Evolutionary history of versatile-lipases from Agaricales through reconstruction of ancestral structures

Triacylglycerol lipases (EC 3.1.1.3) and sterol esterases (EC 3.1.1.13) are defined as enzymes that hydrolyze acylglycerols and sterol esters as their natural substrates, respectively [1]. They are able not only to catalyze hydrolysis reactions under aqueous conditions, but also to carry out synthesis reactions, such as esterification and transesterification, in the presence of organic solvents [2]. All of them display the α/β-hydrolase fold, with their catalytic machinery formed by a catalytic triad (serine, histidine and glutamic acid) and the oxyanion hole [3]. These enzymes are present from microbes to plants and animals, and are widely applicable in industrial processes, such as biofuels, oleochemical, food, detergents, cosmetics, pharmaceutical, textile and paper industry, due to their versatility and stability to harsh conditions [4].

Among filamentous fungi and yeasts, some extracellular proteins show wide substrate specificity and combine the properties from lipases and sterol esterases, being active towards acylglycerols and sterol esters [2, 5]. These enzymes belong to the Candida rugosa-like lipase family (abH03.01), and their reclassification as “Versatile lipases” has been recently proposed [6]. Structurally, they present a hydrophobic cavity covered by a mobile, amphipathic α-helix, named “lid” or “flap”, fixed by a disulfide bond. The difference in activity and substrate affinity towards triglycerides and sterol esters among the proteins of this family is explained by small changes in the hydrophobicity of these regions [7, 8]. Most of the fungi producing these versatile lipases are saprophytic ascomycetes, isolated from forest, agricultural, or composting soils [9]. The only lipase from this family reported in basidiomycetes belongs to a white-rot fungus from the order Agaricales, Pleurotus sapidus. This enzyme acts on alcohol esters, cinnamyl esters and xhantophyl esters [10].

Considering the potential of these enzymes in different biotechnological applications there is an increasing interest in their study and evolution. In this sense, the development of massive DNA sequencing and new bioinformatics tools, have allowed the mining of fungal genomes and metagenomes, especially those from public repositories, such as that from the Joint Genome Institute (JGI) from the US Department of Energy (DOE) (http://www.jgi.doe.gov/), which contains more than 563 accessible genomes from different fungi with potential biotechnological interest [8, 11].

Agaricales is the largest clade of mushroom-forming fungi [12]. This order comprises ectomycorrhizal species and wood saprophytes, such as white rot fungi, which are the only organisms capable of degrading lignin efficiently [13] leaving accessible wood carbohydrates, a major pool of organic carbon in the planet [14]. In addition, Agaricales are one of the most ancient fungal orders, with the last common ancestor of the Agaricomycetidae clade probably dating between 125 and 150 million years ago [15].

On the other hand, exploring the evolutionary history and resurrecting intermediate ancestral forms of enzymes can help to explain the mechanistic basis of enzymes function, to disclose new functionalities, and even to evolve artificially these ancestral proteins in laboratory conditions [16, 17]. Evolutionary studies have been used to reconstruct a growing number of ancestral proteins, including hormone receptors [18, 19], visual pigments [20, 21], carbohydrate binding proteins [22], and elongation factors [23, 24]. In contrast, few ancestral enzymes have been reconstructed [25–31].

Since the number of molecular fossils is very scarce, ancestral sequences are calculated using novel computational methods [16]. Phylogenetic approaches are the most frequently applied for this purpose and, at present, the maximum likelihood method is one of the most widely used [32–34]. Different software applications have been developed to conduct this kind of analysis, as FastML [35], ANSESCON [36], or GASP [37], but the most extensively reported in the literature is PAML [38].

In the present work we carried out a bioinformatics screening of public fungal genomes to explore the presence of genes encoding putative “Versatile lipases” from the order Agaricales. We reconstructed the molecular evolution of these enzymes and inferred the sequence of their ancestral intermediate forms. The potential properties of the candidates are discussed on the basis of their three-dimensional (3D) model structure, the presence and hydrophobicity of the lid, and the substrate binding tunnel. To our knowledge this is the first report on the reconstruction of sequences from ancestral lipases.

