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In silico perceptions in structural elucidation of Exo-β-1,4-glucanase and Endo-β-1,3-glucanase from Streptomyces spp

β-glucanases are industrially significant glycosyl hydrolases which hydrolyze the glycosidic bonds in insoluble or partially soluble β-glucan molecules by acting as exoand endo-hydrolases. Molecular characterization and structural elucidation are vital for uncovering the structural and functional relationship of β-glucan degrading enzymes. In the present study, full-length gene sequences of exo-β-1,4-glucanase (EXO-14) and endo-β-1,3-glucanase (ENDO-13), isolated from Streptomyces althioticus TBG-MR17 and Streptomyces cinereoruber subsp. cinereoruber TBG-AL13, respectively, were obtained. The primary sequence analysis and sequence based structural analysis were performed in silico. The obtained 1737 bp EXO14 gene and 1293 bp ENDO13 gene encoded 578 and 438 putative amino acids, respectively. The in silico functional domain predictions showed a multi-domain architecture of both enzymes and showed diverse physiochemical properties. The substrate binding analysis through molecular docking delivered the possible architecture of active sites, the tunnel type in EXO-14 prominently cleaves external β-1,4linkages and open cleft like structure in ENDO-13 has more propensity to cut internal bonds. Overall, the results generate valuable information for continuing the understanding of β-glucanase enzymes especially from Streptomyces spp., and provide significance for upgrading their catalytic properties through enzyme engineering.


INTRODUCTION
Genes encoding β-glucanase enzymes have been widely explored in various organisms because of enormous importance in industrial applications. β-glucanase gained commercial importance for its role in beer fermentation and poultry feed formulation [1,2], the enzyme was also weighted for its ecological significance [3,4]. Considering commercial applications, molecular redesigning of the enzyme may enhance its properties at industrial scale. This can be achieved by understanding the enzyme at proteomic and genomic level, which can guide the manipulation of the enzyme to suit industrial process.
Protein motifs of β-glucanases have been annotated in the carbohydrateactive enzymes (CAZy) modules as sequence related families [5] and the activities were annotated according to sequence similarities, protein structural folding, and recognized enzyme activities [6]. Understanding molecular structure of enzymes is essential to unfold the functional properties. The molecular structure predominantly controls the chemical and physical properties of enzymes which retains their particular roles in nature and biological systems [7]. Bioinformatics aid in understanding the functional role of proteins based on available sequence at the genomic or proteomic level. Computational biology can help in constructing the three dimensional structure of a protein, that further helps to predict functional roles and these predictions are helpful in outlining required modifications for activity enhancement [8]. Structural and functional characterization of several β-glucanase in the past has helped in defining the functional and structural -relationship of this protein [9,10]. Molecular docking is another bioinformatic tool that serves to identify binding affinity and strength of association between two molecules, especially in drug designing. In enzyme studies, the tool recognizes catalytic domains for binding of substrate molecules [11].
Many molecular in silico characterization reports are available in regard to β-glucanases from prokaryotes and eukaryotes [12], among this source of fungi and bacteria are more prominent; however, studies in Streptomyces are rarely reported [13,14]. In this report, we have isolated complete sequence of exo-β-1,4-glucanase (EXO14) and endo-β-1,3-glucanase (ENDO-13) genes from Streptomyces althioticus TBG-MR17 and Streptomyces cinereoruber subsp. cinereoruber TBG-AL13, respectively. The potent β-glucanase producers were isolated from Western Ghats of Kerala and detailed molecular characterization studies were conducted in silico. With an aim to understand the enzyme substrate interaction, homology modeling and docking analysis of predicted EXO14 and ENDO13 proteins were deliberated.

MATERIALS AND METHODS
S. althioticus TBG-MR17 and S. cinereoruber subsp. Cinereoruber TBG-AL13 were isolated from Western Ghats regions in Kerala, India. The strains were grown and maintained in International Streptomyces Project 4 agar at 28°C. Escherichia coli strain DH5α was purchased from Invitrogen™, (USA) and grown in Luria Bertini (LB) agar (HiMedia, India) at 37°C. GoTaq ® G2 Hot Start Green PCR Master Mix and pGEM ® -T Easy Vector were purchased from Promega (USA). The primers were synthesized in Eurofin Genomics, Bengaluru. DNA sequencing reaction was done in ABI PRISM 3730xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using BigDye® Terminator v3.1 Cycle Sequencing Kit from Applied Biosystems based on Sanger's dideoxy chain termination method.

