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Sep 12, 2016 - Department of Biological Sciences, Konkuk University, Hwayang-dong, ... Department of Life Science, Sangmyung University, 7 Hongji-dong...
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Crystal Structures of Peptide Deformylase from Rice Pathogen Xanthomonas oryzae pv. oryzae in Complex with Substrate Peptides, Actinonin, and Fragment Chemical Compounds Ho-Phuong-Thuy Ngo,†,∇ Thien-Hoang Ho,†,∇ Inho Lee,† Huyen-Thi Tran,† Bookyo Sur,† Seunghwan Kim,§ Jeong-Gu Kim,§ Yeh-Jin Ahn,∥ Sun-Shin Cha,⊥ and Lin-Woo Kang*,† †

Department of Biological Sciences, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 05029, Korea Genomics Division, National Institute of Agricultural Sciences, Rural Development Administration (RDA), Jeonju 54874, Korea ∥ Department of Life Science, Sangmyung University, 7 Hongji-dong, Jongno-gu, Seoul 03016, Korea ⊥ Department of Chemistry & Nano Science, Ewha Womans University, 52, Ewhayeodae-gil, Seodaemun-gu, Seoul 03760, Korea §

S Supporting Information *

ABSTRACT: Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight on rice; this species is one of the most destructive pathogenic bacteria in rice cultivation worldwide. Peptide deformylase (PDF) catalyzes the removal of the N-formyl group from the N-terminus of newly synthesized polypeptides in bacterial cells and is an important target to develop antibacterial agents. We determined crystal structures of Xoo PDF (XoPDF) at up to 1.9 Å resolution, which include apo, two substrate-bound (methionine-alanine or methionine-alanine-serine), an inhibitor-bound (actinonin), and six fragment chemical-bound structures. Six fragment chemical compounds were bound in the substrate-binding pocket. The fragment chemical-bound structures were compared to the natural PDF inhibitor actinonin-bound structure. The fragment chemical molecules will be useful to design an inhibitor specific to XoPDF and a potential pesticide against Xoo. KEYWORDS: bacterial blight, Xanthomonas oryzae pv. oryzae, peptide deformylase, pesticide, fragment chemical

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INTRODUCTION

pneumoniae, Staphylococcus aureus, and Mycobacterium tuberculosis.17−20 PDFs function usually as a monomer and require metal ions such as Fe2+, Co2+, or Ni2+ for hydrolytic activity.16,21−25 Three highly conserved short motifs, motif 1 (GΦGΦAAXQ), motif 2 (EGCΦS), and motif 3 (HEΦDH), where Φ is a hydrophobic amino acid and X is any amino acid,26 are adjacent to each other in the PDF three-dimensional structure, forming an active site pocket with the metal ion.20 The N-formyl group of substrate N-terminus methionine is directly bound to the metal ion in the active site for hydrolysis. Thus, far, no plant pathogen PDF structure has been determined. Structure-based inhibitor/drug screening study has been limited mostly to human pathogen targets.27−30 The present study reports crystal structures of XoPDF as apo and in complexes with the substrates (methionine-alanine, MA, or methionine-alanine-serine, MAS), actinonin, and six fragment chemical compounds (FCCs) and systematic approaches to screen inhibitors against XoPDF using structural studies. The FCCs will provide useful information for the development of XoPDF inhibitors.

Rice is the most widely consumed staple food, especially in Asia. Bacterial blight on rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a devastating disease worldwide.1 Although many pesticides were developed against fungal rice diseases, only limited pesticides such as azoxystrobin, tecloftalam, and tiadinil are available against bacterial blight.2 As the world population grows, rice demand is expected to increase by at least 25% by 2030.3 The complete genome sequence of Xoo has been determined;4,5 systematic efforts have been made to find Xoo pathogenicity genes and rice resistance genes.6 In addition, crystal structures of several Xoo target proteins, which are essential for bacterial cell wall synthesis, lipid synthesis, and peptide synthesis, are published.7−9 In bacteria, the biosynthesis of proteins begins with N-formyl methionine, which comes from the N-terminal formylation of methionine-tRNA catalyzed by a formyl-transferase.10,11 Nformyl methionine is frequently removed from the nascent protein chain via two consecutive cleavage reactions by peptide deformylase (PDF; EC 3.5.1.31) and methionine aminopeptidase.12 PDF removes the N-formyl group from N-formyl methionine at the N-terminus of the proteins, and methionine aminopeptidase cleaves the remaining deformylated Nmethionine.13,14 The inhibition of bacterial PDF can disrupt the protein maturation process and eventually inhibit cellular processes, causing bacterial cell death.15,16 The essential role of PDF in protein maturation has been demonstrated for pathogenic bacteria such as Escherichia coli, Streptococcus © 2016 American Chemical Society