Predicted proteins from the genomes of 39 Agaricales available at JGI (November 2015; Additional file 1) were screened using the terms “esterase” or “lipase”, obtaining a total of 6772 and 2631 candidates, respectively. Furthermore, Blast analysis of the same genomes using C. rugosa Lip3 and P. sapidus lipase as a query produced 1055 and 1031 matches, respectively. Putative lipases obtained for each genome were subjected to further sequence and phylogenetic analysis; those belonging to the versatile-lipases from the C. rugose-like family were selected for the construction of homology 3D models.

Two hundred and thirty four putative lipases matched the selected criteria (prediction of signal peptide, sequence length and sequence identity higher than 30% to C. rugosa Lip3) (Additional file 1). The software for 3D structural modeling of these proteins automatically selected as templates the structures of C. rugosa Lip1, 2 or 3 (PDBs: 1cle, 1clr, 1gz7, 1llf, 1llp, 1thg, 1trh), or the Ophiostoma piceae lipase/sterol-esterase (PDBs: 4be4, 4be9, 4upd). All models presented the typical α/β-hydrolase fold, and the catalytic triad and oxyanionic hole were well orientated in the space.

The average number of lipases in the different genomes was analyzed. Ectomycorrhizal fungi and plant parasites would present the lowest number of total lipases (4.20 ± 1.05), while a significantly higher quantity (7.18 ± 0.64; P = 0.039) was found in species associated to wood habitats and litter-associated fungi had an intermediate number of these enzymes (4.83 ± 0.38).

Among the putative lipases, 107 presented less than 20 amino acids in the lid region and hence there was not an α-helix forming the flap. These were discarded to continue the study since all the active lipases described from the C. rugosa-like family have a lid (Additional file 1). The 127 remaining sequences from putative lipases were selected for further analysis (Additional file 2). These candidates were subjected to phylogenetic analysis, and ancestral sequences reconstruction was performed in all nodes of the tree (Fig. 1). In most of the cases, the candidates grouped independently of their fungal family or habitat, however, there was a clear clustering according to the number of residues in the lid region. The outgroup presented the lowest number of amino acids (23), while the most ancestral branch was formed by 26 putative proteins with a short lid, ranging from 24 to 38 residues (average length 32 amino acids). In the next branch on the evolutionary tree (Fig. 1) there was a very heterogeneous group of enzymes with lids ranging from 25 to 52 residues (average 39 amino acids). In addition, the most evolved groups were mainly formed by lipases with a lid of 37 amino acids (ranging from 36 to 38 amino acids).

Ancestral nodes 128 and 129, and those from 155 to 162 were selected as key intermediates in the evolution as origin of new groups (Fig. 1). The sequences of these ancestors were carefully studied, especially the lid region that presented strong polymorphisms. The most ancestral nodes presented the shorter lids when deleting residues with probability levels below 65, 70, 75, 80, 85 or 90%. Additional file 3 shows the sequences of nodes with residues in the lid region with a probability higher than 85%. The lid of the most ancient ancestor (128) was short, only 12 amino acids, however, the lid was longer in the following ancestors (129 and 155–162), containing sequences of 21, 26, 33, 35, 35, 36, 36, 35, and 35 residues, respectively. Moreover, when the number of hydrophobic amino acids in the lid region was analyzed, an increased percentage of these residues during evolution from ancestor 128 to 162 was observed: 41.6, 52.4, 50.0, 57.6, 57.1, 60.0, 61.1, 63.8, 62.8, and 62.8%, respectively.

The typical conserved motifs of the C. rugosa-like lipase family corresponding to the oxyanionic hole and the catalytic serine (GGGF–GESAG), were both present in all ancestral nodes, except for node 128 that contains the sequences GGGL–GQSAG.