PCR Amplification, Cloning, and Sequencing
High purity genomic DNAs were isolated from the mycelium of Streptomyces strains using the method of Murray and Thompson [15] with adequate modifications. PCR amplification of EXO-14 and ENDO-13 genes was carried out using the primers EX3f/EX3r (EX3f-AACCCGCACAATGAGTCGCACCA; EX3r-GTCAGCCGGTCACGACACCGG) and EN3f/EN3r (EN3f-CACCCGACGGAAGAGAGAGYCATGMCC; EN3r-CAGCGTGTCACCGTCCACTTCTGG), respectively. The primers designed based on the conserved regions of EXO-14 and ENDO-13 genes retrieved respectively from the phylogenetic neighboring species of S. althioticus TBG-MR17 and S. cinereoruber subsp. Cinereoruber TBG-AL13 available in the NCBI database. PCR reaction mixtures (25 µL) contained approximately 25 ng of DNA, 10 ng of each primers and 12.5 µL GoTaq ® G2 Hot Start Green PCR Master Mix. The thermal program included an initial denaturation at 95°C for 2 min followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 66°C for 30 sec, and extension at 72°C for 1.30 sec, and a final extension at 72°C for 5 min. The amplicons were purified using Wizard ® SV Gel and PCR clean-up system (Promega, USA), cloned into pGEM ® -T Easy Vector and transformed to DH5α E. coli cells. The purified plasmids from positive colonies were sequenced using primers TvectF/TvectR (TvectF-GGGTTTTCCCAGTCACGACGT; TvectR-CGCCAAGCTATTTAGGTGACAC).

Homology Modeling
Three dimensional models of EXO-14 and ENDO-13 protein were generated using I-TASSER webserver of University of Michigan, USA maintained by Zhang Lab [24]. It used multiple threading approaches for selecting the templates and predicted full-length models of EXO-14 and ENDO-13 proteins by iterative structure assembly. Based on Z-score, one template was selected from each threading program to predict the target model of each protein. The program selected ten best structural analogs from protein data bank based on closest structural similarity and generated five models by clustering the stimulated structural confirmations based on pair-wise structural similarity with the template. The three dimensional models were evaluated based on TM-score (template modeling Score), C-score (confidence score), and RMSD (Root-mean-square deviation). The confidence of models was measured quantitatively by C-score, normally in range of −5-2. The higher C-score value signified a high confidence model. The RMSD and TM-score calculated mainly based on the C-score. Finally using the TM-align structural alignment program I-TASSER aligned the best model to all available PDB structures. The models were visualized by the Maestro 11.5 element tool of Schrödinger Suites 2018-1 (Schrödinger, LLC, New York, NY, 2018) and finally validated by assessing the model stereochemical quality using BioLuminate module of Schrödinger Suites 2018-1. Superimposition of models with corresponding templates was performed using Chimera 1.12.

Molecular Docking
Protein structures in pdb file format were directly imported to molecular docking software (Glide, Schrödinger Suites 2018-1). Protein structural minimizations and optimizations were carried out accordingly the Protein Preparation Wizard (PrepWizard) in SchrödingerSuites 2018-1. The preparation protocol built loops and side chains with missing atoms, added hydrogen molecules, optimized H-bonding network and finally a restrained minimization was done to get a refined protein structure for docking studies. Active sites (ligand binding sites) of proteins were predicted using SiteMap application of Schrödinger Suites 2018-1 [25]. The binding site within the catalytic domain was considered for docking study.
Two dimensional structures of ligand molecules in sdf format were retrieved from PubChem compound database (https://pubchem. ncbi.nlm.nih.gov/) of NCBI. The ligands were prepared by adding hydrogen, eliminate the discrepancies between bond length and angle and finally converted to the 3D structure using the LigPrep module of Schrödinger Suites 2018-1.
Receptor grid of 15Å × 15Å × 15Å grid points and spacing 0.25 was generated at the center of predicted active site, using the Grid Generation module of Schrödinger Suites 2018-1. The prepared grid was selected and docked with a single best conformation of the ligands using Glide dock-XP mode in Schrödinger Suites 2018-1. Subsequently, the best gliding pose of ligand to protein with maximum glide score (G score) and glide energy (G energy) was generated [26].