Received: Revised: Accepted: Published: 7307

July 4, 2016 September 8, 2016 September 12, 2016 September 12, 2016 DOI: 10.1021/acs.jafc.6b02976 J. Agric. Food Chem. 2016, 64, 7307−7314

Article

Journal of Agricultural and Food Chemistry

Figure 1. Native structure of XoPDF. (a) Overall structure of XoPDF: Signature motif 1 is shown in blue, motif 2 in cyan, and motif 3 in purple; the CD loop is colored orange. The red sphere is a cadmium ion. (b) Metal binding site: The cadmium ion is bound by three signature motifs. Metal coordination is shown with black dashed lines.

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by restrained refinement in the Refmac5 program. All structures were validated using WHATIF37 and SFCheck.38 Graphic presentations were created using PyMOL.39 Crystallization, Data Collection, and Structure Determination of Fragment Chemical Compound-Bound XoPDF. FCCs were chosen from Molsoft’s MolCart Compound Database, which is a collection of vendor compound databases including ChemBridge Corporation (San Diego, CA, USA) and Enamine LLC (Monmouth Jct., NJ, USA), via the Molcart Chemical Cartridge using chemical property filters of molecular weight and empirical drug-likeness.40 A library of 342 compound fragments (20 mg each) was purchased from ChemBridge Corporation (San Diego, CA, USA) and Enamine LLC (Monmouth Jct., NJ, USA). The compounds were dissolved in 100% DMSO to final concentrations of 1 M. FCC cocktails were prepared by mixing five different FCCs of distinct shapes and chemical properties to a final concentration of 200 mM per compound. Native crystals were first isolated and equilibrated in mother liquors consisting of 0.05 M cadmium sulfate, 0.1 M HEPES (pH 7.5), and 3.0 M sodium acetate trihydrate. Fragment cocktails were soaked to the drop at a final concentration of approximately 10 mM. All soaking steps were carried out at room temperature, while incubation steps were carried out at 286 K from 6 to 24 h. The FCC cocktail-soaked crystals were flash-cooled at 100 K in liquid nitrogen using the same cryoprotectant as that used for the native crystals. X-ray diffraction data were collected up to 2.0 Å resolution. The FCC-bound structures were determined in the same way as the MA-, MAS-, and actinoninbound structures were determined. Thereafter, we found extra positive electron density of putative chemical compound in the FCC cocktailsoaked crystal structure, and the identity of the positive density was confirmed again by separately soaking PDF crystals in each of the constituent compounds and determining the structures of the resulting complexes.

