Amino acid sequence based in silico analysis of β-galactosidases

Amino acid sequences of β-galactosidase enzyme belonging to different families of bacteria, fungi and plants retrieved from GenPept database were analyzed for multiple sequence alignment, cluster analysis, conserved motif discovery and their Pfam analysis using different bioinformatics tools. The multiple sequence alignment revealed different conserved residues of amino acids exclusively for each groups except fungi. The cluster analysis for different groups uniformly showed three major clusters based on the closeness of the β-galactosidase protein sequences irrespective of the source organisms. Seven conserved motifs belonging to different families were assessed. These identified motifs showed the evolutionary closeness among species at the molecular level.


Introduction
β-galactosidases are hydrolase enzymes which are involved in the hydrolysis of β-galactosides into monosaccharides.It is widely distributed enzyme among bacteria, fungi and plants.
Sequencing and analysis of amino acid sequences of β-galactosidases originates many ideas about their structural and functional activity.In bacteria, the 1024 amino acids of E. coli β-galactosidase were first sequenced [1] and its structure determined after twenty-four years [2].The protein is a 464-kDa homotetramer.Each unit of β-galactosidase consists of five domains; domain 1 is a jelly-roll type barrel, domain 2 and 4 are fibronectin type III-like barrels, domain 5 a β-sandwich, while the central domain 3 is a TIM-type barrel.The third domain contains the active site [3].In fungi a genomic copy of the β-galactosidase gene of Hypocrea jecorina was cloned [4], and this copy encodes a 1,023-amino-acid protein with a 20-amino-acid signal sequence.This protein has a molecular mass of 109.3 kDa, belongs to glycosyl hydrolase family 35, and is the major extracellular β-galactosidase during growth on lactose.In Plants the relationship between fruit softening and beta-Gal during banana fruit ripening, a beta-Gal cDNA fragment, named MA-Gal, has been cloned from banana fruit pulp using RT-PCR in this study.The results of sequence analysis showed that MA-Gal contained 927 bp, encoding a polypeptide of 309 amino acids, the deduced protein was highly homologous to plant beta-galactosidase expressed in fruit ripening.The MA-Gal putative amino acids have five homologous domains [5].In light of above, the study of β-galactosidase amino acid sequences from various sources is very important.In the present analysis, we performed the In-silico analysis including conserved motif assessment their family identification, MSA, and cluster analysis of β-galactosidase amino acid sequences from bacteria, fungi and plants.

Multiple sequence alignment
The multiple sequence alignment of the individual profiles was performed using MUSCLE at the European Bioinformatics Institute [10].

Conserved Motif identification
Motifs were identified in profiles using the expectation maximization approach implemented in Multiple EM for Motif Elicitation server [11].

Conserved Motif family identification
Motif families were identified by sequence searching in Pfam database [12]

Cluster analysis
The UPGMA approach implemented in the Mega program was employed for constructing phylogenetic relationships among sequences [13] 3. Results

Sequence retrieval
All the sequences belonging to different families of bacteria, fungi and plants were searched and retrieved from NCBI protein database (GenPept) and listed in Table 1 along with their accession number, species name, family and origin.

Multiple sequence alignment
MSA showed the presence of some conserved residues in all the sequences from different sources, while others were restricted only to their groups [14].Four tryptophan, four phenylalanine, three tyrosine, two proline, two alanine, one glycine, one aspartic acid, one isoleucine and one glutamic acid were found to be identically conserved residues in all analysed species of plant.One proline and one glycine were found to be identically conserved residues in all analyzed sequences of bacteria while no residue was found to be conserved in fungal profile.

Conserved motif identification
Seven conserved motifs were identified after the analysis of bacterial, fungal and plant profiles separately.Three conserved motifs were observed in bacterial profile, three in plant profile whereas a single conserved motif was identified in fungal profile (Table .2).

Conserved motif family identification
The seven identified conserved motifs were applied for their family identification in Pfam data base using sequence search option.First two conserved motifs identified in bacterial profile belonged to Glyco hydro 42 domain family while the Pfam entry of third bacterial conserved motif was not found.All the three conserved motifs identified in plant profile belonged to Glyco hydro 35 domain family while a single conserved motif identified in fungal profile belonged to Beta Gal dom2 domain family (Table .2).

Cluster analysis of bacterial profile
Cluster analysis of bacteria showed two major clusters as shown in Figure 1.Cluster A consisted of six species which was further divided into two sub-clusters.Sub-cluster A contains three species (Thermus thermophilus, Meiothermus ruber and Streptomyces flavogriseus).Sub-cluster B contains two species (Bacteroides salanitronis and Bacteroides ovatus).Niastella koreensis was found to be distantly related and therefore outgrouped from both sub-clusters.Cluster B consisted of two species namely Xanthomonas axonopodis, and Streptomyces coelicolor.Frateuria aurantia and Niabella soli were outgrouped from both clusters.

Cluster analysis of fungal profile
Cluster analysis of fungi showed a single major cluster as shown in Figure 2.This cluster consisted of seven species which was further divided into two sub-clusters.Sub-cluster A contains five species (Metarhizium anisopliae, Metarhizium acridum, Penicillium decumbens, Beauveria bassiana and Aspergillus kawachii).Sub-cluster B contains two species (Verticillium dahlia and Verticillium albo-atrum).Colletotrichum orbiculare, Cordyceps militaris and Colletotrichum higginsianum were found to be outgrouped from both sub-clusters and therefore these were distantly related.

Cluster analysis of plant profile
Cluster analysis of plant showed two major clusters as shown in Figure 3. Cluster A consisted of eight species which was further divided into two sub-clusters.Sub-cluster A contains three species (Prunus salicina, Pyrus communis and Cicer arietinum).Subcluster B contains two species (Solanum lycopersicum and Capsicum annuum).Oryza sativa, Brassica oleracea, Medicago truncatula were found to be distantly related and therefore outgrouped from both sub-clusters.Cluster B consisted of two species namely Arabidopsis thaliana and Aegilops tauschii.

Cluster analysis of joint bacterial, fungal and plant profile
Three major clusters were obtained by Cluster analysis of joint bacterial, fungal and plant profile (Figure 4).Cluster A consisted of seventeen species which were further divided into two subclusters.Subcluster A contained eight species of plants, and one species of bacteria.Subcluster B consisted of seven species of fungi and one species of bacteria.Cluster B consisted of six species of bacteria.One species of bacteria was outgrouped from Cluster B. Cluster C consisted of two species of plant and two species of fungi.One bacterial species and one fungal species were outgrouped from all three clusters.

Conclusions
Identification of conserved regions in a profile of protein sequences determines common ancestry combined with conservative evolutionary pressure to maintain important residues at functionally important parts of the protein.MSA revealed the presence of some conserved residues in plant and bacterial profile separately while no residue was found to be conserved in fungal profile.This suggests that the analyzed sequences of fungi showed high variability when compared to bacteria and plants.Seven conserved motifs belonging to different families were identified.Three major sequence clusters were obtained by cluster analysis of all retrieved sequences from different sources indicating the evolutionary history of β-galactosidases.

Figure 1 .
Figure 1.Phylogenetic tree of bacterial profile using UPGMA method

Figure4.
Figure4.Phylogenetic tree of joint profile of bacteria, fungi and plants using UPGMA method

Table . 1
Retrieved sequences, source, species name, family and their accession number

Table . 2
Motifs identified using MEME program and their Pfam analysis using Pfam database