Turkish Journal of Biology Turk J Biol (2016) 40: 623-633 http://journals.tubitak.gov.tr/biology/ © TÜBİTAK Research Article doi:10.3906/biy-1501-4

Modeling of PrnD from Pseudomonas fluorescens RajNB11 and its comparative structural analysis with PrnD expressed in Burkholderia and Serratia

1 2 3 1, Ram Nageena SINGH , Raghvendra Pratap SINGH , Anjana SHARMA , Anil Kumar SAXENA * 1 Division of Microbiology, Indian Agricultural Research Institute, Pusa, New Delhi, India 2 Microbial Genomics Resource Repository, National Bureau of Agriculturally Important Microorganisms, Kushmaur, Maunath Bhanjan, India 3 Department of Post Graduate Studies and Research in Biological Science, Rani Durgavati Vishwavidyalaya, Saraswati Vihar, Pachpedi, Jabalpur, India

Received: 02.01.2015 Accepted/Published Online: 17.08.2015 Final Version: 18.05.2016

Abstract: Pyrrolnitrin is produced by Pseudomonas fluorescens and plays an important role in control of pathogenic fungi. The prnABCD cluster codes for enzymes involved in biosynthesis of pyrrolnitrin. Among the four , prnD is very important as it codes for arylamine N-oxygenase, the only biochemically characterized oxygenase involved in oxidation of the amino group of aminopyrrolnitrin to a nitro group to form pyrrolnitrin. A strain of Pseudomonas fluorescens RajNB11 efficient for antifungal activity was found to produce pyrrolnitrin. The prnD gene product of P. fluorescens RajNB11 that catalyzes the final step of production of pyrrolnitrin was characterized in the present study with regards to its stability and catalytic activity. The prnD gene was amplified from RajNB11, sequenced, translated, and 3-D modeled to its protein structure. The modeled protein of the test organism was compared to PrnD proteins of Burkholderia sp. and Serratia sp. The protein from P. fluorescens was found to be basic, hydrophilic with higher thermal stability, and easily separated after expression and purification. The protein of P. fluorescens was found to have a better folded structure, more serine residues in the active site, and better active site properties and isoelectric point value in comparison to PrnD proteins of Burkholderia and Serratia.

Key word: Pseudomonas fluorescens, pyrrolnitrin, cloning, protein modeling

1. Introduction from tryptophan at four different steps, which involves The members of the genus Pseudomonas are significant conversion of L-tryptophan to 7-chlorotryptophan due to their widespread distribution in soils, ability to followed by formation of monodechloroaminopyrrolnitrin colonize the rhizosphere of host plants, and capacity to at the second step, processed for chlorination to form produce a large number of compounds antagonistic to aminopyrrolnitrin at the third step and finally converted various plant pathogens (Haas et al., 1991; Thomashow to pyrrolnitrin (Hammer et al., 1997). One of the 4 genes, et al., 1997). Several strains of fluorescent Pseudomonas prnD, has a nucleotide length range between 1098 and have been successfully demonstrated to control Fusarium 1140 bp and codes for a protein that shows homology to wilts, take-all disease of wheat, and tobacco root rot 3-chlorobenzoate-3,4-dioxygenase (cbaA) of Alcaligenes (Weller et al., 2002; Lemanceau et al., 2006). The sp., phthalate 4,5-dioxygenase (pht3) of Pseudomonas production of antifungal secondary metabolites such putida, and vanillate demethylase (vanA) of Pseudomonas as 2,4-diacetylphloroglucinol, pyoluteorin, phenazines, sp. (Hammer et al., 1997; de Souza and Raaijmakers, 2003). pyrrolnitrin (PRN), or hydrogen cyanide is a prominent The product of the prnD gene catalyzes the oxidation of feature of many biocontrol fluorescentPseudomonas spp. the amino group of aminopyrrolnitrin to a nitro group (Dowling and O’Gara, 1994; Thomashow and Weller, 1996; to form pyrrolnitrin (Kirner et al., 1998; Hammer et al., Raaijmakers and Weller, 1998). Pyrrolnitrin, a secondary 1999; Lee et al., 2005, 2006) in the last step of the PRN metabolite with strong antifungal activity, was first isolated biosynthetic pathway. The PrnD protein has a very specific and described from Pseudomonas pyrrocinia (Arima and important function in the oxidation of amino group, et al., 1964, 1965). The prnABCD gene cluster codes for whereas similar structural proteins perform oxidation four enzymes involved in the biosynthesis of pyrrolnitrin in different synthetic pathways (Mason and Cammack, * Correspondence: [email protected] 623 SINGH et al. / Turk J Biol