Amplification, Cloning, and Sequencing
The PCR amplification of ENDO-13 and EXO-14 genes yielded ~1400 bp and ~2000 bp corresponding intense amplicons [Supplementary Figure

Sequence Analysis
BLASTp search revealed that both proteins exhibited sequence similarity and identity to specific β-glucanases of Streptomyces spp. The amino acid sequence of EXO-14 showed 99% of sequence identity with Streptomyces sp. 4F cellulose 1,4-β-cellobiosidase (GenBank Accession No. WP058917325) and Streptomyces griseorubens cellulose 1,4-β-cellobiosidase (GenBank Accession No. WP037642617). The ENDO-13 protein amino acid sequence exhibited 82% identity with Streptomyces globisporus ENDO13 (GenBank Accession No. WP030690772) and Streptomyces sp. TLI-105 laminarinase like protein (GenBank Accession No. WP093872583). Multiple sequence alignments of EXO-14 protein with homologous sequence revealed a consensus sequence VIYNLPGRDCAALASNG, which is the key characteristics consensus pattern, (2)-x-G, of all other EXO14 (cellulose 1,4-β-cellobiosidase) protein [ Figure 1]. This confirmed that the catalytic domain of EXO14 resembled that of cellulose 1,4-β-cellobiosidase (EXO14). MSA of ENDO-13 protein showed the presence of consensus sequence EIDVMENVGFEP, similar to the characteristic consensus pattern, E- , of all other ENDO13 (laminarinase) proteins [ Figure 2]. The computed physicochemical analysis of the predicted EXO-14 and ENDO-13 proteins is shown in Table 1. The EXO14 has high number of negative amino acid residues than ENDO13. The instability index <40 is predicted as stable, the values obtained showed that both EXO-14 and ENDO-13 proteins are considered as stable. Low GRAVY values of EXO-14 (−0.446) and ENDO-13 (−0.424) indicated both have the property to interact with water. The nucleotide sequence for EXO14 and ENDO13 was deposited in GenBank database under the accession number of MG983485 and MH719000 respectively.

Phylogenetic Analysis, Motif Architecture, and Secondary Structure Predictions
The phylogenetic tree constructed based on the BLASTp revealed that the EXO-14 protein grouped into a clade with Streptomyces sp. 4F cellulose 1,4-β-cellobiosidase and Streptomyces griseorubens cellulose 1,4-β-cellobiosidase. The sequence showed more close similarity to the Streptomyces sp. 4F cellulose 1,4-β-cellobiosidase [ Figure 3a]. Constructed neighbor-joining phylogenetic tree of ENDO-13 protein identified that it was grouped in a first clade and showed more similarity to S. globisporus ENDO13 [ Figure 3b].
However, it displayed a prominent discrepancy from other sequences, this evidently confirmed the ENDO-13 protein from S. cinereoruber subsp. cinereoruber TBG-AL13 is a novel one.  Supplementary Figures 6 and 7]. Based on the results of signal analysis, CDD search and dbCAN analysis a schematic representation of EXO14 and ENDO13 proteins are shown in Figure 4. Secondary structures of EXO-14 and ENDO-13 proteins were predicted with high confidence using the PSIPREDv3.3 based on local sequence similarity. Both proteins have varied secondary structures containing several alpha-helices and beta-sheets with loop structural intervals, while both have a predicted N-terminal helical structure which indicated N-terminal leader peptide [ Figure 5].

Homology Modeling
The best 3D structural model of EXO14 predicted by I-TASSER using protein 4b4fA as the protein model target showed C-score = −2.07, estimated TM-Score = 0.47 ± 0.15, and RMSD = 12.7 ± 4.3   structural model predicted using 3dgtA as template protein, showed C-score = −1.01, estimated TM-Score = 0.59 ± 0.14, and RMSD = 9.3 ± 4.6 Å [ Figure 6b]. The identified EC classification of ENDO-13 by I-TASSER was EC 3.2.1.39. The predicted structures were further validated using the superimposition of their corresponding templates. Superimposed structures of EXO-14 and ENDO-13 with its corresponding templates are shown in Figure 6c and d, respectively.