MATERIALS AND METHODS

Gene Cloning, Protein Expression, And Protein Purification. XoPDF protein from the Xoo def (Xoo1075; XoPDF) gene was produced and crystallized as previously published.31 Briefly, the XoPDF gene was cloned into the pET11a expression vector (Novagen). XoPDF was expressed and purified in a four-step procedure: first by precipitation with 45% saturated ammonium sulfate, followed by a Bio-Gel P60 column (2.5 × 50 cm, Bio-Rad) equilibrated in the lysis buffer with 150 mM NaCl, then by ionexchange chromatography using a UNO Q6 column (Bio-Rad), and finally by a Bio-Gel P100 column (2.5 × 50 cm, Bio-Rad). Purified XoPDF protein was dialyzed for 4 h against a buffer of 25 mM Tris (pH 7.5), 15 mM NaCl, and 3 mM β-mercaptoethanol; this sample was concentrated to 8 mg mL−1 in a Vivaspin 20 10000 MWCO (Vivascience). Crystallization, Data Collection, and Structure Determination of Substrate- or Inhibitor-Bound XoPDF. Crystals of XoPDF were grown using the hanging-drop vapor-diffusion method in a 24 well plate (SPL Life Sciences, Korea). The reservoir solution contained 0.05 M cadmium sulfate, 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5, and 2.0 M sodium acetate trihydrate. The hanging drops were made of 0.9 μL of protein solution mixed with 0.9 μL of reservoir solution. After 1 day, hexagonal-pillar-shaped crystals with adequate dimensions were obtained. For cryoprotection, the fully grown crystals were added to a mixture of reservoir solution and 20% (v/v) glycerol and subsequently flash-cooled at 100 K in liquid nitrogen.31 For MA-, MAS-, and actinonin-bound structures, native crystals were soaked in the reservoir solution with 10 mM of substrate (N-formylmethionine-alanine, fMA or N-formylmethioninealanine-serine, fMAS) or actinonin for less than 1 h and flash-cooled at 100 K in liquid nitrogen using the same cryoprotectant as that used for the native crystals. X-ray diffraction data were collected at beamline 4A at the Pohang Light Source (PLS), South Korea and at beamline BL6A at the Photon Factory, High Energy Accelerator Research Organization (KEK), Japan. Native crystals diffracted to 2.6 Å resolution, while MA-, MAS-, and actinonin-bound crystals diffracted up to 1.9 Å resolution. The XoPDF protein was crystallized in the hexagonal space group P6122. Data were integrated and scaled using DENZO and SCALEPACK, respectively.32 The phase of native XoPDF was obtained by molecular replacement (MR) with Phaser in the CCP4 software package.33 PDF from Leptospira interrogans (PDB ID: 1SV2, 42.3% sequence identity)34 was used as a search template. Model building and electron density interpretation were performed using the program COOT.35 The structure was refined using the CCP4 program Refmac5.36 The determined native XoPDF structure was used as a template to solve the substrate-bound complex structures

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RESULTS Overall apo-XoPDF Structure. The XoPDF protein was purified as a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The crystal structure of XoPDF was determined at 2.6 Å resolution (Table S1). There was one molecule in the asymmetric unit, and electron density maps for all residues of XoPDF were clearly observed, except for several residues in the C-terminus. Similar to other PDF structures, XoPDF adopted an α + β fold with two α-helices, eight β-strands, and four 310 helices (Figure 1a). Helix α2 was at the central position and was surrounded by eight antiparallel β-strands forming three sides. 7308

DOI: 10.1021/acs.jafc.6b02976 J. Agric. Food Chem. 2016, 64, 7307−7314

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Journal of Agricultural and Food Chemistry

Figure 2. Superimposed apo-, MA-bound, and MAS-bound XoPDF structures: (a) Substrate MA is shown as a cyan stick, and the CD loop of the MA-bound XoPDF structure is shown in salmon. Substrate MAS is shown as a purple stick, and the CD loop of the MAS-bound XoPDF structure is shown in dark brown. The apo-XoPDF structure is shown as in Figure 1. (b) The MAS substrate in the MAS-bound XoPDF structure is superimposed on the apo-XoPDF structure with the closed CD loop.