1992). This highly active metabolite (pyrrolnitrin) has pH (5’-AAGGAGGTGATCCAGCCGCA-3’) (Edwards et been used as a clinical antifungal agent for the treatment al., 1989). The PCR mixture was the same as that described of skin mycoses (Umio et al., 1986; Tawara et al., 1989). A by Singh et al. (2013). The amplified product was resolved phenylpyrrole derivative of PRN has also been developed, in 2.0% agarose gel and a digitalized image was obtained which is used as an agricultural fungicide (Nevill et al., as described above. 1988; Gehmann et al., 1990). In this study, we have analyzed 2.2. Sequencing of pyrrolnitrin D (prnD) and 16S rRNA the structural model of the PrnD protein of Pseudomonas gene fluorescens RajNB11 and compared it with PrnD protein Amplified fragments of theprn D gene were cloned in models of two other genera in order to find out the relation T/A vector pCR2.1 (Invitrogen, USA) and transformed and percent similarity of protein with regards to structure into E. coli strain DH5-α. Transformants were screened and composition in 3 different bacterial genera as they are on Luria Bertani agar medium amended with kanamycin specific for production of pyrrolnitrin. (75 µg/mL) and X-Gal (20 µg/mL). Plasmid DNA was extracted by the Plasmid Mini Kit (QIAGEN, the 2. Materials and methods Netherlands) and used for amplification. T7 and SP6 2.1. Bacterial strain, DNA extraction, and amplification promoter primers (5’-GCTAGTTATTGCTCAGCGG- of prnD and 16S rRNA gene 3’/5’-ATTTAGGTGACACTATAGAA-3’) were used for 2.1.1. Bacterial strain and genomic DNA extraction amplification of the cloned prnD gene and pA/pH primers A bacterial strain was isolated from a sediment sample for the 16S rRNA gene for sequencing. Cycle sequencing collected from Bramhkund (pH 5.8), Rajgir thermal was performed using the Big Dye v3.0 chain termination springs, India, through a serial dilution plating technique cycle sequencing kit following the manufacturer’s protocol using King’s B medium following incubation at 37 °C. A (ABI, USA). In brief, amplification of gene prnD and 16S purified single colony was used for Gram staining test and rRNA was carried out using T7/SP6 and pA/pH primers, observed under a microscope. The culture was streaked on respectively. The sequencing master mix was used for the King’s B medium plates and observed under UV light for amplification reaction in a thermal cycler with conditions production of fluorescent pigments. A single colony of the of initial denaturation of 96 °C for 30 s followed by 25 bacterial strain was inoculated in Luria broth (Sambrook cycles of denaturation at 96 °C for 10 s, annealing at 50 °C et al., 1989) and incubated overnight at 37 °C for growth. for 5 s, and extension at 60 °C for 4 min. While performing Culture broth was harvested for genomic DNA extraction amplification of the 16S rRNA gene, annealing was done at by the method described by Raaijmakers et al. (1997). 53 °C for 5 s. Amplification was followed by purification of Genomic DNA was quantified and assayed on 0.8% cycle-sequenced PCR products with the ethanol/sodium agarose gel (Singh et al., 2013). acetate (3 M, pH 5.2) precipitation method. The purified 2.1.2. Amplification of pyrrolnitrin D (prnD) gene product was vacuum-dried and dissolved in 10 µL of HiDi A set of primers, PRND1 formamide (ABI), vortexed smoothly, and denatured (5’-GGGGCGGGCCGTGGTGATGGA-3’) and PRND2 at 95 °C for 2 min followed by snap-chilling on ice. The (5’- YCCCGCSGCCTGYCTGGTCTG-3’) (de Souza and denatured sample was loaded on an automated sequencer Raaijmakers, 2003), were used to amplify the pyrrolnitrin (3130XL Genetic Analyzer, ABI) for sequencing. The prnD D (prnD) gene from Pseudomonas fluorescens strain gene and 16S rRNA gene were sequenced from both sides RajNB11. The PCR cocktail contained 100 ng of DNA and assembled to get the full sequences. Gene sequences template, 1X Taq buffer, 0.2 mM each of dNTPs, 20 were identified through similarity search against the database through BLASTn and BLASTx (Table 1) for the pmol of each primer, 1.5 mM MgCl2, and 2 U of Taq DNA polymerase (Promega). The PCR was performed 16S rRNA and prnD genes, respectively. Gene sequences in a thermocycler (GeneAmp PCR System 9700, Applied of prnD (1101 bp) and the 16S rRNA gene were deposited Biosystems Inc., USA) using the following conditions: in the NCBI database and accession numbers (KC847080 initial denaturation of 5 min at 94 °C, followed by 40 cycles and KM679401) were obtained. consisting of 30 s at 94 °C (denaturation), 45 s at 62 °C 2.3. Protein modeling (annealing), and 1 min at 72 °C (extension) with a final Protein models were prepared from amino acid sequences extension of 5 min at 72 °C. The amplified product was of prnD expressed by Pseudomonas fluorescens RajNB11, resolved in 2.0% agarose gel and stained with ethidium Burkholderia sp., and Serratia sp. Three protein sequences bromide (1 µg/ml), and the gel image was digitalized in were modeled; the first sequence was from the lab organism Alpha-Imager. (P. fluorescens RajNB11) and the other two were retrieved 2.1.3. Amplification of 16S rRNA gene from the NCBI database (http://www.ncbi.nlm.nih.gov/ Amplification of the 16S rRNA gene region was done by ). The gene sequences fromprn D of Burkholderia primers pA (5’-AGAGTTTGATCCTGGCTCAG-3’) and sp. and Serratia sp. were used to find the related protein

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Table 1. BLASTx results for the prnD gene from Pseudomonas fluorescens strain RajNB11.

