27218.1, KDQ27220.1, and KDQ26299.1), two of Pleurotus eryngii var. Ferulae (AKE48164.1 and ACI62810.1), 2 of Pleurotus eryngii (ADG01835.1 and CAO79914.1), four of Pleurotus salmoneostramineus (BAI66147.1, BAI66142.1, BAI66139.1, and BAI66145.1), one of Pleurotus sp. Florida (CAA80305.1), two of Pleurotus pulmonarius (AAX40733.1 and AAX40732.1), one of Pleurotus sapidus (CAJ00406.1), one of Coprinus comatus (AFD97049.1), three of Lentinus sajor-caju (CAD45379.1, CAD45380.1, and CAD45381.1), one of Stereum hirsutum FP-91666 SS1 (EIM79648.1), two of Heterobasidion irregulare TC 32-1 (XP_009545278.1 and ETW82985.1), one of Hypsizygus marmoreus (KYQ44884.1), and one of Leucoagaricus sp. SymC.cos (KXN81384.1).
For bacteria, two of Halovivax ruber (WP_015301801.1 and WP_015300460.1), one of Haloferax sp. ATCC BAA-644 (WP_008572767.1), one of Haloferax volcanii (WP_013034979.1), and one of Halorubrum saccharovorum (WP_004045604.1).
For plants, three of Malus domestica (XP_008357173.1, XP_008363777.1, and XP_008362821.1) and seven of Solanum tuberosum (XP_006363100.1, XP_006367545.1, XP_015163413.1, XP_015163415.1, XP_015159950.1, XP_006360308.1, and XP_006363101.1). For insects, one of Bactrocera cucurbitae (XP_011179084.1), one of Microplitis demolitor (XP_014297903.1), one of Musca domestica (XP_011290564.1), one of Pimpla hypochondriaca (CAD20461.1), one of Plutella xylostella (XP_011555576.1), one of Polistes canadensis (XP_014599609.1), and two of Zootermopsis nevadensis (KDR15437.1 and KDR11754.1).
Analysis of the Sequences
The Pfam database suggests that the laccase enzymes of all organisms used in this study belong to the large family of multicopper oxidases; in addition, it showed the position of the active site and the domains of each of the sequences. Figure 1 shows the alignment of the sequences in the regions conserved in the histidine channels, which are the amino acids bound to the copper atoms and catalytic site of the enzymes. This analysis also allowed for the observation of changes in the amino acids adjacent to the histidine channels.
Phylogenetic Analysis
The phylogenetic analysis allowed for the construction of a tree with the 63 sequences of laccases. In phylogenetic construction, two well-defined groups, one belonging to prokaryotes (bacteria) and the other to eukaryotic cells (fungi, plants, and insects), were observed. Additionally, in eukaryotes, a grouping corresponding to the phylum was observed; on the one hand the laccases of the fungi, on the other side of the plants and one more of insects. An evolutionary selection was observed in the laccase enzymes of insects and fungi before that of the plants; in contrast, the tree made it possible to observe that there was a common ancestor that gave rise to insect laccases and diverged in the laccases of plants and fungi. It was also possible to observe that the Lacc 6 enzyme of Pleurotus ostreatus appeared approximately 5.57 million years ago (MYA) and has a phylogenetic relationship with the enzymes AKE48164 and ACI62810 of Pleurotus eryngii var. Ferulae and with the isoenzyme KDQ26265 of Pleurotus ostreatus, as shown in Fig. 2. Figure 3 shows the phylogenetic analysis constructed with 40 laccase sequences of basidiomycete fungi, showing the estimated time of appearance of the common ancestor of all fungal laccases used in this study. The time that was used to make the calculation was proposed by Floudas et al. (2012), suggesting that the appearance of agarycomicetes was 290 MYA. It can be observed that there was a common ancestor of laccase in all the fungi and that approximately 278.48 MYA was divided to give place to a laccase of Phanerochaete chrysosporium and to a group of the rest of fungal laccases used in this study. It can be seen that the laccase of Phanerochaete chrysosporium has not changed over time, and the other group showed a separation into two groups 148.87 MYA. The divergence of laccase enzymes over time suggests that the fungi evolved, obliged by the changing conditions of their environment, becoming more efficient for the degradation of lignocellulosic compounds present in their natural substrates (Heckman et al. 2001; Honrubia 2009). Jones and Dangl (2006) suggested a co-evolution between plants and host pathogens that affects their growth and reproduction. Plants respond to infection using a two-branched innate immune system, the first branch recognizes and responds to molecules common to many kinds of microorganisms, even non-pathogens. The second branch responds to the virulence factors of the pathogens, either directly or through their effects on the host targets.
