In silico characterization and over-expression of squalene hopene cyclase from Pseudomonas mendocina

Abstract

Pseudomonas mendocina was identified as a novel endophytic isolate of Murraya koenigii with squalene cyclase activity. The PCR amplification of squalene hopene cyclase (shc) gene from the isolate Pseudomonas mendocina with the primers PA1/PA2 showed a band at 1980 bp specific for the enzyme squalene hopene cyclase. The in silico translation of the squalene hopene cyclase gene showed 96% sequence similarity with squalene hopene cyclase of Pseudomonas agarici (WP-060782422). Docking studies of the template and the modeled protein with the ligand squalene showed that the main interacting residues were Asp376 and Asp377. Squalene hopene cyclase template 1 sqc.1A sequence from Alicyclobacillus acidocaldaruis was used as the template for docking experiments. The gene coding for squalene hopene cyclase from Pseudomonas mendocina has been cloned in pET-28a vector to produce recombinant vector and was expressed in E.coli BL21 (DE3) expression system. Squalene hopene cyclase enzyme was isolated, purified and the molecular weight was confirmed by SDS-PAGE as 75 KDa.

Electronic supplementary material

The online version of this article (10.1007/s13205-019-1901-7) contains supplementary material, which is available to authorized users.

Introduction

Squalene hopene cyclase (SHC) catalyzes the cyclization of the linear triterpenoid squalene to hopene or hopanol, and is considered as one of the most complex single-step reactions known in biochemistry. These enzymes have a broad substrate range and its active site cavity can accommodate substrates with varying carbon chain length. This property of the enzyme can be exploited in pharmaceutical industries for drug biotransformation on a large scale replacing chemical transformation. Also enzyme-mediated biotransformations produce less toxic wastes and these processes are ecofriendly too. The eukaryotic counterpart of this enzyme is oxidosqualene cyclase (OSC) which cyclizes 2,3-oxidosqualene to lanosterol in fungi and mammals and to cycloartenol and variety of pentacyclic triterpenes in plants (Racolta et al. 2012). Hopanoids and sterols are integral membrane proteins and they impart structural stability to the phospholipid bilayer by reducing the permeability of the membrane. The high similarity of cyclization reaction catalyzed by SHCs and OSCs makes triterpene cyclases good candidates for evolutionary studies on phylogenetic relatedness of these enzymes in prokaryotic and eukaryotic organisms (Siedenburg and Jendrossek 2011). In bacterial system, squalene gets converted into hopene by the enzyme squalene hopene cyclase. Bacteria do not epoxidize squalene prior to π-cation cyclization. Rather, the enzyme catalyzes an enantioselective and diastereoselective polycyclization initiated by proton transfer to 2, 3-alkene. It is believed that this bacterial cyclase is the ‘primitive cyclase’ from which all other oxidosqualene cyclases have been evolved (Yoder and Johnston 2005). The analysis of crystal structure of SHC confirmed that these enzymes are partially integrated in the membrane and do not span the lipid bilayer (monotopic membrane protein). The enzyme is anchored to the negatively charged phospholipid membrane by positively charged amino acids. A protruding part in the center of the enzyme contained a lipophilic channel which targeted the substrate to the active site cavity inside the protein (Oliaro-Bosso et al. 2005). The crystallization and structural elucidation of enzyme from only one thermophilic bacterium Alicyclobacillus acidocaldarius had been looked into for the study of active site residue (Lenhart et al. 2002). The sequence of human OSC and bacterial SHC has 17% identical residues and could be used as sequence finger prints. Both cyclases carried conserved repeating motif rich in glutamine and tryptophan residues (QW motif) (Dang and Prestwich 2000).

The binding of substrate with the active site of the enzyme can be predicted by in silico studies. The in silico approach acts as fast and accurate prediction method for the interaction. The over-expression system for SHC in Alicyclobacillus acidocaldarius was constructed by using pET3a vector with high expression from the strong T₇ promoter when incorporated into E.coli BL21 (DE3) expression system (Sato et al. 1998). The shc gene from Streptomyces peucetius was cloned into pET32a (+) and introduced into E.coli BL21 (DE3) pLySs by heat-pulse transformation (Ghimire et al. 2009). Here, we report the isolation of a shc gene from Pseudomonas mendocina, a novel endophyte from the leaves of Murraya koenigii. The present study also focussed on the in silico characterization of the protein by homology modeling to understand the interaction of the active site of the modeled structure as well as the template protein with the substrate. The over expression of the enzyme was attempted by cloning the shc gene from Pseudomonas mendocina in pET 28a cloning vehicle. The plasmid containing the shc gene and other genes for expression, the T7 promoter, lac Z sequence and the antibiotic resistance gene was transformed into E.coli BL 21 (DE3) cells.