S. no. Sequence description Length Score Coverage Identity E value Accession

Aminopyrrolnitrin oxidase 1 365 aa 769 99% 100% 0.0 AGN519491.1 (P. fluorescens) 2Fe-2S ferredoxin 2 363 aa 765 98% 100% 0.0 WP_011061884.1 (P. protegens) 2Fe-2S ferredoxin 3 363 aa 761 98% 99% 0.0 WP_041118840.1 (P. protegens) 2Fe-2S ferredoxin 4 363 aa 760 98% 99% 0.0 WP_019094365.1 (P. fluorescens) 2Fe-2S ferredoxin 5 363 aa 721 98% 93% 0.0 ERO55431.1 (Pseudomonas sp.) Aminopyrrolnitrin oxidase PrnD 6 363 aa 720 98% 93% 0.0 AFI26234.1 (P. fluorescens) structure to be used as a template by the BLAST (Basic ucla.edu/Verify_3D/) (Eisenberg et al., 1997), 3-D profile Local Search Alignment Tool) program (http://blast.ncbi. analysis (Suyama et al., 1997), and Errat (MacArthur et nlm.nih.gov/Blast.cgi) against the al., 1994; Gundampati et al., 2012) to check the quality of (PDB) database. the overall fold/structure, errors over localized regions, 2.3.1. Primary and secondary structure analysis and stereochemical parameters such as bond lengths and Analysis of the primary structure of PrnD proteins was angles. Structural validations of target protein models performed on the ProtParam server (http://us.expasy.org/ were done by PROCHECK (Laskowski et al., 1993), which tools/protparam.html) (Gasteiger et al., 2005) for the study determines stereochemical aspects along with main chain and side chain parameters with a comprehensive analysis. of physicochemical properties like theoretical isoelectric Distribution of amino acid residues of PrnD proteins in point (pI), molecular weight, molecular formula, total allowed, favored, and disallowed regions was described number of positive and negative residues, instability index by Ramachandran plot analysis (Morris et al., 1992) (II) (Guruprasad et al., 1991), extinction coefficient (Gill performed by the PROCHECK server (Laskowski et al., and Hippel, 1989), aliphatic index (AI) (Ikai, 1980), and 1993). Functional sites of proteins based on structural grand average of hydropathy (GRAVY) (Kyte and Doolittle, conformations were predicted by the Q-site finder (Laurie 1982). The sulfide bond pattern (S-S) and linkages were and Jackson, 2005). Visualization of proteins and a protein predicted by the CYS_REC (http://linux1.softberry.com/ contact map of target proteins were made with the Accelrys berry.phtml/topic) tool. PSIPRED v3.3 (http://bioinf. Discovery Studio Visualizer v2.5 (Accelrys Software Inc., cs.ucl.ac.uk/psipred/) (Buchan et al., 2013) was employed USA). for the enumeration of the secondary structural features (Jones, 1999) of PrnD protein sequences. PSI-BLAST 3. Results and discussion (Position Specific Iterated-Basic Local Search Alignment 3.1 Bacterial strain, genomic DNA, PCR, and sequencing Tool) (Altschul et al., 1997) of analyzed protein sequences analysis was performed against the database to get the position- The bacterial isolate RajNB11 showed medium size, with specific scoring matrix. The output results of PSI-BLAST flat and off-white colored colonies with smooth margins (Altschul et al., 1997) were used as input data to perform on King’s B growth medium. It showed production of a analysis using PSIPRED v3.3 (Buchan et al., 2013). greenish yellow (Figure 1a) pigment that fluoresced under 2.3.2. Homology modeling and identification of UV light (Figure 1b). The isolate was found to be gram- functional site negative and rod-shaped (Figure 2). PCR amplification The 3-D models of PrnD protein sequences of using specific primers yielded a product of 1.5 kb for the Pseudomonas fluorescens RajNB11, Burkholderia sp., and 16S rRNA gene and about 1.0 kb for the prnD gene. The Serratia sp. were generated by the I-TASSER web server sequencing of the 16S rRNA gene from RajNB11 followed (Wu, 2007; Zhang, 2008; Roy et al., 2010; Yang et al., by BLASTn search showed similarity index of >97% with 2013) and evaluated by VERIFY 3D (http://services.mbi. Pseudomonas fluorescens in the RDP database (Cole et al.,

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Figure 1. (a) Bacterial isolate showed pigment production on King’s B medium; (b) fluorescence test under ultraviolet light.

2009). The BLASTx analysis of the sequenced prnD gene presence of high concentrations of tryptophan, tyrosine, showed its similarity with the putative aminopyrrolnitrin and cystine residues, as these three amino acids are protein (PrnD) (Table 1). important for determination of the extinction coefficient 3.2. Protein structure analysis (Edelhoch, 1967; Pace et al., 1995). Cystine content is The primary structures of representative protein sequences identical to the number of disulfide bonds in the protein of PrnD of Pseudomonas fluorescens RajNB11, Burkholderia as it is formed when two cysteine residues are ligated with sp., and Serratia sp. were speculated and compared (Table a disulfide bond. This information could be helpful in the 2). The computed isoelectric point (pI) was 8.77 for P. chemical and physical stability of proteins (Trivedi et al., fluorescens, 7.67 for Burkholderia sp., and 6.39 for Serratia 2009) as well as the quantitative study of protein–protein sp., indicating the basic nature of the target protein. This and protein–ligand interactions in solution. The sulfide was further supported by the ratio of negative and positive (S-S) bonding pattern showed that the PrnD proteins of residues of the proteins (Table 2). The estimated protein Burkholderia sp. and Serratia sp. have 2 cysteine residues at extinction coefficient (Gill and Hippel, 1989) of the PrnD positions 145 and 174, while P. fluorescens RajNB11 has 3 protein of Burkholderia sp. was highest among the three cysteine residues at positions 3, 146, and 175. These results (Table 2), indicating that it can absorb more light than showed that the PrnD protein of P. fluorescens RajNB11 the other two at a given wavelength. It also indicates the has higher enthalpy and thermodynamic stability in the folded state (Dill, 1990; Tiedge et al., 2000) as compared to Burkholderia and Serratia. It is known that the disulfide bond increases the entropy of proteins in the folded state by stabilizing local interaction (Wedemeyer, 2000). The folded protein structure is a biologically functional molecule, which is resistant to the next rearrangement and has higher stability (Walker and Gilbert, 1997). The thermal stability of the PrnD protein was also determined by II (Table 2), which was found to be 34.32, 27.19, and 34.13 for P. fluorescens RajNB11, Burkholderia sp., and Serratia sp., respectively. All the three PrnD proteins are thermostable, as values of II smaller than 40 make them stable while those above 40 lead to instability (Guruprasad et al., 1991). The calculated AI of the proteins were 79.53 (P. fluorescens RajNB11), 76.70 (Burkholderia sp.), and 77.66 (Serratia sp.) (Table 2). These values reflect the higher relative volume occupied by aliphatic side Figure 2. Gram staining showing rod-shaped cells of P. fluorescens chains (alanine, valine, isoleucine, and leucine) (Gasteiger strain RajNB11 under a microscope.