The sequences from the ten ancestral nodes selected were subjected to 3D homology modeling (Fig. 2). All of them used Lip2 from C. rugosa as template (PDB 1gz7) except node 128 that used O. piceae lipase/sterol-esterase (PDB 4be4), with a Qmean value ranging from −6.20 to −9.86. All models had the typical α/β-hydrolase fold and the catalytic triad and oxyanionic hole correctly orientated in the space (Fig. 2). However, there were differences at the lid region; the older ancestors presented a loop instead of an α-helix, while more modern ancestors displayed the typical flap (Fig. 2).

Finally, the prediction of intramolecular tunnels that could be responsible for the different catalytic properties reported in the C. rugosa-like proteins [8] was studied in detail in the 10 selected ancestors. The models depicted in Fig. 3 show that tunnels with a reduced size are expected in the more ancient enzymes, while longer tunnels were predicted for more modern proteins. Likewise, Table 1 shows that the length of the intramolecular tunnel, measured from the catalytic serine to the end of the tunnel, increases proportionally to the evolution time.

Node	Lenght (Å)	Max. radius (Å)	Min. radius (Å)
128	17,8	2,88	1,55
129	18,5	3,2	2
155	17,8	2,7	1,85
156	21	2,4	1,75
157	21,5	3,2	2,1
158	23,5	2,7	1,95
159	19	3	2,1
160	32,5	2,8	1
161	37	2,8	1
162	29,8	2,65	1,5

The outcomes from the present work reveal the presence of a great number of putative proteins belonging to the C. rugosa-like family in Agaricales, an extensive group of fungi where only one lipase with activity on alcohol esters, cinnamyl esters and xanthophyll esters has been characterized [10]. The use of the well know versatile lipase from the ascomycete C. rugosa, with wide activity on triglycerides and sterol esters, or the lipase from the basidiomycete P. sapidus in the Blast analysis of the genomes, rendered a similar number of matches despite their low sequence identity (41%). After screening the candidates, the number of total lipases in each individual genome seems to be correlated to the habitat of the fungus, rather than with the family to which they belong. Wood decay fungi presented the highest number of putative versatile lipases. These microorganisms are essential to initiate lignocellulose degradation and complete the carbon cycle [41]. In particular, primary colonizers may need a battery of lipases with different affinities, as these substrates have different composition in lipids and extractives [42]. The presence of versatile lipases in litter-degrading fungi was lower, probably because soil litter has previously been colonized and partially degraded by other microorganisms [43].

On the other hand, ectomycorrhizal fungi and plant pathogens may have specialized during evolution for colonization of a specific host, with a defined cuticle composition, thus needing a smaller set of genes encoding lipases. This has already been observed in other groups of enzymes such as plant cell wall-degrading hydrolases [14].

Variations in the number of lipases in fungi with the same life-style may be related to their adaptations to more than one specific habitat. This is the case of Hebeloma cylindrosporum that is both a wood colonizer and a mycorrhizal fungus [44]. Furthermore, these intrinsic variations may also be due to different origins of evolution, i.e. ectomycorrhizal Agaricales may have had up to 11 events of specialization [45].

On the other hand, although all the known active lipases from the C. rugosa-like family have an α-helix in the lid region, the abundance of sequences (107) in the genomes screened with the absence of this structure, and its potential relationship with fungal habitat, may indicate some specific biological function for these putative proteins. Further studies on these enzymes are necessary to determine this hypothesis, which is a plausible one, as other lipase families include enzymes without lid with high activity on typical lipase substrates [4, 5, 46].

Phylogenetic analysis of the selected lipases showed that they clustered according to the length of their lid rather than to the genome or family where they belonged (Fig. 1). The group of lipases presenting a heterogeneous lid length (between 25 and 52 amino acids) could be evolutionary intermediates, a mixture of short- and long-lid enzymes, that further stabilized this sequence to 37 residues, as seen in the following branches of the tree. Furthermore, the longer lids (48–52 amino acids) may correspond to candidates with wrong introns prediction in the automatic annotation pipeline at the JGI.

Reconstruction of ancestral sequences of proteins has been used as a strategy to understand molecular evolution, and to test hypotheses about protein function [16]. However, protocols for ancestral sequence reconstruction require a precise model of evolutionary processes, being the accuracy of the reconstructed ancestral sequences critical for such studies [47]. In this work we reconstructed the internal nodes of the tree, inferring the sequences of the ancestral putative lipases that gave rise to the different evolutionary branches. Ten ancestral sequences were studied attending to their relevance generating new groups. The tridimensional models of the ancestral proteins rendered a typical α/β-hydrolase fold with the catalytic triad and the oxyanionic hole correctly orientated in the space, indicating the possible functionality of these enzymes (Fig. 2). The oldest protein, in node 128, is the origin of the sequence selected as outgroup, in accordance with its shorter flap (12 amino acids). In this sense, it is worthy to mention that the 107 lipases with a lid shorter than 20 amino acids grouped as a new branch from node 128 when included in the phylogenetic analysis (data not shown). The next ancestors (129, 155 and 156) had longer lids (21, 26 and 33 amino acids, respectively). Node 129 is the precursor of the group of lipases with short lids (average 32 amino acids) while nodes155 and 156 are the origin of the rest of the groups in the tree (Fig. 1). The number of residues in the lid of proteins in nodes 157 to 162 stabilizes between 35 and 36, being the origin of all “modern” lipases with 37 residues in the flap region. The gradual expansion in length of this sequence seems to lead to the formation of the α-helix in the lid across evolution from node 128 to 162 (Fig. 2).

In the same way, the hydrophobicity of the lid seems to increase during evolution, ranging from 41% in node 128 to 63% in the last three nodes (160, 161 and 162). These data suggest that hydrophobic insertions were selected and finally stabilized. In this sense, it is known that the lid participates in substrate recognition and that the number of hydrophobic amino acids in this region is related to enzyme-substrate specificity [7, 8]. Bulky substrates interact with the lid while the acyl-chain of the substrate is being processed in the hydrophobic pocket. Therefore, the flap’s length may be crucial for substrate recognition and specificity. This theory is in good agreement with others postulating the promiscuity of ancestral enzymes, i.e. processing several different substrates during the very first steps of their evolution [48, 49].

Comparison of the structures of different lipases from the C. rugosa-like family indicates the importance of both the shape and amino acid composition of the internal tunnel, and their relationship with the kinetic properties of these proteins [3, 7, 8]. The analysis of putative intramolecular tunnels in the ancestral lipases showed that its size increased along evolution (Fig. 3 and Table 1). The most ancestral lipases seem to have a smaller substrate-binding pocket, which is getting bigger in more evolved proteins. Particularly, the length of the predicted tunnels increases from node 128 to node 162 with only two exceptions (nodes 159 and 162). The minimum radius of the tunnels seems to increase from node 128 to 159, but decrease dramatically in the last three nodes. This could be explained by reasoning that in some enzymes of the family, the tunnel extension is long enough to communicate the end of the substrate-binding pocket with the outside of the protein, creating a narrow exit tunnel to release the reaction products [6, 8]. The evolutionary advantage of having a longer tunnel, as those found in some of the current proteins, could be related to a better accommodation of the acyl moiety of the substrate, increasing the specificity of the enzyme as reported for C. rugosa lipase isoenzymes and the lipase/esterase from O. piceae [2, 46].

The search of “Versatile lipases” in public genomes from Agaricales has brought to light that the fungi with different habitats and life styles have different lipases arsenals. Analysis of the evolutionary history of these putative proteins revealed the increased length of the lid region and size of the substrate-binding pocket area across the evolution time, suggesting the specialization of these enzymes for certain substrates and their loss of promiscuity. Despite the consistency of the data showed by ancestral sequences reconstruction, it is often difficult to obtain a highly accurate sequence. Thus, the heterologous expression of some of these candidates would be interesting not only as a way to corroborate their real activities, but also to establish a platform for their artificial evolution in order to create enzymes endowed with novel capabilities.