Materials and methods

Source of bacterial strain and culture conditions

Pseudomonas mendocina, an endophyte of Murraya koenigii with Gen Bank accession number MFO99411 (Nair and Jayachandran 2017), was grown in 5-mL LB broth for 24 h at 28 °C with 200-rpm agitation until the culture growth reached to an OD of 1.0 at 600 nm. The genomic DNA was isolated using Chromous Biotech Bacterial Genomic DNA Mini Spin Kit (RKT 17).

Selective amplification of shc gene

Genomic DNA from Pseudomonas mendocina was used as template for the amplification of genes specific to squalene hopene cyclase by PCR primers PA1 (5′-GAT GGT TCY TGG TAY GGT′-3′) and PA2 (5′-CCC CAR CCR CCR TCY TCG TTC TG-3′). PCR was carried out in a 50 μL reaction volume containing 50 ng of genomic DNA, 20 pico-moles of each primer (both forward and reverse), 1 X Emerald Amp GT PCR Master Mix (Takara Bio Inc.). PCR was carried out for 35 cycles in a Mycycler™ (Bio-Rad, USA) with the initial denaturation at 94 °C for 3 min, cyclic denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s and extension at 72 °C for 2 min with a final extension of 7 min at 72 °C. The specific size of the product has been confirmed by conducting agarose gel electrophoresis with standard molecular weight markers (100 bp ladder, Himedia, India). The amplified product was further purified from the gel and was subjected to sequencing using Big Dye Terminator Sequence Reaction Ready Mix (Applied Biosystem).

In silico translation of the gene sequence

The DNA sequence was translated using EXPASY online translation tools (http://www.expasy.ch/tools/dna.html). From the six translational frames for the prediction of most possible open reading frames (ORF), amino acid without any interrupting stop codons was selected for further analysis. Protein BLAST with previously reported SHC sequences was used for the confirmation of the sequence. Physico-chemical properties like molecular weight, isoelectric point (pI), aliphatic index, half-life, amino acid property, instability index and Grand Average Hydropathy (GRAVY) were obtained by the EXPASY tool Prot Param (http://web.expasy.org/protparam/). The GRAVY of the linear polypeptide sequence was calculated as the sum of hydropathy values of all amino acids, and the amino acid variation of the sequences was analyzed by multiple sequence alignment and comparative sequence analysis by Clustal W program of Bio Edit (Hall 1999).

The translated squalene hopene cyclase gene along with the sequence obtained from pBLAST analysis was used for phylogenetic analysis. The selected sequences were aligned by Clustal W and the aligned data were used for phylogenetic analysis using MEGA 5 (neighbor-joining method) with 1000 boot strap replicates (Tamura et al. 2011).

Homology modeling of squalene hopene cyclase

The three-dimensional homology modeled structure of the enzyme squalene hopene cyclase was generated through SWISS-MODEL (http://swissmodel.expasy.org/workspace) (Arnold et al. 2006). The built model was visualized under molecular visualization software (Accelrys BIOVIA Discovery Studio 3.5). The template1sqc.1.A sequence used for modeling was squalene hopene cyclase from Alicyclobacillus acidocaldarius (PDB ID: P33247). Energy minimization of the built model was performed using the GROMOS96 implemented in the Swiss PDB viewer program.

Structure validation of the predicted model of the protein

The quality of the predicted model was analyzed by PROCHECK and Ramachandran plot using the RAMPAGE web server (http://mordred.bioc.cam.ac.uk) (Lovell et al. 2003). The phi–psi torsion angles for the residues in structure were plotted in Ramachandran plot at RAMPAGE. Root mean square deviation (RMSD) values were calculated for finding the potential structural deviations between template and modeled structure using SUPERPOSE webserver (http://wishart.biology.ualberta.ca/cgi-bin/). Visualization and analysis of the model were performed using the Swiss PDB viewer and PyMOL programs. The secondary structure functional parameters were predicted using ProFunc (http://www.ebi.ac.uk/thronton-serv/ProFunc).