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Table 2. Details of primary structural properties of PrnD proteins.

Organism Acc. number Molecular formula M.W. pI. –R +R EC II AI GRAVY

P. fluorescens RajNB11 KC847080 C1842H2809N515O506S17 40.80 8.77 35 40 78840 34.32 79.53 –0.245

Burkholderia sp. ZP04891205 C1864H2845N527O522S18 41.57 7.67 42 43 77350 27.19 76.70 –0.285

Serratia sp. WP_013814510 C1844H2805N511O522S19 41.09 6.39 42 38 82850 34.13 77.66 –0.728 et al., 2005) and showed that the proteins could be stable estimating the quality of predicted models), TM-score at a wide range of temperatures. This is because higher AI (template modeling score: topological similarity between values indicate increased thermostability while lower AI two protein structures) (Zhang and Skolnick, 2004), and values indicate increased flexibility in the protein structure root mean square deviation (RMSD) values for better (Singh et al., 2011). GRAVY values of the PrnD proteins alignment with the template (Table 4). Results showed (Table 2) were found negative (–0.245, –0.285, and –0.264 that the protein model of Burkholderia sp. is closer to its respectively for the three strains), indicating the hydrophilic template than those of P. fluorescens RajNB11 and Serratia, nature of the proteins. Hydropathy (hydrophobicity vs. as a TM-score closer to 1.0 and RMSD value closer to 0.0 hydrophilicity or lipophobicity vs. lipophilicity) is usually describe closeness between the models. A TM-score of expressed by numbers (hydrophobic moments) ranging >0.5 indicates that both the proteins are in the same fold from –7.5 (arginine) to 3.1 (isoleucine). More positive (Xu and Zhang, 2010). The positive values of the C-scores values indicate that the majority of the amino acid residues of all three models showed that they are significantly will tend to push out of aqueous environment. In contrast, closer to the template used. This also showed that the negative values indicate hydrophilic side chains having protein model of Burkholderia is closer to its template. The greater affinity for water (Eisenberg et al., 1984). developed 3-D model (Figure 4) of the PrnD protein of P. The secondary structures of the PrnD proteins fluorescensRajNB11, Burkholderia sp., and Serratia sp. were (Figure 3) of target genera were predicted and analyzed deposited in the PMDB database (PM0078950). Predicted by the PSIPRED v3.3 server (Buchan et al., 2013) and are models (Figure 4) were further analyzed with the Accelrys shown in Table 3. Currently, the algorithms mostly used Discovery Studio Visualizer v.2.5. The generated structural for protein secondary structure prediction are based on forecast reports of the PrnD protein models of all three machine learning techniques in which PSIPRED v3.3 organisms contained different bonding patterns and the has been shown to be capable of achieving an average details are given in Table 5. The predicted central atom Q3 score of 81.6%, which is a maximum score for any of the PrnD protein is Fe, a positively charged metal that method published to date (Buchan et al., 2013). α-Helices partakes in various properties of protein like stabilization are the most common secondary conformation in natural of the protein structure, ligand bonding, electron proteins and about 40% of amino acids adopt helical transport, and substrate/coenzyme activation (Lee et al., conformations (Almeida et al., 2012). Our results showed 2005). The generated contact maps of the PrnD protein that the numbers of helices (Table 3) that are dominant in of the three genera explain the reduced representation of the predicted structures (Figure 3) support the stability of the target structure that helps in the accurate prediction all three studied proteins, by both enthalpic and entropic of the protein structure from sequence data (Glasgow et contributions (Qin and Buehler, 2010). The tertiary al., 2006). The quality of predicted structures (Figure 3) of structure prediction was performed by the I-TASSER the PrnD protein were further assessed and confirmed by server (Wu, 2007; Zhang, 2008; Roy et al., 2010; Yang et al., VERIFY 3D (Eisenberg et al., 1997), 3-D profile (Suyama 2013) using the best aligned template (2zylA, 3gkeA: http:// et al., 1997), and Errat (Gundampati et al., 2012). The www.rcsb.org/pdb/explore/explore.do/structureId=3gke analysis report of VERIFY 3D (Table 4) showed that and 3gobA: http://www.rcsb.org/pdb/explore/explore.do/ 90.36% of the residues of the protein model of P. fluorescens structureId=3gob). The templates were selected to analyze RajNB11 had average 3D-1D scores above 0.2, whereas the 3-D structure with a higher sequence similarity that models of Burkholderia and Serratia had only 85.14% and would guarantee a more accurate alignment between 71.98% residues. A protein model needs at least 80% of the target sequence and template structure (Sekhar et the amino acids with a score of >0.2 in the 3D-1D profile al., 2006). Out of five generated similar models of the to pass the analysis (Eisenberg et al., 1997). According target sequence, the best one was chosen after employing to the analysis by Errat (Table 4), the model structure different criteria like the C-score (a confidence score for of P. fluorescens RajNB11 has an overall quality factor of

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Figure 3. Secondary structural analysis of all three proteins from the prnD gene.

40.653, Burkholderia of 21.823, and Serratia sp. of 21.910 process using Ramachandran map calculations with (McArthur et al., 1994). the PROCHECK program (Laskowski et al., 1993). The 3.3. Ramachandran plot analysis Ramachandran plot (Figure 5) (Ramachandran et al., 1963) The stereochemical quality and accuracy of the predicted obtained showed a tight clustering of phi~ –50 and psi~ –50. models of PrnD were evaluated after the refinement In the plot analysis, the residues were classified according

Table 3. Comparative details of secondary structure of proteins of P. fluorescens RajNB11, Burkholderia sp., and Serratia sp.

S. no. Organism name Alpha helix Strand Random coil 1. Pseudomonas fluorescens RajNB11 222 97 44 2. Burkholderia sp. 225 101 44 3. Serratia sp. 222 98 44

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Table 4. Analysis details of tertiary structures of proteins of P. fluorescens RajNB11, Burkholderia sp., and Serratia sp.

No. of Cluster Organism name Template C-score Exp.TM-score Exp. RMSD Verify 3D Errat decoys density P. fluorescens RajNB11 2zylA –0.55 0.64 ± 0.13 7.8 ± 4.4 2098 0.1561 90.36% 40.56 Burkholderia sp. 3gkeA 0.25 0.75 ± 0.11 6.1 ± 3.8 2093 0.3420 85.14% 21.82 Serratia sp. 3gobA –0.53 0.65 ± 0.13 7.8 ± 4.4 –2076 –0.1569 71.98% 21.91

Figure 4. The 3-D models of PrnD proteins of P. fluorescens RajNB11, Burkholderia sp., and Serratia sp.

Table 5. Type of bonds and their distribution in protein structures of P. fluorescens RajNB11, Burkholderia sp., and Serratia sp.

Partial Aromatic Organism name Single bond Double bond Sp2 Sp3 double bond bond P. fluorescens RajNB11 2204 458 118 186 1715 1165 Burkholderia sp. 2234 460 142 180 1731 1165 Serratia sp. 2205 588 132 186 1731 1165 to regions in the quadrangle. The red regions in the graph (protein–ligand binding sites) in the modeled PrnD (Figure 5) point out the most allowed regions, whereas the proteins. This can be beneficial for molecular docking, yellow regions represent allowed regions. Glycine is shown de novo drug design, and structural identification and by triangles and other residues are represented by squares. comparison of functional sites prospectively for the The analysis report of the Ramachandran plot concluded further study of PrnD proteins. Analysis resulted in 11, that phi and psi angles contributed in the conformation 13, and 14 putative functional site residues with significant of amino acids (Table 6) with 76.8%, 83.8%, and 84.4% matches in the modeled proteins of P. fluorescensRajNB11, residues in most favored regions; 19.0%, 12.7%, and 12.1% Burkholderia sp., and Serratia sp., respectively. The (16 amino acids) in the additional allowed region; 3.2%, comparison of amino acid contents of PrnD proteins of 2.9%, and 2.2% in the generously allowed region; and P. fluorescens, Burkholderia sp., and Serratia sp. are given 1.0%, 0.6%, and 1.3% residues in the disallowed region in in Table 6. In comparison to the other two, P. fluorescens P. fluorescens RajNB11, Burkholderia sp., and Serratia sp. RajNB11 protein reflected fewer acidic atoms. The respectively (Figure 5), indicating good quality plots (Filiz information on protein group atoms could be important and Tombuloğlu, 2015), excluding glycine and proline. for further studies on protein interactions and/or activity 3.4. Functional site analysis of PrnD. Among the predicted 3-D structures (Figure 4) of The Q-Site Finder server (Laurie and Jackson, 2005) was the targeted three strains, Pseudomonas showed, based on employed for the prediction of functional site residues TM-score, RMSD value, VERIFY 3D analysis, and Errat

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Ramachandran Plot Ramachandran Plot

Pseudomonas fluorescens RajN B11 MET 33 (A) Burkholderia sp. 180 180 B PHE ~2b34 (A) B ~b b b

TYARS1N92(4A7) (A) VAL 302 (A) ILE 226 (A) MET 239 (A) 135 b ~b 135 b ~b

~l ~l

90 90 l l PRO 259 (A)

PHE 255 (A) MET 264 (A) 45 L 45 L

a a ees ) ees ) A A VAL 29192 (A) 0 ARG 232 (A) 0 deg r deg r (

( SER 362 (A) ~a ~a i

i SER 108 (A)

s s P P HIS 191 (A) SER 249 (A) –45 –45

–90 LYS 343 (A) –90

ARG 39 (A) PRO 259 (A) ~b ~p ~b ASP 10 (A) ARG 131 ~(Ap ) –135 –135 PHE 203 (A) CYS 262 (A) b p b PHpE 48 (A) GLN 8 (A) ~b ~b