DISCUSSION
Streptomyces sp. from soil has been a reservoir of compounds and enzymes that gained scientific, medical, and industrial importance. Streptomyces isolates are acknowledged as chief producers of extracellular enzymes. However, current understanding of the glucanolytic enzymes from Streptomyces is very limited. As per our studies, two soil isolates S. althioticus TBG-MR17 and S. cinereoruber subsp. cinereoruber TBG-AL13 were potent producers of extracellular EXO14 and ENDO13, respectively. In this study, we have isolated the complete gene sequence of EXO-14 from S. althioticus TBG-MR17 and ENDO-13 from S. cinereoruber subsp. cinereoruber TBG-AL13, and scrutinized the biological structure and interaction topographies with particular substrates using in silico approach.
PCR amplification provided 1737 bp complete gene sequence of EXO-14 which encoded a putative protein of 578 amino acids. BLASTp analysis and phylogenetic tree construction confirmed that EXO-14 protein has very close resemblance to the Streptomyces sp. 4F cellulose 1,4-β-cellobiosidase. The functional domain and motif architectures were identified using CDD search and dbCAN annotation, this was vital for determining the molecular functions of proteins. The EXO-14 protein has N-terminal CBM2 domains and C-terminal GH6 domain, the characteristics functional domains of enzyme EXO14 (cellulose 1,4-β-cellobiosidase, or cellobiohydrolase) [27]. According to Tomotsune et al., [28] cellulose producing Streptomyces have been assigned to the protein families of GH5, GH6, GH9, GH12, and GH48 in CAZy database. Annotation of complete genome sequence of a pineboring wood wasp associated Streptomyces sp. SirexAA-E (ActE) predicted 1,4-β-cellobiohydrolase with GH6 and CBM2 domains, but structural information was not elucidated [29]. In fungi and plants, GH modules are accompanied by CBM modules [30,31]. Mostly, CBM2 are fused on the C-terminal side of the catalytic domain in all glycoside hydrolase families, whereas in GH5 and GH6 domain families it is located on the N-terminal end of domain architecture [32]. Both GH and CBM domains are separated by a linker sequence, rich with Pro/ Thr residues [33,34]. In CBM2/GH6, the average length of linker is 35 amino acids [35]. In our case, linker with 45 amino acids was found in between these two domains. As per report of Gilkes et al. [36], the catalytic site of EXO14 (cellulose 1,4-β-cellobiosidase) has a key characteristics consensus pattern, of V-x-Y-x(2)-P-x-R-D-C-[GSAF]x(2)-[GSA](2)-x-G. The EXO-14 protein alignment also showed similar catalytic core pattern, VIYNLPGRDCAALASNG, in between amino acid 236 and 254.
The complete sequence of ENDO13 has 1293 bp that encodes a putative protein of 438 amino acids. Based on the multiple sequence alignment, phylogenetic analysis and domain annotations the ENDO-13 protein came under GH16 family of ENDO13 and showed more similarity to S. globisporus ENDO13. However, it displayed a prominent divergence from other sequences, which evidently confirmed the ENDO-13 protein a novel one. Functional annotation of ENDO13 protein confirmed that it has a N-terminal Ricin-B-lectin such as GH16 and C-terminal CBM13 domains which is typical for ENDO13 enzymes. Both domains are separated by a 16 amino acid long linker sequence. GH16 is one of the largest CAZyme family, including endoglucanases involved in glucan degradation [37]. Endo-1,3-β-glucanase from Cellulosimicrobium cellulans reported a N-terminal GH16 catalytic domain and C-terminal CBM13 regions separated by a Gly/Ser-rich linker [38]. Catalytic region of ENDO-13 protein showed a consensus sequence WPNSGEIDVMENVGFEP, similar to the characteristic consensus pattern, W-P- a b x- [PSTA], found in all other ENDO13 (laminarinase) proteins. The two glutamic acid residues, Glu-175 and Glu-180, within this conserved region are thought to be involved in catalysis [39], though the aspartic acid residue (Asp-177) is significant in preserving the relative position of above catalytic amino acids [40]. In addition, minor differences were revealed in active site sequences, WPKCGEIDIMELLG, of Clostridium thermocellum ENDO13, Lic16A [41]. Our Streptomyces ENDO-13 also showed such some differences in the catalytic site regions when comparing with Laminarinase genes of Rhodothermus marinus, Thermotoga neapolitana, Bacillus circulans, and Strongylocentrotus purpuratus [42].