Fig. 1. Alignment of the amino acid sequences of the active site region of laccase enzymes. The three conserved regions are between amino acids 328 to 330, 377 to 379, and 1030 to 1032. The left column shows the laccases access numbers that were mentioned above. The differences in the sequences are indicated with the sign (-). The conserved regions of the histidine channel bound to the copper atoms and active sites are highlighted in blue.
Fig. 2. Phylogenetic tree of the amino acid sequences of laccase enzymes of bacteria (fonts in red), basidiomycetes (fonts in blue), plants (fonts in green), and insects (fonts in orange). The values in the nodes represent the Bayesian posterior probabilities in percentage.
These two scenarios predict different results with respect to the timing and amplitude of the responses that are triggered and also causes a rise in multiple evolutionary pressures in both the host and the pathogen. In agreement with this study, it was suggested that the fungi co-evolved with respect to their carbon source, promoting an adaptation and greater catalytic effectiveness of the enzymes involved in their degradation, including laccases.
Hoegger et al. (2006) performed a phylogenetic comparison of the laccase enzymes of fungi, insects, plants, and bacteria to develop a classification and relation between the sequences of these type of enzymes. The comparison resulted in an observation that these multicobre oxidases appear grouped according to the type of organism and also that some species encode for proteins of the ferroxidase type, while other proteins have different oxidative characteristics. As such, this observation differed from this study, which only worked with proteins classified as multicobre oxidase. Given the importance of these enzymes and the wide range of organisms capable of producing them, it is interesting to know the phylogenetic relationship with a common ancestor that can correlate their pathogenic, saprophyte, or synthesis functions of producing organisms. The phylogeny described in this research showed the clustering of the protein sequences according to the order of each phylum. A common ancestor was observed for prokaryotic and eukaryotic cells. From this ancestral protein, a divergence occurred to give origin to the proteins of bacteria, where it was observed that during millions of years they have not undergone considerable changes in structure, and to a protein from which the laccase enzymes emerge from the other three kingdoms (fungi, plants, and insects). From this second group, it was observed that a protein gave rise to fungi and plant laccases and also to insect enzymes. From these enzymes, it was observed that insect and fungal proteins were formed earlier than those of plants, possibly because they are used in the degradation of substrates in insects and fungi; whereas in plants, they are mainly used for structure synthesis (Hoegger et al. 2006; Floudas et al. 2012).
Floudas et al. (2012) conducted a phylogenetic analysis of the paleozoic origin of ligninolytic enzymes from 31 fungal genomes, including the basidiomycete and ascomycheta families. The study predicted an approximate time of 290 Ma for the agaricomycetes, which was used to determine the approximate time of appearance of the different laccases through the use of the phylogenetic analysis of the fungi sequences. The results observed coincided with those reported by Floudas et al. (2012).
Relationship of the structure of laccase enzymes with respect to phylogeny
The structures of all laccase enzymes were analyze and showed conserved regions related to the active site that did not have changes in the type of amino acid bound to the copper atoms. However, there were changes in the sequence of the rest of the peptide chain, causing differences in the domains of proteins, and thus, in the tertiary structure. Through the Swiss-Model program (Biasini et al. 2014; Waterhouse et al. 2018), it was observed that some enzymes contained a different number of copper atoms in the active site, and in some cases, contained molecules of oxygen, hydroxyl ion, or zinc. In the structures of the laccases of plants, the hydroxyl ion was preferentially found; in those of bacteria and insects, the oxygen molecule existed, and only in some cases of fungal enzymes zinc was present. However, the presence of these molecules could not be correlated evolutionarily with respect to the phylogeny.
The conserved regions of the active site and the amino acids where the copper atoms are attached were observed in all the laccase structures of this study.
For the design of the mutants, the laccase sequence Lacc 6 of Pleurotus ostreatus was used, which has shown a high and efficient enzymatic activity for the degradation of lignocellulosic compounds (Díaz et al. 2013), as well as the sequences of laccases of the bacteria Halorubrum saccharovorum, Haloferax volcanii, Haloferax sp. ATCC BAA-644, and Halovivax ruber(with two sequences) because they grow in environments of high temperature and low humidity.