–180 –135 –90 –45 0 45 90 135 180 –180 –135 –90 –45 0 45 90 135 180 Phi (degrees) Phi (degrees)

Plot statistics Plot statistics

Residues in most favoured regions [A,B,L] 238 76.8% Residues in most favoured regions [A,B,L] 264 83.8% Residues in additional allowed regions [a,b,l,p] 59 19.0% Residues in additional allowed regions [a,b,l,p] 40 12.7% Residues in generously allowed regions [~a,~b,~l,~p] 10 3.2% Residues in generously allowed regions [~a,~b,~l,~p] 9 2.9% Residues in disallowed regions 3 1.0% Residues in disallowed regions 2 0.6% –––– –––––– –––– –––––– Number of non–glycine and non–proline residues 310 100.0% Number of non–glycine and non–proline residues 315 100.0% Number of end–residues (excl. Gly and Pro) 2 Number of end–residues (excl. Gly and Pro) 2 Number of glycine residues (shown as triangles) 31 Number of glycine residues (shown as triangles) 33 Number of proline residues 20 Number of proline residues 20 –––– –––– Total number of residues 363 Total number of residues 370

Based on an analysis of 118 structures of resolution of at least 2.0 Angstroms Based on an analysis of 118 structures of resolution of at least 2.0 Angstroms and R–factor no greater than 20%, a good quality model would be expected to and R–factor no greater than 20%, a good quality model would be expected to have over 90% in the most favoured regions. have over 90% in the most favoured regions.

Ramachandran Plot

Serratia sp. 180 B ~b b PRO 260 (A)

TRP 222 (A) 135 b ~b PHE 235 (A) TYR 234 (A) LEU~l73 (A) VAL 300 (A) 90 l

45 L

a ees ) A 0 deg r ALA 19 (A) ( ~a ASN 109 (A) i s P –45 ASP 270 (A)

–90

THR 266 (A) ~b ARG 132~(pA) –135 ALA 219 (A) b p ~b

–180 –135 –90 –45 0 45 90 135 180 Phi (degrees)

Plot statistics

Residues in most favoured regions [A,B,L] 265 84.4% Residues in additional allowed regions [a,b,l,p] 38 12.1% Residues in generously allowed regions [~a,~b,~l,~p] 7 2.2% Residues in disallowed regions 4 1.3% –––– –––––– Number of non–glycine and non–proline residues 314 100.0% Number of end–residues (excl. Gly and Pro) 2 Number of glycine residues (shown as triangles) 30 Number of proline residues 18 –––– Total number of residues 364

Based on an analysis of 118 structures of resolution of at least 2.0 Angstroms and R–factor no greater than 20%, a good quality model would be expected to have over 90% in the most favoured regions.

Figure 5. Ramachandran plot analysis of PrnD protein models of P. fluorescens RajNB11, Burkholderia sp., and Serratia sp. analysis, a comparatively superior structural model. The 3.5. Comparative analysis of protein models overall 3-D structure of PrnD proteins is well conserved The 3-D structure of the PrnD protein of the 3 different among the bacteria of the studied genera. genera was predicted, superimposed (Figure 6), and

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Table 6. Comparison of amino acid content of PrnD protein of P. fluorescens RajNB11, Burkholderia sp., and Serratia sp.

Organism name Backbone Side chain Hydrophobic Hydrophilic Acidic Basic Disulfide P. fluorescens RajNB11 1453 1427 140 116 35 55 2 Burkholderia sp. 1481 1450 141 119 42 56 2 Serratia sp. 1457 1439 139 119 42 52 2

Figure 6. Superimposition study of 3-D models: A (model of P. fluorescens RajNB11 with Burkholderia sp.), B (model of P. fluorescens RajNB11 with Serratia sp.), and C (model of Burkholderia sp. and Serratia sp.). compared. The comparative protein structure analysis have similarity in their structural attributes (Tables 2 and of these genera is still incomplete. The physicochemical 3) with respect to TM-score and RMSD value (Table 4). results showed that molecular weights for the PrnD On the basis of the TM-score and RMSD value (Table 7), proteins expressed by the 3 bacterial genera are almost it could be proposed that protein models of P. fluorescens identical (Table 2). However, Pseudomonas fluorescens RajNB11 and Burkholderia sp. are closer to each other RajNB11 showed a significantly higher pI value of 8.77 and have similar folded states as compared to that of as compared to Burkholderia sp. (7.67) and Serratia sp. Serratia sp. Active site prediction analysis showed that (6.37), which would play a key role during the purification the P. fluorescens RajNB11 PrnD protein is catalytically from the protein mixture. P. fluorescensRajNB11 protein more efficient than the other two proteins as it has 13 exhibited more sulfur bonding sites in comparison to the serine residues at its active sites, while Burkholderia other 2 protein molecules, which provide extra stability and Serratia have 9 and 10 serine residues. Based on all to the protein in in vitro expression. The II suggests that the above analysis, it could be concluded that the PrnD although all three proteins are stable, the protein of P. protein expressed by P. fluorescens RajNB11 is more stable fluorescens RajNB11 is comparatively more stable than (higher II, higher AI, higher GRAVY) and functionally the other two. The 3-D model ofBurkholderia protein more efficient, and it could be more easily separated from had a higher C-score; however, all three proteins studied a mixture than the other two compared proteins.