Docking of the modeled protein squalene hopene cyclase

After validation of the homology modeled protein structure, docking of the substrate squalene (Chem Sketch Software) to the active site of the modeled structure as well as the template protein1sqc.1.A sequence (PDB ID P33247) was performed using GOLD Suite docking software. GOLD (Genetic optimization for ligand docking) (Jones et al. 1995) is a genetic algorithm for docking flexible ligands into binding sites. Prior to docking, the bound ligand and water molecules were removed from the template crystal structure and missing hydrogen bonds were added to the structure. In docking simulations, the amino acid residues of the protein were held rigid and the ligand was kept flexible. All rotatable bonds in the ligands were allowed to rotate during the docking trials. The docking poses obtained were analyzed, and the best scoring poses with least binding energy were selected.

DNA manipulation and overexpression of squalene hopene cyclase gene

The PCR-amplified shc gene from Pseudomonas mendocina after supplementation with suitable cloning sites was ligated into pET 28a vector using BamHI and XhoI restriction sites (plasmid constructed at CCAMP, NCBS, Bangalore). The positive clones were analyzed for sequence confirmation. The 1980 bp shc gene bearing recombinant vector was transformed into E.coli BL21 (DE3) for expression studies (modified procedure Ghimire et al. 2009). The transformants were selected on LB-kanamycin plates.

E.coli BL21 (DE3) pLySs harboring pET28a-SHC were grown overnight in 5-mL LB medium supplemented with kanamycin (100 μg/mL) at 37 °C with shaking at 150 rpm. These pre-cultures were used to inoculate two independent 200-mL LB cultures with antibiotic in two 1-L Erlenmeyer flasks and the cultures were inoculated overnight at 37 °C. When OD₆₀₀ reached 0.6, SHC expression was induced by the addition of IPTG in a final concentration of 0.2 mM. An induction time of 4–5 h was given and the cultures were mixed. The cells were harvested by centrifuging at 10,000 rpm for 20 min at 4 °C. The cells were then pooled and washed twice with cell washing buffer (0.1-M sodium phosphate, pH 7.0). After the washing step, cells were transferred and stored at − 20 °C for enzyme extraction.

Extraction and purification of membrane bound squalene hopene cyclase

Thawed cells (OD₆₀₀ = 100) were resuspended in cell disruption buffer (0.2-M sodium citrate, 0.1-M EDTA, pH 6.0). The cells were disrupted by ultrasonic treatment for six times, with cooling on ice for 1 min in between the sonication steps. The cell debris were collected by centrifuging at 16,000 rpm for 60 min at 4 °C. The supernatant was then discarded. Cell debris were resuspended in solubilization buffer (0.05 M sodium citrate, 0.01% MgCl₂, 1% Triton-X 100, pH 6.0) and incubated for an hour by gently mixing at 4 °C, to solubilize the membrane-bound protein with detergent (1% Triton-100). After centrifugation at 16,000 rpm for 60 min at 4 °C, the supernatant was collected and the soluble protein from the supernatant was purified by Ni²⁺ affinity chromatography (Ni sepharose beads).

The over-expressed protein was run on denaturing conditions in SDS gel having 12% resolving gel consistency to confirm the subunits of the protein (Laemmli 1970).

Results

Selective amplification of squalene hopene cyclase gene

The PCR amplification of shc gene was positive for Pseudomonas mendocina (MKE5), representing a band with 1980-bp size with the primers PA1/PA2 specific for the enzyme squalene hopene cyclase Fig. 1. The PCR product of size 1980 bp was gel eluted, subjected to sequencing and the sequence data were analyzed. A negative control containing primers and PCR mix was kept as control.

Fig. 1.

PCR amplification of the gene coding squalene hopene cyclase (shc) in Pseudomonas mendocina. Lane 1: MKE5; lane 2: control; lane 3: marker DNA

In silico characterization

In silico translation analysis of the DNA sequences obtained after the selective amplification of the shc of the isolate Pseudomonas mendocina confirmed that the DNA sequence obtained was error free with uninterrupted ORF (supplementary Fig. 1). The obtained amino acid sequence was subjected to protein BLAST analysis which confirmed that the sequence was having similarity towards squalene hopene cyclase. The sequence showed 96% similarity towards squalene hopene cyclase of the organism Pseudomonas agarici (WP_060782422). This was further confirmed using phylogenetic analysis Fig. 2.