PDFs have three conserved short stretches of amino acids (motif 1, motif 2, and motif 3), which are physically close to each other; this proximity enables them to coordinate an active site metal ion (Figure 1b). Helix α2 carried conserved motif 3, HEXXH (HEYDH in XoPDF), which is essential for metal coordination and substrate activation (Figure S1). The strong electron density map indicated that a metal ion was found at the active site. As cadmium ions were present at a high concentration in the crystallization buffer, we interpreted the strong density as a cadmium ion. This cadmium ion was coordinated by three conserved residues of Cys99, His141, and His145, as well as two water molecules with the distances of 2.4, 2.2, 2.2, 2.3, and 2.5 Å, respectively (Figure S2a). The CD loop (Glu64-Ala74) of XoPDF occurred at the mouth of the substrate-binding pocket (Figure 2). The extended conformation of the CD loop closed the substratebinding pocket. In order for substrates to bind to the substratebinding pocket, the closed conformation of the XoPDF CD loop should be opened. The CD loops of most other PDFs were open in the native structures, and substrate-binding pockets were exposed to the solvent (Figure S3). Substrate−Complex Structure. Two substrates, fMA and fMAS, were separately soaked in the crystals of apo-XoPDF. An unambiguous positive electron density for each substrate was observed in the complex structures, but the N-formyl group was cleaved and is not shown (Figure 3a,b). Overall structures of apo-, MA-bound, and MAS-bound PDF were well conserved except for the CD loop; the root-mean-square deviation (RMSD) was calculated in COOT35 as 0.44 between apo and MA-bound structures based on Cα atoms of 168 residues and 0.52 between apo and MAS-bound structures based on Cα atoms of 166 residues. The CD loop of XoPDF exhibited three distinct conformations upon substrate binding: the closed conformation of apo-XoPDF, the moderately open conformation of the MA complex, and the wide-open conformation of the MAS complex (Figure 2a). Larger substrates forced the CD loop further outward from XoPDF. Upon binding of MA, the CD loop was shifted outward by up to 1.6 Å relative to its native position. In the MAS complex structure, the CD loop was shifted further outside; the residues from Glu67 to Tyr69 (the central part of the CD loop) were disordered and are not shown.

Figure 3. Refined electron density map of bound substrates and inhibitor actinonin: (a) substrate MA, (b) substrate MAS, and (c) inhibitor actinonin. Refined maps show the 2Fo−Fc electron density map (contoured at 1.0 σ, blue mesh). The proposed positions of the cleaved-off N-formyl group of substrates are shown as red dotted circles. The pyrrolidine ring, the pentyl hydrocarbon chain, and the hydroxamate group of actinonin are shown in green, orange, and blue shades, respectively.

The methionine side chain of the MA substrate was located in the hydrophobic pocket, which consists of two layers of surrounding residues. The lower layer, the mouth of the channel, was formed by His141, Val45, and Val138. The upper layer, including the top of the channel, was formed by Phe134, Glu97, Arg137, and Tyr69 (Figure 4). The amino group of the MA substrate methionine was H-bonded with Gly46 from motif 1 and Glu142 from motif 2. The alanine side chain of the MA substrate had van der Waals interactions with Arg68 and Leu100. In the MAS complex structure, the position and 7309

DOI: 10.1021/acs.jafc.6b02976 J. Agric. Food Chem. 2016, 64, 7307−7314

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Journal of Agricultural and Food Chemistry

Figure 4. Substrate methionine-binding site: (a) the side view of two layers of residues in the substrate methionine-binding site of the MA-bound XoPDF structure. Residues in the lower layer, the mouth of the channel, are labeled with black letters, and residues in the upper layer, the top of the channel, are labeled with white letters. Residues having a H-bond with the substrate methionine are labeled with orange letters. (b) The bottom view of the substrate methionine-binding site.

conformation of the substrate Met residue were almost identical to those of the MA complex structure. However, the MAS substrate Ala residue was rotated toward the CD loop, and the substrate residues of Ala and Ser pushed out and disordered the central part of CD loop. The hydroxyl side chain of Ser66 in the CD loop formed a H-bond with the terminal serine residue of MAS. Inhibitor−Complex Structure. Actinonin, a pseudotripeptide hydroxamate derivative, is a naturally occurring antibacterial agent from Streptomyces Cutter C/241 and a potent inhibitor of E. coli PDF (IC50 = 10 nM).42 We determined the structure of actinonin-bound XoPDF (Figure 3c). The pyrrolidine ring and pentyl hydrocarbon scaffold of actinonin showed van der Waals interactions, forming a bent U-shape. The pentyl hydrocarbon scaffold of actinonin mimics the side chain of Met; however, it was one carbon longer. This extra carbon pushed the hydroxamate group farther down toward the metal ion at the active site, to coordinate the metal directly (Figure 5a). The resulting shifted carbon, directly connected with the pentyl and hydroxamate groups, rotated the other main backbone of actinonin from the substrate peptide bond backbone. The pyrrolidine ring of actinonin was observed to bind Phe134, which replaced the previous benzene ring of Tyr69 in the CD loop having hydrophobic interactions with Phe134. The CD loop including Tyr69 was opened and is not shown due to its flexible conformation, as is the case for the MAS-bound structure. Fragment Chemical Complex Structures. We soaked XoPDF crystals with FCCs from a library of 342 chemicals under ∼300 Da and determined six different FCC-bound structures (Figure 6). All six FCCs were at least partially bound to the hydrophobic substrate methionine-binding site with Val45 and His141. Five out of six FCCs had π-bonds of aromatic ring structures sandwiched between Val45 and His141 (Figure 6a−e). Only FCC6 had a methyl amino group instead of an aromatic ring to bind parallel to the imidazol ring of the His141 side chain (Figure 6f). Compounds FCC1, FCC2, and FCC3 have simple hydrophobic structures with at least one ring of conjugated π-bonds constituting more than half of the structures; this ring bound at the substrate methionine-binding site (Figure 5b). The five-member ring of FCC4 was also bound at the methionine-binding site; the elongated nine-atom