Table 7. Comparative and superimposition analysis of modeled 3D protein structures from P. fluorescens RajNB11, Burkholderia sp., and Serratia sp.

Organism name, Organism name, TM-score RMSD value MaxSub- score GDT-TS score model 1 model 2 P. fluorescens Burkholderia sp. 0.7419 6.784 0.4901 0.5331 RajNB11 P. fluorescens Serratia sp. 0.6454 7.020 0.1864 0.3558 RajNB11

Burkholderia sp. Serratia sp. 0.7296 8.135 0.2104 0.4595

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References

Altschul SF, Madden TL, Schaffer A, Zhang J, Zhang Z, Miller W, Gundampati RK, Chikati R, Kumari M, Sharma A, Pratyush DD, Lipman DJ (1997). Gapped BLAST and PSI-BLAST: a new Jagannadham MV, Kumar CS, Debnath Das M (2012). generation of protein data base search programs. Nucleic Acids Protein-protein docking on molecular models of Aspergillus Res 25: 3389-3402. niger RNase and human actin: novel target for anticancer Arima K, Imanaka H, Kousaka M, Fukuta A, Tamura G (1964). therapeutics. J Mol Model 18: 653-662. Pyrrolnitrin, a new antibiotic substance, produced by Guruprasad K, Reddy VP, Pandit MW (1991). Correlation between Pseudomonas. Agr Biol Chem Tokyo 28: 575-576. stability of a protein and its dipeptide composition: a novel Arima K, Imanaka H, Kousaka M, Fukuta A, Tamura G (1965). Studies approach for predicting in vivo stability of a protein from its on pyrrolnitrin, a new antibiotic. I. Isolation and properties of primary sequence. Protein Eng 4: 155-161. pyrrolnitrin. J Antibiot 18: 201-204. Hamill RL, Elander RP, Mabe JA, Gorman M (1967). Metabolism of Buchan DW, Minneci F, Nugent TC, Bryson K, Jones DT (2013). tryptophans by Pseudomonas aureofaciens. V. Conversion of Scalable web services for the PSIPRED Protein Analysis tryptophan to pyrrolnitrin. Antimicrob Agents Ch 7: 388-396. Workbench. Nucleic Acids Res 41: W349-357. Hamill RL, Elander RP, Mabe JA, Gorman M (1970). Metabolism Chang CJ, Floss HG, Hook DJ, Mabe JA, Manni PE, Martin LL, of tryptophan by Pseudomonas aureofaciens. III. Production Schroder K, Shieh TL (1981). The biosynthesis of the antibiotic of substituted pyrrolnitrins from tryptophan analogues. Appl pyrrolnitrin by Pseudomonas aureofaciens. J Antibiot 34: 555- Microbiol 19: 721-725. 566. Hammer PE, Burd W, Hill DS, Ligon JM, van Pée KH (1999). Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam SA, Conservation of the pyrrolnitrin biosynthetic gene cluster McGarrell DM, Marsh T, Garrity GM et al. (2009). The Ribosomal among six pyrrolnitrin producing strains. FEMS Microbiol Database Project: improved alignments and new tools for rRNA Ecol 180: 39-44. analysis. Nucleic Acids Res 37: D141-145. Hammer PE, Hill DS, Lam ST, van Pée KH, Ligon JM (1997). de Souza JT, Raaijmakers JM (2003). Polymorphisms within the prnD Four genes from Pseudomonas fluorescens that encode the and pltC genes from pyrrolnitrin and pyoluteorin-producing biosynthesis of pyrrolnitrin. Appl Environ Microb 63: 2147- Pseudomonas and Burkholderia spp. FEMS Microbiol Ecol 43: 2154. 21-34. Ikai AJ (1980). Thermostability and aliphatic index of globular Dowling DN, O’Gara F (1994). Metabolites of Pseudomonas involved proteins. J Biochem 88: 1895-1898. in the biocontrol of plant disease. Trends Biotechnol 12: 133-141. Koehl P, Levitt M (1999). Structure-based conformational preferences Edwards U, Rogall T, Blöcker H, Emde M, Böttger EC (1989). Isolation of amino acids. P Natl Acad Sci USA 96: 12524-12529. and direct complete nucleotide determination of entire genes. Kyte J, Doolittle RF (1982). A simple method for displaying the Characterisation of a gene coding for 16S ribosomal RNA. hydropathic character of a protein. J Mol Biol 157: 105-132. Nucleic Acids Res 17: 7843-7853. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993). Eisenberg D, Luthy R, Bowie JU (1997). VERIFY3D: assessment of protein models with three dimensional profiles. Method PROCHECK-a program to check the stereochemical quality of Enzymol 277: 396-404. protein structures. J Appl Crystallogr 26: 283-291. Filiz E, Tombuloğlu H (2015). Genome-wide distribution of superoxide Laurie A, Jackson R (2005). Q-SiteFinder: an energy-based dismutase (SOD) gene families in Sorghum bicolor. Turk J Biol method for the prediction of protein-ligand binding sites. 39: 49-59. Bioinformatics 21: 1908-1916. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel Lemanceau P, Maurhofer M, Defago G (2006). Contribution of RD, Bairoch A (2005). Protein identification and analysis tools studies on suppressive soils to the identification of bacterial on the ExPASy server. In: Walker JM, editor. The Proteomics biocontrol agents and to the knowledge of their modes of Protocols Handbook. New York, NY, USA: Humana Press, pp. action. In: Gnanamanickam SS, editor. Plant-Associated 571-607. Bacteria. Dordrecht, the Netherlands: Springer, pp. 231-267. Gehmann K, Neyfeler R, Leadbeater A, Nevill D, Sozzi D (1990). Morris AL, MacArthur MW, Hutchinson EG, Thornton JM CGA173506: A new phenylpyrrole fungicide for broad-spectrum (1992). Stereochemical quality of protein structure disease control. In: Proceedings of the Brighton Crop Protection coordinates. Proteins 12: 345-364. Conference: Pests Diseases, pp. 399-406. Nevill D, Nyfeler R, Sozzi D (1988). CGA142705: a novel fungicide Gill SC, Von Hippel PH (1989). Calculation of protein extinction for seed treatment. In: Proceedings of the Brighton Crop coefficients from amino acid sequence data. Anal Biochem 182: Protection Conference: Pests Diseases, pp. 65-72. 319-328. Pace CN, Vajdos F, Fee L, Grimsley G, Grey T (1995). How to Glasgow J, Kuo T, Davies J (2006). Protein structure from contact maps: measure and predict the molar absorption coefficient of a a case-based reasoning approach. Inform Syst Front 8: 29-36. protein. Protein Sci 4: 2411-2423.

632 SINGH et al. / Turk J Biol

Qin Z, Buehler MJ (2010). Molecular dynamics simulation of the Tawara S, Matsumoto S, Hirose T, Matsumoto Y, Nakamoto S, -helix to -sheet transition in coiled protein filaments: evidence Mitsuno M, Kamimura T (1989). In vitro antifungal synergism for a critical filament length scale. Phys Rev Lett 104: 198304. between pyrrolnitrin and clotrimazole. J Med Mycol 30: 202- 210. Raaijmakers J, Weller DM, Thomashow LS (1997). Frequency of antibiotic producing Pseudomonas spp. in natural Thomashow LS, Weller DM (1996). Current concepts in the use of environments. Appl Environ Microb 63: 881-887. introduced bacteria for biological disease control: mechanisms and antifungal metabolites. In: Stacey G, Keen NT, editors. Raaijmakers JM, Weller DM (1998). Natural plant protection by Plant-Microbe Interactions. Dordrecht, the Netherlands: 2,4-diacetylphloroglucinol-producing Pseudomonas spp. in Springer, pp. 187-235. take-all decline soils. Mol Plant Microbe In 11: 144-152. Trivedi MV, Laurence JS, Siahaan TJ (2009). The role of thiols and Ramachandran GN, Ramakrishnan C, Sasisekharan V (1963). disulfides in protein chemical and physical stability. Curr Stereochemistry of polypeptide chain configurations. J Mol Protein Pept Sc 10: 614-625. Biol 7: 95-99. Umio S, Kamimura T, Kamishita T, Mine Y (1986). Antifungal Sambrook J, Fritsch EF, Maniatis T (1989). Molecular Cloning: A composition employing pyrrolnitrin in combination with an Laboratory Manual. 2nd ed. Cold Spring Harbor, NY, USA: imidazole compound. U.S. Patent 4: 636-520. Cold Spring Harbor Laboratory Press. van Pee KH, Salcher O, Lingens F (1980). Formation of Sekhar PN, Kishor PB, Reddy LA, Mondal P, Dash AK, Kar M, pyrrolnitrin and 3-(29-amino-39-chlorophenyl) pyrrole from Mohanty S, Sabat SC. (2006). In silico modelling and hydrogen 7-chlorotryptophan. Angew Chem Int Ed Engl 19: 828. peroxide binding study of rice catalase. In Silico Biol 6: 435- 447. Wedemeyer WJ (2000). Disulfide bonds and protein folding. Biochemistry-US 39: 703. Singh RN, Kaushik R, Arora DK, Saxena AK (2013). Prevalence of opportunist pathogens in thermal springs of devotion. J Appl Weller DM, Raaijmakers JM, Gardener BBM, Thomashow LS Sci Environ Sanit 8: 195-203. (2002). Microbial populations responsible for specific soil suppressiveness to plant pathogens. Annu Rev Phytopathol 40: Singh RP, Singh RN, Srivastava AK, Kumar S, Dubey RC, Arora DK 309-384. (2011). Structural analysis and 3D-modeling of FUR protein from Bradyrhizobium japonicum. J Appl Sci Environ Sanit 6: Wu S, Skolnick J, Zhang Y (2007). Ab-initio modeling of small 357-366. proteins by iterative TASSER simulations. BMC Biol 5: 17. Singh RP, Singh RN, Srivastava M, Srivastava AK, Kumar S, Dubey Zhou P, Mocek U, Seisel B, Floss HG (1992). Biosynthesis of RC, Sharma AK (2012). Structure prediction and analysis of pyrrolnitrin. Incorporation of 13C 15N double-labelled D- and MxaF from obligate, facultative and restricted facultative L-tryptophan. J Basic Microb 32: 209-214. methylobacterium. Bioinformation 8: 1042-1046. Suyama M, Matsuo Y, Nishikawa K (1997). Comparison of protein structures using 3D profile alignment. J Mol Evol 44 (Suppl. 1): S163-173.

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