Fig. 2.

The phylogenetic analysis of shc gene sequence of Pseudomonas mendocina (MKE5)

Homology modeling and structure validation of the predicted model of the protein

The comparative alignment analysis and 3D structure model of squalene hopene cyclase proteins were obtained by homology modeling using SWISS-MODEL workspace. The squalene hopene cyclase template 1sqc.1.A sequence from Alicyclobacillus acidocaldarius with an average sequence identity of 96% with the target protein and the E value together with the Q mean Z score value of the selected sequence showed the reliability of the model (Table 1). The modeled structures were then subjected to structural reliability analysis of physico-chemical properties, predicted by ProtParam tool. By comparing the structural parameters with the selected template, the structural reliability of the modeled proteins was calculated.

Table 1.

Physiochemical parameters of the squalene hopene cyclase of the isolate Pseudomonas mendocina in docking studies

Properties	MKE5
Amino acid number	622
Molecular weight	68.7 kDa
PI	6.22
Number of negatively charged residues	62
Number of positively charged residue	54
Instability index	43.99
Aliphatic index	88.17
GRAVY	− 0.093
Q mean	− 0.07

The homology modeled protein Fig. 3 and the template were subjected to Ramachandran plot analysis to confirm the reliability of the structure obtained. More than 95% of the residues from both the template and the isolated protein were in the most favored region and the rest of the residues in the favored region confirmed the integrity of the modeled structure Fig. 4. When the model was further analyzed using Super Pose for the comparison of potential deviation of the modeled protein with that of the template, RMSD value was Cα: 0.04 Å, backbone: 0.04 Å and 0.26 Å for all atoms which were found very low for superimposition with the template protein. This indicated that the structure was having high similarity with that of the template structure.

Fig. 3.

Homology-modeled SHC protein of the isolate Pseudomonas mendocina (MKE5)

Fig. 4.

Validation of the 3D protein structure obtained by homology modeling of the SHC protein sequence using Ramachandran plot. a Isolate Pseudomonas mendocina; b template (1sqc.1.A)

Docking of the modeled squalene hopene cyclase

Docking studies showed that the substrate squalene has almost comparable energies with the modeled protein (− 130.47 k cal mol⁻¹), as well as the template protein (− 112.16 k cal mol⁻¹). The docking score obtained for the target protein was 5190 when compared to the template protein of 5270. The interactions of molecules were observed by selecting the docking poses with more energetically favored ligand binding. The substrate receptor complexes for the template and target proteins are shown in Fig. 5a and b, respectively. The docked active sites of the molecules were carefully analyzed for identifying the amino acids involved. The results showed that the amino acid residues which interacted with the complex were Leu35, Met42, Ile261, Gln262, Pro263, Ala306, Ser307, Trp312, Phe365, Asp374, Asp376, Asp377, Trp409, Tyr420, Gly600, Phe601, Phe605, Leu607, Tyr609, and Tyr612. The comparison of the 3D structure and active site residues of the modeled and the template protein showed high similarities. The residues Asp376, Asp377, Phe601, Phe605 were conserved in both template and modeled structure. Strikingly, some of these amino acids were found to be conserved in SHC of other organisms also. The predicted model suggested the presence of shc gene in the novel isolate Pseudomonas mendocina from Murraya koenigii.

Fig. 5.

Detailed molecular interaction of the substrate at the docked active site of the homology modeled squalene hopene cyclase. a Isolate Pseudomonas mendocina; b template (1sqc.1.A) sequence of Alicyclobacillus acidocaldarius

Expression studies

The shc gene was cloned successfully into pET28a vector, and both the gene construct and the vector were digested with Bam H1 and Xho1 enzymes. The recombinant plasmid digests were subjected to gel electrophoresis Fig. 6. Further proof for the existence of the protein squalene hopene cyclase was provided by expression studies. The purified protein was subjected to SDS-PAGE at 12% resolving gel concentration with suitable molecular weight markers. A 75-KDa protein band was observed on staining with Coomassie brilliant blue 250 Fig. 7. The purified protein was confirmed as squalene hopene cyclase.