Figure 5. Comparison of actinonin-bound and FCC-bound XoPDF structures: (a) Actinonin (yellow) and MAS (purple) in the corresponding complex structures were superimposed on the apoXoPDF structure. The carbon atom, directly connected with the pentyl and hydroxamate groups, is indicated by the pale blue arrow. The closed CD loop in the apo structure is shown in orange. (b) FCC1- (green), FCC2- (pink), and FCC3-bound (orange) structures were superimposed on the actinonin-bound (yellow) structure. (c) Actinonin (yellow) was superimposed on the FCC4-bound (salmon) structure. (d) FCC5- (pale blue) and FCC6-bound (light silver) structures were superimposed on the actinonin-bound (yellow) structure.

aliphatic chain of FCC4 initially followed the substrate peptide backbone conformation and was then exposed to solution (Figure 5c). FCC5 has two rings of conjugated π-bonds, which are directly bound to each other. The six-member benzene ring was bound at the methionine-binding site, and the additional five-member ring had van der Waals interactions with Phe134 (Figure 5d). These two rings were tilted toward each other by 42° due to hydrogen bond and steric hindrances from nearby residues. The binding of FCC6 is different from that of others. Instead of a hydrophobic ring, it has an aliphatic structure 7310

DOI: 10.1021/acs.jafc.6b02976 J. Agric. Food Chem. 2016, 64, 7307−7314

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Journal of Agricultural and Food Chemistry

Figure 6. Chemical structures and refined electron density maps of bound FCCs. Each panel depicts a chemical structure (left) and a 2FoFc map (right), (a) FCC1, (b) FCC2, (c) FCC3, (d) FCC4, (e) FCC5, and (f) FCC6. The 2Fo−Fc electron density maps (contoured at 1.0 σ) of bound FCCs are shown as blue mesh.

that of Ni2+, Co2+, and Zn2+ ions in EcPDF structures47,49 (Figure S2c). Even though Cd2+ is not a native metal cofactor of XoPDF, the binding site and coordination geometry of the Cd2+ ion will be similar to that of the native metal ion. Further studies will be necessary to identify the native metal cofactor of XoPDF and the native metal-dependent catalytic activity of XoPDF. Rice has two PDF genes of OsPDF1A and OsPDF1B. AtPDF1A and AtPDF1B genes of Arabidopsis thaliana are the orthologs of OsPDF1A and OsPDF1B; the sequence identity is 60.1% between AtPDF1A and OsPDF1A and 55.6% between AtPDF1B and OsPDF1B (Figure S5). XoPDF has the sequence identity of 33.3% with OsPDF1A and 29.1% with OsPDF1B. The crystal structures of AtPDF1A and AtPDF1B were determined with substrate MAS and inhibitor actinonin, respectively.50,51 The longer CD loop region of AtPDF1A formed the more rigid secondary structure including a helix rather than a flexible loop of XoPDF (Figure S6a). The CD loop of AtPDF1B showed the wide open conformation (Figure S6b). In the substrate-binding site, the upper layer of the hydrophobic pocket for the substrate methionine side chain showed differences between XoPDF and AtPDFs (Figure S6c,d). The top surface of the hydrophobic channel of AtPDF1A and AtPDF1B was tightly filled with bulkier residues of Trp146 in AtPDF1A and Phe87 in AtPDF1B. However, smaller residues of Gly95 and Phe134 in XoPDF generated a free unoccupied space in the top surface, which could make the XoPDF binding site more flexible and might allow a bulkier molecule to bind. The structural differences between XoPDF and AtPDFs will provide useful information to develop a specific inhibitor against XoPDF. We determined crystal structures of XoPDF in complex with six FCCs in the substrate-binding pocket. All FCCs were at least partially bound at the hydrophobic substrate methioninebinding site, which could accommodate bulkier structural motifs such as rigid aromatic rings with conjugated π-bonds than the aliphatic side chain of methionine. The ring structures

bound at the methionine-binding site (Figure 5d). At the opposite end of FCC6, the chlorinated benzene ring is stacked on the peptide backbone of the β4-strand.

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DISCUSSION

There are two putative PDF genes for Xoo1075 (XoPDF) and Xoo0585 in Xoo. XoPDF and Xoo0585 genes have sequence identities of 31.5 and 51.2% to E. coli PDF (EcPDF), respectively. Both genes have three strictly conserved signature motifs of PDF. Recently, an in vitro assay system was developed to activate the pathogenicity of Xoo by mimicking the initial interactions between rice and Xoo, and the assay system activated the expression and secretion of pathogenic Xoo effector proteins from Xoo.43,44 Combining the in vitro assay with RNA-Seq enabled analysis of Xoo genome-wide transcriptional gene expression upon the pathogenic interaction with host rice in a time-resolved manner.45 In the pathogenic interaction with rice, the immediate transcriptional activation of the XoPDF gene was found; the Xoo0585 gene did not exhibit a change in the expression level (Figure S4). PDF’s native metal cofactor is known as an Fe2+ ion.46 However, the native Fe2+ ion can be easily replaced by other more stable metal ions such as Ni2+, Co2+, and Zn2+.22,47,48 Initially, we suspected the bound metal ion in XoPDF structures to be a Zn2+ ion. When we put a Zn2+ ion in the metal site, and the strong positive electron density still remained in the FoFc map, contoured even at 5.0 σ (Figure S2b). We searched for possible alternative metal ions to bind in the XoPDF metal site from the solutions used in protein purification and crystallization. The Cd2+ ion (50 mM) existed in the crystallization reservoir solution. With the Cd2+ ion in the metal site, the extra-positive electron density in the FoFc map disappeared (Figure S2a). The temperature factor of the Cd2+ ion was reasonably low at 38.0, 30.7, and 24.1 in apo-, MA-bound, and MAS-bound XoPDF structures, respectively. The binding site and trigonal-pyramidal coordination geometry of the Cd2+ ion in XoPDF structures was well conserved with 7311

DOI: 10.1021/acs.jafc.6b02976 J. Agric. Food Chem. 2016, 64, 7307−7314

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Journal of Agricultural and Food Chemistry Author Contributions

were mainly stacked between one plane of His141 and Glu97 and another plane of Val45 (Figure S7). The binding of elongated ligand-like substrate MAS and actinonin in XoPDF caused opening of the CD loop, which revealed the concealed hydrophobic Phe134 in solution (Figure S8). The revealed Phe134 looked for a compensating hydrophobic moiety. The terminal five-member pyrrolidine ring of actinonin provided the compensating hydrophobic moiety and bound to Phe134. The Tyr69 residue in the CD loop had hydrophobic interactions with Phe134 in the closed conformation of the apo structure. The angle between two aromatic rings of Phe134 and Tyr69 was approximately 42°, of which the partial and unparalleled interaction had an interaction of moderate strength and allowed plausible switching of the CD loop from closed to open conformation. Teh PDF structure from Leptospira interrogans also showed the closed conformation of the CD loop in the apo structure,34 and the tyrosine residue in the CD loop corresponding to XoPDF Tyr69 was conserved (Figures S1 and S2). Most other PDF structures exhibited an open CD loop conformation in the apo structure52−54 (Figure S2). The position of Phe134 could also be an important site for the development of a specific inhibitor against XoPDF. FCC5 and FCC6 exhibited different binding than other FCCs. Both compounds bound from the methionine-binding site to the exposed Phe134 site as a direct line, which implies that the two binding sites are directly connected (Figure 5d). In the actinonin-bound XoPDF structure, the pentyl group and the other chain structure with the pyrrolidine ring of actinonin were ∼4 Å apart, which is a good distance for van der Waals interaction (Figure S9). The direct hydrophobic van der Waals interaction could hold actinonin as the same U-shape in water before binding to XoPDF, and the conformational rigidity of actinonin could increase the binding affinity for PDFs. In summary, the hydrophobic substrate methionine-binding site is mainly involved in FCC binding via hydrophobic interaction and can accommodate bulky rigid aromatic ring structures. The Phe134 residue provides an additional hydrophobic interaction site for ligands. Crystal structures of XoPDF in complex with substrates and various FCCs may be useful for the development of new pesticides against Xoo.

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Funding

This work was supported by WTU Joint Research Grants of Konkuk University. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the beamline staffs for their assistance at beamline 4A at the Pohang Light Source (PLS), South Korea and at beamline BL-6A at the Photon Factory, High Energy Accelerator Research Organization (KEK), Japan.

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ABBREVIATIONS USED Peptide deformylase, (PDF); Xanthomonas oryzae pv. oryzae, (Xoo); PDF from Xanthomonas oryzae pv. oryzae, (XoPDF); fragment chemical compound, (FCC)

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REFERENCES

(1) Ezuka, A.; Kaku, H. A historical review of bacterial blight of rice. Bull. Natl. Inst Agrobiol Resour (Japan) 2000, 15, 53−54. (2) PPDB: Pesticide Properties DataBase. http://sitem.herts.ac.uk/ aeru/ppdb/en/ (2016). (3) Li, J. Y.; Wang, J.; Zeigler, R. S. The 3,000 rice genomes project: new opportunities and challenges for future rice research. GigaScience 2014, 3, 8. (4) Lee, B. M.; Park, Y. J.; Park, D. S.; Kang, H. W.; Kim, J. G.; Song, E. S.; Park, I. C.; Yoon, U. H.; Hahn, J. H.; Koo, B. S.; Lee, G. B.; Kim, H.; Park, H. S.; Yoon, K. O.; Kim, J. H.; Jung, C. H.; Koh, N. H.; Seo, J. S.; Go, S. J. The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice. Nucleic Acids Res. 2005, 33, 577−86. (5) Ochiai, H.; Inoue, Y.; Takeya, M.; Sasaki, A.; Kaku, H. Genome Sequence of Xanthomonas oryzae pv. oryzae Suggests Contribution of Large Numbers of Effector Genes and Insertion Sequences to Its Race Diversity. JARQ 2005, 39, 275−287. (6) Zhang, H.; Wang, S. Rice versus Xanthomonas oryzae pv. oryzae: a unique pathosystem. Curr. Opin. Plant Biol. 2013, 16, 188−95. (7) Doan, T. T.; Kim, J. K.; Ngo, H. P.; Tran, H. T.; Cha, S. S.; Min Chung, K.; Huynh, K. H.; Ahn, Y. J.; Kang, L. W. Crystal structures of d-alanine-d-alanine ligase from Xanthomonas oryzae pv. oryzae alone and in complex with nucleotides. Arch. Biochem. Biophys. 2014, 545, 92−99. (8) Natarajan, S.; Kim, J. K.; Jung, T. K.; Doan, T. T.; Ngo, H. P.; Hong, M. K.; Kim, S.; Tan, V. P.; Ahn, S. J.; Lee, S. H.; Han, Y.; Ahn, Y. J.; Kang, L. W. Crystal structure of malonyl CoA-Acyl carrier protein transacylase from Xanthomanous oryzae pv. oryzae and its proposed binding with ACP. Mol. Cells 2012, 33, 19−25. (9) Kim, J. K.; Natarajan, S.; Park, H.; Huynh, K. H.; Lee, S. H.; Kim, J. G.; Ahn, Y. J.; Kang, L. W. Crystal structure of XoLAP, a leucine aminopeptidase, from Xanthomonas oryzae pv. oryzae. J. Microbiol. 2013, 51, 627−32. (10) Becker, A.; Schlichting, I.; