Molecular Docking Analysis and Biochemical Evaluation of

(8) Five mutants were created by site-directed mutagenesis, where active site residue Gln275 (corresponding to the His296 in LevU of Z. mobilis) was s...
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Research Article Cite This: ACS Comb. Sci. XXXX, XXX, XXX−XXX

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Molecular Docking Analysis and Biochemical Evaluation of Levansucrase from Sphingobium chungbukense DJ77 Soo Youn Lee,†,‡,# Woo-Ri Shin,†,# Simranjeet Singh Sekhon,†,# Jin-Pyo Lee,† Young-Chang Kim,† Ji-Young Ahn,*,† and Yang-Hoon Kim*,† †

School of Biological Sciences, Chungbuk National University, 1 Chungdae-Ro, Seowon-Gu, Cheongju 28644, Korea Climate Change Research Division, Korea Institute of Energy Research, 152 Gajeong-Ro, Yuseong-Gu, Daejeon 34129, Korea

‡

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S Supporting Information *

ABSTRACT: Bacterial exopolymer Levan (β-(2,6) polyfructan) synthesized by levansucrase has attracted interest for various applications due to its low intrinsic viscosity compared with other polysaccharides. We report a novel levansucrase (Lsc) isolated from Sphingobium chunbukense DJ77 and verify its biochemical characteristics by comparative analysis of molecular docking analysis (MOE) and catalytic residue analysis. The complete sequence of the Lsc encoding gene (lsc) was cloned under the direction of the T7 promoter and purified in an Escherichia coli BL21 (DE3) protein expression system. The enzyme activity analysis and ligand docking MOE study of S. chungbukense DJ77 Lsc revealed that Arg 77, Ser112, Arg 195, Asp196, Glu257, and Gln275 were involved in the sucrose binding and splitting as well as transfructosylation activity. A catalytic comparison of Lsc of S. chungbukense DJ77 with the results of site-directed mutational analysis indicated that Gln275 may coordinate a favorable substrate binding environment, offering broad pH resistance in the range of 5−10. The results suggest that the recombinant E. coli carrying S. chungbukense DJ77 Lsc might produce levan under the regular growth conditions with less need for pH manipulation. KEYWORDS: exopolymer, Sphingobium chungbukense DJ77, levansucrase, pH resistance, MOE-ligand docking analysis

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INTRODUCTION The bacterial extracellular macropolymer levan has commercial importance as a fructose sweeter, thickening agent in foods, and in pharmaceutical applications as potential anticancer and antioxidant reagents. 1 Levansucrases (EC 2.4.1.10) are reportedly involved in the synthesis of levan from sucrose by transfructosylation while releasing glucose and in the hydrolysis of levan to monosaccharides of fructose.2,3 Several bacterial genera including Bacillus subtilis,4 Streptococcus mutants,5 Zymomonas mobilis,6−8 Acetobacter diazotropicus,9 Erwinia amylovora,10 and Pseudomonas syringae pv Phaseolicola11 have been reported to belong to family 68 glycoside hydrolases (GH68) and have been biochemically characterized using sucrose as the substrate to synthesize levan, a fructose macropolymer. On the basis of multiple sequence analysis of GH68 family proteins, critical amino acid residues have been identified, and their role in acceptor and product specificity has been reported.3,10,12 Although levan has beneficial applications, the amount of levan produced is relatively low as compared to © XXXX American Chemical Society

the yields of other biopolymers including dextran and xanthan.13 A levansucrase gene was isolated from the novel strain S. chungbukense DJ77 using the genome project approach, and the putative open reading frame (ORF) with an initiation codon was determined by contig extension and primer walking.14 A comparative study showed that the genes and gene orders for sucrose hydrolysis of strain DJ77 were similar to the corresponding genes of Sphingobium species, such as Novosphingobium aromaticivorans F199.15,16 The complete gene coding levansucrase (lsc) from S. chungbukense DJ77 has been reported and deposited with GenBank under Accession No. DQ060376. Sequence alignments and substitution studies reveal that the conserved catalytic side residues of levansucrase are functionally important in both sucrose hydrolysis and fructosyl transfer in Received: January 8, 2018 Revised: May 10, 2018 Published: May 29, 2018 A