Fig. 6.

Cloning of shc gene in pET28a vector. M molecular weight marker; lane 1: 1 Kb plus DNA ladder; lane 2: uncut pET28a vector; lane 3: linearised pET28a vector (BamHI and XhoI); lane 4: uncut shc synthesis vector; lane 5: cut shc vector showing release of 1980 bp shc gene

Fig. 7.

SDS-PAGE analysis of purified recombinant overexpressed Pseudomonas mendocina SHC enzyme after Coomassie blue staining. Lane 1: molecular weight markers; lane 2: purified elution fraction containing recombinant SHC

SHC is the key enzyme involved in hopanoid biosynthesis. PCR-based screening methods were employed for identification of shc gene in different organisms like Bradyrhizobium japonicum (Perzl et al. 1997), Streptomyces peucetius (Ghimire et al.2009), Zymomonas mobilis (Reipen et al. 1995) and Methylococcus capsulatus (Tippelt et al. 1998). The in silico methods of protein–ligand interactions acted as fast and accurate methods for the prediction of best interaction models. The homology modeling and docking studies gave deep insight into the detailed mechanism of interaction and also about the participating amino acids.

The docking studies using GOLD suite docking software helped in selecting docking poses with low binding energy. The active site residues which interacted with the complex were validated. The amino acids Asp376 and Asp377 played a crucial role in the interactions. These residues were located on the top of the active site cavity and Glu45 at the bottom of the cavity. Trp169 and Trp489 determined the termination of cyclization. The active site cavity accommodated an extended squalene molecule between the two carboxylate group of Asp376 and Glu45 (Dang and Prestwich 2000). Asp376 protonated the terminal isopropylidene bond of squalene (Wendt et al. 1997) and Glu45 was involved in cyclization termination. The in silico studies provided a very interesting platform to study the ligand–protein interactions. The structural model of previously reported SHC revealed that the Asp376 and Asp377 residues were highly conserved. Different strains of E.coli were used as expression system because it was an ideal host as it did not synthesize hopanoids or sterols. Since E.coli lacked triterpene cyclases, the over-expressed protein, from the cell extracts were purified and confirmed for cyclase activity (Ochs et al. 1990).

The amplified shc gene from the specific organism was cloned into suitable vector and expressed in E. coli expression system. For cloning experiment, usually pET vectors were preferred as they could carry large fragments of DNA. The shc gene from Zymomonas mobilis was cloned in pUC 19 (Sma I digested) vector and expressed in E. coli K12 DH5 cells (Reipen et al. 1995). SHC from Methylococcus capsulatus was over-expressed along with OSC in E. coli DHα using pUC 19 vectors (Tippelt et al. 1998).

Cyclization reactions catalyzed by squalene hopene cyclase are an interesting reaction since it can catalyze the conversion of a linear compound to a cyclic derivative in a single step. Many reports are there for the existence of this enzyme in thermophilic, acidophilic and other bacterial species which live in extreme conditions. Squalene hopene cyclases provide stability to the lipid membrane and reduce permeability leading to an up-regulation of this enzyme in extreme environments. Since these enzymes were terpene cyclases, the prediction of their existence in terpene producing plants was an interesting hypothesis. Endophytic bacteria residing in these plants, contributed to the synthesis of terpene cyclases. Understanding the endophytic microbial flora within the selected plant and exploration of the mechanism involved can open up new areas of research in the field of microbial bio-transformations.

Conclusion

The present study involving the comparative modeling, multiple alignment and docking studies of the protein squalene hopene cyclase provided a reliable platform for exploring the protein–ligand interaction. The gene sequence coding for squalene hopene cyclase was successfully cloned into pET-28 a vector and expressed in E.coli BL21 (DE3) expression system. Pseudomonas mendocina is being reported for the first time as an endophyte of Murraya koenigii. The strain exhibited squalene hopene cyclase activity which was confined only to a few bacterial strains as per the present available information. The existence of SHC in an endophyte could be co-related with the evolution of OSC in higher eukaryotes like plants and fungi for the formation of sterols.

Electronic supplementary material

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Author contributions

The author IMN has contributed to the execution of the work and the preparation of the manuscript. The corresponding author Dr. JK contributed to the planning of the work and correction of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.