SignalP program detected the EXO-14 protein has a TAT signal peptide sequence of 32 amino acid in length. It helps to cleave the mature protein in between the amino acids Ala-32 and Ala-33. TAT pathway mostly secretes proteins in native folded state, found in both Streptomyces and E. coli [43,44]. 35 amino acids long N-terminus TAT family signal peptide domain was also found in ENDO-13 protein. It cleaves the mature protein in between Ala-35 and Ala-36 positions.
The analysis of physicochemical property showed that the isoelectric point (pI) of EXO14 was 4.34; this detailed the need to develop buffer system for pH based purification [45]. At pI, proteins are compact and stable [46]. Estimated half-life of EXO-14 in E. coli was more than 10 h and the designated instability index value of 24.65; this predicted that the protein was stable. The high aliphatic value of 66.26 showed that the protein was stable at varying range of temperatures since the aliphatic index is directly related with thermostability of protein [47]. Very low GRAVY value −0.446 concludes that the protein makes good interaction with water molecules. Secondary structure of EXO14 protein has 11 helixes and 15 sheets. Physicochemical properties deduces that the ENDO-13 has a pI value of 8.1 (pI>7.0), this reveals the basic nature of protein. Estimated half-life in E. coli was more than 10 h and the protein predicted as stable with instability index of 29.37. The aliphatic value of 59.35 showed that the ENDO-13 was less thermostable than EXO-14. Low GRAVY value, −0.424 revealed the good hydrophilic interaction. Predicted secondary structure has only 2 helixes and 24 sheets. Greater β-sheet content above other consistent secondary structures is a collective feature of β-1,3 glucanases [45].
Predicted three-dimensional structure of EXO-14 and ENDO-13 proteins using I-TASSER proved it as good quality with proper fold confirmation. Structural modeling in I-TASSER is based on the secondary structure enhanced profile-profile threading alignment [48]. Initially, it predicts secondary structure and then goes to tertiary structure. In the current study, for EXO-14 protein modeling, 4b4fA (Thermobifida fusca Cel6B) [49] was selected as best template with Z-score 2.46 and model 1 was selected as top ranked model based on high C-score (−2.07), signified the model was good. This was additionally validated by highest TM-Score (0.57 ± 0.15) and RMSD (12.7 ± 4.3 Å). For the molecular modeling of ENDO-13 protein, the template 3dgtA with Z-score= 8.44 (endo-1,3-β-glucanase from Streptomyces sioyaensis) [50] was selected as a best template and the best model was built with high C-score value 1.01, estimated TM-Score = 0.59 ± 0.14 and estimated RMSD = 9.3 ± 4.6 Å. The superimposed models of EXO-14 and ENDO-13 confirmed that the active sites of both enzymes are completely overlapped with corresponding templates. These two models were selected for further molecular docking studies to understand the enzyme substrate interaction.
The EXO-14 docking studies used cellotriose as ligand. It forms a favorable interaction with active site groove (site score 1.008, size 102, Dscore 1.052, volume 407.484) of protein and produced better docking score of −7.393 and Emodel energy −79.575. As mentioned by Wu et al. [51], we also found that a tunnel-type active site within the protein structure allows the polysaccharide chain to enter the active site from one entrance of tunnel in an "exo" fashion and release the products from other side while firmly clamping the chain.
The key amino acid residues involved in the binding of cellotriose were found to be Tyr239,

CONCLUSION
In short, the study enlighten the theoretical overview of EXO14 and ENDO13 encoding genes and the corresponding predicted putative proteins from Western Ghats isolates S. althioticus TBG-MR17 and S. cinereoruber subsp. cinereoruber TBG-AL13, respectively. It delivers an overall idea about protein structural domain architecture that eventually governs their stability, function, and interaction with particular substrates. The study of structure-function relationships exclusively uncovers the complete potential of both enzymes. Overall, it provides new information into the Streptomyces β-glucanases and moreover delivers a basis for enhancing the expression studies and protein stability toward industrial applications.

ACKNOWLEDGMENTS
Authors would like to acknowledge Woman Scientist Division, Kerala State Council for Science, Technology and Environment, Kerala for financial support.

CONFLICTS OF INTEREST
Authors declared that they do not have any conflicts of interest.