DOI: 10.1021/acscombsci.8b00002 ACS Comb. Sci. XXXX, XXX, XXX−XXX

Research Article

ACS Combinatorial Science the formation of levan.4,6,17 Levansucrase (LevU) of Z. mobilis has been extensively studied to understand their catalytic functional sites for levan production.18 A previous study demonstrated that the replacement of His296 in LevU of Z. mobilis attenuated its transfructosylation activity; however, the substitutions of His296, which are situated with its side chain orienting toward the entrance of the active site cavity, did not change the central pocket of the protein structure.7 In light of our findings, it was of interest to verify whether the mutations in levansucrase (Lsc) of S. chungbukense DJ77 affect the active site pocket and its catalytic capability to synthesize levan. In this study, the levansucrase gene (lsc) of S. chungbukense DJ77 was overproduced and purified on the basis of recombinant E. coli protocols. The Lsc of S. chungbukense DJ77 was biologically active over a broad pH range of 5−10, whereas in other studies, the levansucrase is shown to be stable under acidic conditions.8 Five mutants were created by site-directed mutagenesis, where active site residue Gln275 (corresponding to the His296 in LevU of Z. mobilis) was substituted with amino acids of different polarity and charge to understand the catalytic mechanism of Lsc of S. chungbukense DJ77. To investigate the role of the catalytic configuration of Lsc, we conducted a protein and interface visualization study. Despite remarkable progress in X-ray crystallography for solving protein structures, significant unsolved problems still remain in the prediction of the noncovalent intermolecular interactions between protein and ligand. The recent development of sophisticated docking methodologies allows an accurate prediction of the biological activity of molecules. Three-dimensional (3D) structural prediction and molecular docking analysis (MOE) was performed to determine conformational changes that take place at the catalytic active site of wild-type/mutant Lsc of S. chungbukense DJ77. The geometric configuration of sucrose binding to the active site was determined and scored based on the binding energy. The MOE results provide useful insights on the structure-based interactions at the active site of Lsc of S. chungbukense DJ77.

recombinant E. coli BL21 (DE3) induced the expression of full length lsc genes from all plasmids (pET21c-Lsc and pET21c-LevU). Bacteria were harvested by centrifugation (6,000 rpm, 10 min), washed with 10 mL of 10 mM TrisHCl (pH 8.0), and resuspended in 5 mL of distilled water. After cell disruption using a Vibra cell (Sonics & Materials, Newtown, Connecticut, USA), the cell-free supernatant and insoluble protein aggregates were separated at 13,000 rpm for 10 min. The cellular extracts were subjected to reducing 12% SDS-PAGE, and the soluble supernatant was kept at 4 °C. The purification process was carried out according to the manufacturer’s protocol (pET His-Tag System, Novagen). The supernatant samples were directly loaded on a Chelating Sepharose Fast Flow resin column for nickel affinity chromatography. The collected proteins from affinity chromatography were then loaded onto a Superdex 200 column (HiLoad 10/300 prep grade; GE Healthcare, Stockholm, Sweden). All purification steps were carried out using an Ä kta Purifier System (GE Healthcare) at 4 °C. The purified enzymes were dialyzed and introduced to the activity assay. Levan-Forming Activity. Levan-forming activity of Lsc was evaluated by showing the effect of pH ranging from 4−10. One milliliter of 10% sucrose solution was mixed with enzyme solution (1 μL of purified enzyme at a final concentration of 50 μg μL−1) and incubated at 37 °C for 1 h. Absorbance (optical density) was measured at 575 nm. The levan products were analyzed by high-performance anionic exchange (HPAE) chromatography using a Carbopac PA1 column (4 by 250 mm; Dionex). Detection was performed with an ED40 electrochemical detector (Dionex). The enzymatic products were eluted with 200 mM NaOH at a flow rate of 1 mL/min. Peaks were identified by comparing their retention times with those of standard levan and sucrose (Sigma). 3D Prediction and Molecular Docking Investigations. The X-ray structure of E. amylovora Ea Lsc (PDB ID: 4D47) has 54 and 34% identities with LevU from Z. mobilis and Lsc from S. chungbukense DJ77, respectively. This structure was used as a template to predict the active sites of LevU and Lsc from Z. mobilis and S. chungbukense DJ77, respectively, using the ModWeb server.20 Molecular docking analysis was carried using the MOE docking software tool (version MOE 2016.0802). The docking of levansucrase−sucrose complexes was performed with the MOE function at standard settings (T = 300 K, pH 7.0). The sucrose binding regions in the active site were also predicted from PDB ID 4D47. 3D structures of Lsc and its variants were predicted by PHYRE II analysis system and PyMOL software.

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EXPERIMENTAL PROCEDURE Site-Directed Mutagenesis of lsc. All strains and plasmids used in this study are listed in Table S1. Site-directed mutagenesis was carried out as previously described.19 The oligonucleotides used for mutagenesis are listed in Table S2. PCR experiments were performed using Pfu DNA polymerase (Thermo Scientific, Waltham, MA, USA). The PCR products were treated with DpnI for 2 h to enhance the overall efficiency and reliability. The plasmids containing the mutant genes ligated into pET-21c were screened on LB agar containing 50 μg mL−1 ampicillin after transformation into E. coli DH5α. The mutant genes were verified by sequencing (Solgent, Daejeon, Korea). The resulting plasmids were transformed into E. coli BL21 (DE3) for protein expression. The resultant enzymes in which Glutamine (Gln, Q) 275 was substituted with aspartate (Asp, D), arginine (Arg, R), serine (Ser, S), glutamate (Glu, E), or histidine (His, H) were designated as Q275D, Q275R, Q275S, Q275E, and Q275H, respectively. Preparation of Levansucrase. The levansucrase genes (lsc and levU) were cloned, and a detailed description is given in the Supporting Information. The recombinant E. coli BL21 (DE3) clones were independently grown in 200 mL of LB medium containing 50 μg mL−1 ampicillin until the optical density at 600 nm reached 1.0. Addition of isopropyl β-D-1-thiogalactopyranoside (IPTG, 1 mM) to the growing cultures of

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RESULTS Analysis of Lsc Coding Gene of S. chungbukense DJ77. A DNA sequence similar to a part of the Lsc coding gene (lsc) has been observed in the clone CU896 from the shot gun library by the genome project of S. chungbukense DJ77.15 As a result, a 1.2 kb DNA fragment containing a functional ORF with an initiation codon was obtained, which was amplified by PCR with the genomic DNA as template and cloned in the pBluescriptII SK (−). In addition, a predicted ribosomal binding site (5′-AATGGG-3′) is 13 bp upstream of the ORF and its GC content amounts to 60%. The nucleotide sequence of the full-length lsc is given in Figure S1 and has been submitted to GenBank under Accession No. DQ060376. Amino Acid Sequence Analysis of Lsc. The deduced amino acid sequence of the S. chungbukense DJ77 Lsc was B

DOI: 10.1021/acscombsci.8b00002 ACS Comb. Sci. XXXX, XXX, XXX−XXX

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ACS Combinatorial Science Table 1. Comparison of Amino Acid Homology among Various Bacterial Levansucrases

S. chungbukense DJ77

Sphingobium chungbukense DJ77

Novosphingobium aromaticivorans

Pseudomonas syringae pv phaseolicola

Zymomonas mobilis

Bacillus subtilis

Bacillus amyloliquifaciens

Erwinia amylovora

Geobacillus stearothermophilus

100

55

46

46

37

34

34

22.54

Figure 1. Conserved regions encompassing catalytic amino acids of levansucrases from S. chungbukense DJ77 (Lsc) and Z. mobilis (LevU). Conserved active residues with Z. mobils are colored with black and boxed in regions 1−4. Numbering refers to the processed form of S. chungbukense DJ77. Accession nos. of the enzymes are noted in Table S1.

of the sucrose fructoside bond.12,23 For example, the P. syringae Lsc3 mutant with Ala substitutions at these positions, Asp62Ala, Asp219Ala, and Glu303Ala variants, were practically inactive. When they were substituted with other amino acids (i.e., Glu, His, Gln, and Ser), sucrose hydrolysis was attenuated or abolished.22 Substitution of His296 in Z. mobilis affected the transfructosylation and formation of a highly polymerized saccharide.7 Gln275 in region 4 of S. chungbukense DJ77 Lsc is located within the bacterial fructosyltransferase moiety. This gene product (S. chungbukense DJ77 Lsc) has been used in the present study for the gene expression and enzyme activity analysis. Cloning and Overexpression of lsc Gene from S. chungbukense DJ77. The levansucrase from S. chungbukense DJ77 was amplified as shown in Figure S2. According to the gene data in NCBI (Accession no. DQ060376, http://www. ncbi.nlm.nih.gov/nuccore/DQ060376), P-lsc-F and P-lsc-R were designed to amplify PCR products from pCU896 of S. chungbukense DJ77. The full-length Lsc was cloned into the same pET-21c vector system (Figure S1 and Figure S2(A)). The exact levansucrase genes were successfully cloned into the E. coli expression system, plasmids pET-Lsc, harboring the wildtype lsc gene as shown in Figure S2(B). The recombinant levansucrases were successfully expressed in E. coli BL21 (DE3)

compared with other bacterial levansucrases (Table 1). BLAST analysis for putative Lsc proteins exhibiting sequence identities with Lsc revealed percent identities ranging from 55 to 22.5% with various levansucrases of bacteria. Lsc exhibited good similarity with levansucrases originating from Gram-negative bacteria including P. syringae pv phaseolicola (46%), E. amylovora (34%), and Z. mobilis (46%) and the hypothetical protein 29.2554 from N. aromaticivorans F199 (55%). This protein showed a slightly low amino acid similarity with proteins from Gram-positive bacteria including B. amyloliquefaciens (34%), B. subtilis (37%), and G. stearothermophilus (22.54%). Among those, LevU of Z. mobilis has been extensively mutated, revealing many catalytically functional positions for levan production.7 Three residues, two Asp (D), and one Glu (E), referred to as the “catalytic triad”, are indispensible for sucrose binding and splitting.21,22 Sequence alignment of the respective enzymes Lsc of S. chungbukense DJ77 and LevU of Z. mobilis revealed that the catalytic active site residues were conserved (Figure 1). In LevU of Z. mobilis and its mutational assay results,7 Asp48 (in region 1 corresponding to Asp42 of S. chungbukense DJ77), Asp194 (in region 2 corresponding to Asp196 of S. chungbukense DJ77), and Glu278 (in region 3 corresponding to Glu257 of S. chungbukense DJ77) were conserved and essential for cleavage C

DOI: 10.1021/acscombsci.8b00002 ACS Comb. Sci. XXXX, XXX, XXX−XXX

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ACS Combinatorial Science and homogeneously purified. The transformants were grown on LB medium containing 1 mM IPTG. For the gene product to be verified, protein samples of E. coli (W, whole E. coli; S, soluble fraction; IS, insoluble fraction; P, purified) were analyzed by SDS-PAGE (12% separating gel). The protein expression in E. coli has been detected at the position corresponding to ∼40.6 kDa for Lsc based on the deduced amino acid sequence as shown in Figure S2(C). In fact, several reports have been attempted for recombinant levansucrase expression but shown to be accumulated in an intracellular area as inclusion bodies when using the E. coli expression system.8,24 Levansucrases of S. chungbukense DJ77 were effectively expressed in a soluble form using the pET-21c plasmid under the T7 promoter (Figure S3). Analysis of Levansucrase Catalytic Activity. In general, levansucrase carries out both sucrose hydrolysis as well as transfructosylation reactions with the sucrose molecule as acceptor. The process involves the protonation of the glycosidic oxygen by an acid/base catalyst (Glu257) and the attack on the anomeric carbon of the substrate by the nucleophile (Asp42). The transition state is believed to be stabilized by a transition state stabilizer (Asp196). As illustrated in Figure 2(A), they can cleave the glycosidic bond of their substrate sucrose and form 6-kestose of the basic trisaccharide, levan precursor, including a chain of two fructose molecules and a terminal glucose molecule. The overall reaction involves the subsequent

hydrolysis of a covalent fructosyl−enzyme intermediate (pingpong mechanism).25 Levan consists of β(2−6)-linked fructosyl units in a linear arrangement. The fructosyl−enzyme intermediates can generate a fructan (polysaccharide formation, levan) chain that can consist of up to 100,000 fructose units.26 The dependence of enzyme activity on pH was investigated. Levansucrase can hydrolyze sucrose utilizing a growing oligosaccharide chain, resulting in high molecular weight of levan and free glucose.27 The dependence of enzyme stability on each pH value was determined by measuring the turbidity at 575 nm, reflecting the accumulation of levan polymer in solution.6 Lsc was prepared and tested with the well-known levansucrase, LevU, from Z. mobilis to compare catalytic activity. For the comparative experiment, the structural gene of LevU was cloned and transformed into E. coli using the pET21c expression vector, and protein expression was confirmed (Table S2 and Figure S3). For the optimal pH range for the enzymatic reaction to be determined, 50 μg of each purified enzyme, Lsc and LevU, was mixed with 1 mL of 10% sucrose (w/w) dissolved in pH variant sodium phosphate buffer (10 mM, pH 4−10) and incubated at 37 °C for 1 h. Enzyme activity related to the formation of high molecular weight levan was monitored (Figure 2(B)). LevU displayed maximum activity at pH 5. With the shift of pH from 5 to 8, pH-driven down-shift in activity was observed in agreement with a previous study.28 In contrast, a gradual increase in levan synthesis activity of Lsc of S. chungbukense DJ77 was shown at neutral-alkaline pH range, and a plateau level was reached at pH 7. Levan formation was alternatively visualized at neutral pH (pH 7). Lsc showed whitish color change activity as evident by the formation of levan, whereas LevU was transparent at pH 7. HPAE chromatography allowed the identification of the levan products by the enzymes Lsc and LevU. The resultant chromatogram revealed peaks of levan (3.5 min) and sucrose (6.0 min), respectively (Figure 3). Lsc synthesized the large amounts of levan from sucrose, which was varied significantly depending on the standard substrate used (Figure S4). Prediction of Active Site. S. chungbukense DJ77 Lsc is related to proteins grouped as GH68, which hydrolyze the

Figure 2. (A) Schematic representation of levan polymerization reaction by levansucrase. The catalytic residues Asp42 (nucleophile), Asp196 (transient state stabilizer), and Glu257 (acid base catalyst) and their hydrolytic performance are represented as a red arrow. (B) pH dependence of S. chungbukense DJ77 Lsc and Z. mobilis LevU. Activity was determined the turbidity at OD575. All tests were performed in triplicate. ▲, Lsc; □, LevU; ●, without enzyme (control). Insert: left, S.chungbukense DJ77 Lsc; right, Z. mobilis LevU.

Figure 3. HPAEC profiles of the Lsc (A) and LevU (B) products. D

DOI: 10.1021/acscombsci.8b00002 ACS Comb. Sci. XXXX, XXX, XXX−XXX

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Table 2. Information Regarding the 3D Predicted Model of S. chungbukense DJ77 Lsc and Z. mobilis LevU Based on the X-ray Structure of E. amylovora proteins

template PDB ID

template PDB source

sequence identity with the template

modeled area (aa)

ligand

Lsc (S. chungbukense DJ77) LevU (Z. mobilis)

4D47 4D47

E. amylovora E. amylovora

34% 54%

1−369 1−405

sucrose sucrose

Figure 4. Structural alignment of the predicted 3D structures of S. chungbukense DJ77 Lsc (green) and Z. mobilis LevU (cyan). The active site residues of both proteins are conserved and are displayed as sticks. The His296 residue in Z. mobilis LevU is substituted by Gln275 in S. chungbukense DJ77 Lsc. The close up view shows conserved active site regions 1−4 with sucrose bound to the active site. The sucrose at the active site (yellow) is modeled from PDB ID 4D47.

enlarged image shot. The structure alignment of these two levansucreases (Lsc and LevU) shows that the active sites display extensive overlap with each other (regions 1−4) (Figure 4). Comparative analysis revealed that the His296 in Z. mobilis is substituted by Gln275 in S. chungbukense DJ77. Green color stick mode denotes active site residues (Asp42, Asp196, Glu257, and Gln275) in Lsc, and blue sticks denote active site residues (Asp48, Asp194, Glu278, and His296) in LevU. The glucosyl moiety was determined to be located at region 1 with the fructosyl unit located immediately adjacent to region 4 of the active site. Amino acid sequence alignment revealed two Asp and one Glu residues highly conserved with sucrose hydrolysis.31 Most likely, the acid/base catalyst Glu in region 3 protonates the glycosidic bond of sucrose, and Asp in region 1 acts as a nucleophile. Asp in region 2 is also known as a transition-state stabilizer, the RDP-motif, for substrate binding and stabilization.26 With regard to pH resistance, several strategies have been proposed for the design of pH stability profiles of enzymes, and they concluded that alkaline adaptation can be modulated by an increased number of neutral hydrophilic amino acids such as Asp and Gln.32 Therefore, we assumed that the strong adaptation of the fructose unit of sucrose at Gln275 in region 4 of S. chungbukense DJ77 Lsc seems to be favorable for the fructan chain elongation under neutral-alkaline pH conditions. Catalytic Activity of Levansucrase Variants. How the Gln275 residue interacts with sucrose, with other binding residues belonging to highly conserved sequence motifs, including Asp42, Asp196, and Glu257, was investigated. For investigating the catalytic role of Gln275 in S. chungbukense

glycosidic bond between two or more carbohydrates or between a carbohydrate and a noncarbohydrate moiety.3,10,12 Multiple sequence alignment of GH68 members revealed three conserved residues, two Asp and one Glu, which are referred to as the “catalytic triad”.21 In particular, His296 is essential for polyfructan synthesis in Z. mobilis. Histidine is found at the equivalent position in Gram-negative levansucrase.29 This site also corresponds to Arg360 in B. subtilis levansucrase.26 Among the already known levansucrase enzymes, LevU of Z. mobilis has been extensively reported, and mutational studies at the active region have revealed numerous catalytically functional sites for the levan production.7 Hence, this enzyme was used as a model for the comparison. The already known X-ray structure of E. amylovora levansucrase (PDB ID: 4D47) was used as a template model to generate the three-dimensional structures of LevU and Lsc from Z. mobilis and S. chungbukense DJ77. As shown in Table 2, the X-ray structure of the E. amylovora levansucrase displayed 34 and 54% identities with S. chungbukense DJ77 Lsc and Z. mobilis LevU, respectively. When we used the crystal structure of E. amylovora levansucrase as a template for modeling, the homology score in their amino acid sequences was high enough based on the general requirement for homology modeling, e.g., sequence similarity of at least 25%.30 Structural alignment of S. chungbukense DJ77 with Z. mobilis LevU revealed a conserved structure (0.0 RMSD value, data not shown) in the 262 structurally equivalent residues with a sequence identity of 46%. Structural alignment of S. chungbukense DJ77 Lsc and Z. mobilis LevU revealed the superimposed conserved active site residues, as shown in Figure 4. The sucrose binding to the active site is highlighted in an E

DOI: 10.1021/acscombsci.8b00002 ACS Comb. Sci. XXXX, XXX, XXX−XXX

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ACS Combinatorial Science DJ77, five mutants were constructed in which Gln275 was substituted with the following residues having different polar as well as electrostatic properties: His and Arg are positively charged, Ser is a polar but uncharged residue, and Asp and Glu are polar but negatively charged residues. Furthermore, levan synthesis activities of purified enzymes including wild-type (wt) Lsc (Q275-wt) and Q-point mutation variants (Q275H, Q275R, Q275S, Q275D, and Q275E) were examined (Figure 5 and Table S3). As compared to the enzyme

activity of Lsc Q275-wt at pH 7, the amino acid substitutions resulted in 49.2−89.7% attenuation of the enzymatic activities. The Q275R variant maintained higher activity (89.7%) compared to Lsc Q275-wt than other variants. The Q275E variant showed ∼69% activity with drastic loss of activity under alkaline (pH 8−10) conditions. The Q275S and Q275E variants had high activity (60 and 69.9%, respectively) only at pH 7.0, and their enzyme activity was significantly reduced at alkaline pH (pH 8−10). The Q275H variant resulted in stable accumulation at acidic-neutral pH (56−58%, pH 6.0−7.0). In contrast, variant Q275D exhibited 47.5−49.2% of activity in the pH range of 7.0−8.0. Molecular docking techniques can play an important role in the elucidation of binding site interaction mechanism. The molecular operating environment (MOE) program can predict favorable protein−ligand complex structures with high accuracy and can be used prior to experimental screening to reduce time and better understanding of bioactivity mechanisms. MOE offers various different scoring functions and options as compared with other docking programs and plays an important role in drug discovery. Protein−sucrose docked complexes formed by Lsc Q275-wt and its Q275-variants (Q275H, Q275R, Q275S, Q275D, and Q275E) were modeled by MOE−ligand interaction analysis at the standard setting mode (T = 300 K, pH 7.0) (Figure 6 and Figure S5). The two-dimensional (2D) ligand interaction map obtained by MOE docking for Lsc Q275-wt revealed that sucrose maintained its interactions with active site residues, such as Asp42 and Glu257, but not Asp196. In addition, the Arg77 and Arg195 residues also interact with sucrose (Figure 6(A)). Interestingly, the similar docking pattern was present in most

Figure 5. Effect of pH on Lsc Q275-wt and its Q275-variants (Q275H, Q275R, Q275S, Q275D, and Q275E). The reaction was started by the addition of enzyme solutions and incubated at 37 °C for 1 h with buffered sucrose ranging from pH 4.0 to 10. Absorbance (optical density) was measured at 575 nm (see Table S3). All tests were performed in triplicate.

Figure 6. Molecular docking results of S. chungbukense DJ77 Lsc (A) and Z. mobilis LevU (B) by MOE show binding pockets for sucrose, and the detailed combinations of each binding site are presented using the “ligand interactions” module of MOE, which obtains binding forces, binding distances, and binding atoms between the sucrose and levansucrase. The direct interactions of active region residues with sucrose are highlighted with dotted green lines. An inset caption describing all the detected interactions is included in the picture. F

DOI: 10.1021/acscombsci.8b00002 ACS Comb. Sci. XXXX, XXX, XXX−XXX

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residues (i.e., Asp42, Asp196, and Glu257) in the active pocket were also characterized through ligand-docking MOE. We can conclude that the residue Asp196 does not directly take part in the catalytic mechanism itself, although it has been known to be conserved and is a key residue for levan polymerization. This residue may play a role in maintenance of mechanism as a transition-state stabilizer.39 Residue Gln275, located behind the general acid/base, was also of major interest. The levan polymerization absolutely requires Gln275 in S. chungbukense DJ77 Lsc or His295 at the corresponding position in Z. mobilis LevU. The analysis of protein structure shows that Q275 variants (Q275D, Q275S, and Q275H) might reduce sucrose binding strength and disrupt the relevant intermolecular orientation for fructan polymerization due to the participation of polar amino acid Ser112. However, the crossover mutation (His for Gln in S. chungbukense DJ77 Lsc, Q257H) did not preserve polymerase activity at the low pH of 5.0 and in neutral-alkaline pH ranges (Figure 5). Finally, the observed MOE ligand-binding interactions between S. chungbukense DJ77 Lsc and sucrose indicate that the high levansucrase activity in S. chungbukense DJ77 may be achieved by introducing Gln275 to the substrate as well as inducing the nucleophile-coordinating network for the transfructosylation activity. Ser112 could be assumed to interact with sucrose/water molecules alternatively, and its interaction was responsible for the low catalytic activity. Our study also indicated that Gln275 should be placed near the substrate docking site with a series of active pocket amino acids (i.e., Asp42, Arg77, Arg 195, and Glu257). As demonstrated for the levan synthesis activity over a broad pH range (pH 5−10) in S. chungbukense DJ77 Lsc, it would be more advantageous for applications in the levan industry. For instance, several versatile progressions in bioreactor applications over the past decades have provided hundreds of recombinant proteins in E. coli.40,41 However, the control of culture pH on microbial production cannot be ignored in these large-scale fermentation processes.42 The levansucrase was also engineered for high yield production in bioreactor manipulation environments to this day.11,16,43 In this context, it presumes that the Lsc of S. chungbukense DJ77 expression system in E. coli would offer the advantageous choice in commercial activities and can also be a useful benchmark for comparison among various biomass applications. Therefore, it is expected that S. chungbukense DJ77 Lsc can produce high levels of levan in an easily handled bioreactor operating environment, without strict pH control, using the E. coli system. Further chimerical strategies can then be applied to manipulate levansucrase that is programmatically active to acidic and akaline environments.

Q275 variants. The Q257R, which showed 89.7% attenuation in levan synthesis, 2D interaction map show sucrose in the active site with a set of hydrogen bonds with the active pocket amino acid Asp42, Glu257, Arg275, Arg77, and Arg195 (Figure S5(A)). The Q257D sucrose docking map depicts interactions with Asp42, Glu257, Asp275, Arg77, and Arg195 (Figure S5(B)), whereas Q257E, Q257S, and Q257H, which exhibited 58−70% attenuation in levan synthesis, present different liganddocking patterns as shown in Figure S5(C−E). The mutations Gln257 to Ser257 (Q257S) and His257 (Q257H) do not interact with their substrate in the active pocket considering the indirect consequence of introducing a polar side chain, such as Ser112, adjacent to the binding pocket. In the case of Q257D, their biological enzymatic activity was low (49.2%), although the docking map for Q257D showed the participation of all relevant amino acids in the active pocket. It is probable that the potential hydrogen-bonding interactions with Ser112 do not generate enough binding geometry, causing them to become unstable in the neutral-alkaline pH range. In fact, one of the reported factors contributing to catalytic activity of levansucrase is the impaired positioning of S173 in Bacillus megaterium33 corresponding to S112 and S119 in S. chungbukense DJ77 Lsc and Z. mobilis LevU, respectively. A substitution study at S173 revealed it was responsible for coordinating the essential role of the interaction with Asp (nucleophile). We therefore decided to take a closer look at the active site network of interactions involving positions Asp48, Asp194, Glu278, and His296 in Z. mobilis LevU (Figure 6(B)). The substrate-docking results for Z. mobilis LevU revealed that sucrose maintained its interactions with catalytic triad residues Asp48 and Glu278 but not Asp194. The substrate binding with residues Arg 83, Ser119, and Arg 193 corresponding to Arg77, Ser112, and Arg195 in S. chungbukense DJ77 Lsc was also observed. This result might suggest that the interaction between Ser119 and the fructose unit in the sucrose-bound state not only disrupts the contact with water molecules for hydrolyzing the substrate but also the nucleophile-coordinating network for the transfructosylation activity in the neutral-alkaline pH range33 (see Figure 2(B), open square).

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DISCUSSION Sphingobium chungbukense DJ77, isolated from contaminated sediment of an industrial complex near Daejon, Republic of Korea, is a Gram-negative bacteria strain. We previously reported that it is useful for the synthesis of sphingolipids and for the degradation of poly- and monocyclic aromatic hydrocarbons such as phenanthrene, anthracene, toluene, mxylene, phenol, salicylate, benzoate, and p-cresol.14,15,34 This bacterium has the potential to produce exopolysaccharides (EPS), which can be used as a gelling agent for various pharmaceutical and industrial processes, because some genes encoding functional enzymes (i.e., phosphomannose isomerase and phosphoglucose isomerase) involved in EPS biosynthesis pathways have been isolated through its genomic analysis.35,36 EPS levan, which is synthesized by levansucrase, is a fructantype homopolysaccharide with β-(2,6) glycosidic linkages.37 It is useful in a wide range of applications including food, feed, cosmetics, and medicine due to its uncommon properties, for example, low intrinsic viscosity from other polysaccharides.37,38 A novel Lsc in S. chungbukense DJ77 was sequenced and overexpressed in this study. Multisequence alignments of the respective enzymes Z. mobilis LevU revealed that the highly conserved catalytic triad was observed. Substrate binding

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CONCLUSIONS Novel levansucrase (Lsc) in S. chungbukense DJ77 was sequenced and successfully overexpressed in an E. coli protein expression system. Multisequence alignments of the respective enzymes Z. mobilis LevU revealed that the highly conserved catalytic triad (Asp42, Asp196, and Glu257) was observed. S. chungbukense DJ77 Lsc exhibits catalytic activity over a broad pH range (5−10) in contrast to Z. mobilis LevU, which lacks catalytic activity at pH levels higher than neutral. Substrate binding residues (i.e., Asp42, Arg77, Arg 195, Asp196, Glu257, and Gln275) in the active pocket were also characterized through the ligand-docking MOE and single amino acid substitution analysis. These observed binding interactions G

DOI: 10.1021/acscombsci.8b00002 ACS Comb. Sci. XXXX, XXX, XXX−XXX

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ACS Combinatorial Science

(6) Goldman, D.; Lavid, N.; Schwartz, A.; Shoham, G.; Danino, D.; Shoham, Y. Two active forms of Zymomonas mobilis levansucrase. An ordered microfibril structure of the enzyme promotes levan polymerization. J. Biol. Chem. 2008, 283, 32209−32217. (7) Li, S. Y.; Chen, M.; Li, G.; Yan, Y. L.; Yu, H. Y.; Zhan, Y. H.; Peng, Z. X.; Wang, J.; Lin, M. Amino acid substitutions of His296 alter the catalytic properties of Zymomonas mobilis 10232 levansucrase. Acta Biochim Polym. 2008, 55, 201−206. (8) Sunitha, K.; Chung, B. H.; Jang, K. H.; Song, K. B.; Kim, C. H.; Rhee, S. K. Refolding and purification of Zymomonas mobilis levansucrase produced as inclusion bodies in fed-batch culture of recombinant Escherichia coli. Protein Expression Purif. 2000, 18, 388− 393. (9) Betancourt, L.; Takao, T.; Hernandez, L.; Padron, G.; Shimonishi, Y. Structural characterization of Acetobacter diazotropicus levansucrase by matrix-assisted laser desorption/ionization mass spectrometry: identification of an N-terminal blocking group and a free-thiol cysteine residue. J. Mass Spectrom. 1999, 34, 169−174. (10) Caputi, L.; Cianci, M.; Benini, S. Cloning, expression, purification, crystallization and preliminary X-ray analysis of EaLsc, a levansucrase from Erwinia amylovora. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2013, 69, 570−573. (11) Hettwer, U.; Jaeckel, F. R.; Boch, J.; Meyer, M.; Rudolph, K.; Ullrich, M. S. Cloning, nucleotide sequence, and expression in Escherichia coli of levansucrase genes from the plant pathogens Pseudomonas syringae pv. glycinea and P. syringae pv. phaseolicola. Appl. Environ. Microbiol. 1998, 64, 3180−3187. (12) Yanase, H.; Fujimoto, J.; Maeda, M.; Okamoto, K.; Kita, K.; Tonomura, K. Expression of the extracellular levansucrase and invertase genes from Zymomonas mobilis in Escherichia coli cells. Biosci., Biotechnol., Biochem. 1998, 62, 1802−1805. (13) Sutherland, I. W. Novel and established applications of microbial polysaccharides. Trends Biotechnol. 1998, 16, 41−46. (14) Yeon, S. M.; Kim, Y. C. Complete sequence and organization of the Sphingobium chungbukense DJ77 pSY2 plasmid. J. Microbiol. 2011, 49, 684−688. (15) Kim, S. J.; Chun, J.; Bae, K. S.; Kim, Y. C. Polyphasic assignment of an aromatic-degrading Pseudomonas sp., strain DJ77, in the genus Sphingomonas as Sphingomonas chungbukensis sp. nov. Int. J. Syst. Evol. Microbiol. 2000, 50, 1641−1677. (16) Vigants, A.; Marx, S. P.; Linde, R.; Ore, S.; Bekers, M.; Vina, I.; Hicke, H. G. A novel and simple method for the purification of extracellular levansucrase from Zymomonas mobilis. Curr. Microbiol. 2003, 47, 198−202. (17) Li, H.; Ullrich, M. S. Characterization and mutational analysis of three allelic lsc genes encoding levansucrase in Pseudomonas syringae. J. Bacteriol. 2001, 183, 3282−3292. (18) Song, K. B.; Joo, H. K.; Rhee, S. K. Nucleotide sequence of levansucrase gene (levU) of Zymomonas mobilis ZM1 (ATCC10988). Biochim. Biophys. Acta, Gene Struct. Expression 1993, 1173, 320−324. (19) Liu, H.; Naismith, J. H. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol. 2008, 8, 91. (20) Pieper, U.; Eswar, N.; Stuart, A. C.; Ilyin, V. A.; Sali, A. MODBASE, a database of annotated comparative protein structure models. Nucleic Acids Res. 2002, 30, 255−259. (21) Lammens, W.; Le Roy, K.; Schroeven, L.; Van Laere, A.; Rabijns, A.; Van den Ende, W. Structural insights into glycoside hydrolase family 32 and 68 enzymes: functional implications. J. Exp. Bot. 2009, 60, 727−740. (22) Mardo, K.; Visnapuu, T.; Vija, H.; Elmi, T.; Alamae, T. Mutational analysis of conserved regions harboring catalytic triad residues of the levansucrase protein encoded by the lsc-3 gene (lsc3) of Pseudomonas syringae pv. tomato DC3000. Biotechnol. Appl. Biochem. 2014, 61, 11−22. (23) Li, S.; Yan, Y.; Zhou, Z.; Yu, H.; Zhan, Y.; Zhang, W.; Chen, M.; Lu, W.; Ping, S.; Lin, M. Single amino acid residue changes in subsite −1 of levansucrase from Zymomonas mobilis 10232 strongly influence

between S. chungbukense DJ77 Lsc and sucrose might explain the structural role and promote the pH stability of levansucrase. Gln275 might coordinate a more favorable conformation for catalysis against a neutral-basic pH environment.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscombsci.8b00002. General experimental section with characterization of the lsc gene from S. chungbukense DJ77, nucleotide and predicted amino acid sequences, genetic construction of lsc genes, SDS-PAGE analyses, HPAEC profiles, molecular docking results, bacterial strains, plasmids, and oligonucleotides used in this study, and turbidity study of levansucrase activity (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Phone: +82-43-261-3575, fax: +82-43-264-9600, e-mail: kyh@ chungbuk.ac.kr. *Phone: +82-43-261-2301, fax: +82-43-264-9600, e-mail: [email protected]. ORCID

Ji-Young Ahn: 0000-0002-3729-2284 Yang-Hoon Kim: 0000-0002-3406-4868 Author Contributions #

S.Y.L., W.-R.S., and S.S.S. contributed equally to this work.

Author Contributions

Conceived and designed the experiments: J.-Y.A. and Y.-H.K. Performed the experiments: S.Y.L. and J.P.L. Analyzed the protein prediction data: W.-R.S., J.P.L., Y.-C.K., S.S.S., and Y.H.K. Wrote the manuscript: S.Y.L., J.Y.A., and Y.-H.K. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The present work was funded by “Cooperative Research Program for Agriculture Science & Technology Development (PJ01191701)”, Rural Development Administration, Republic Korea.

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DOI: 10.1021/acscombsci.8b00002 ACS Comb. Sci. XXXX, XXX, XXX−XXX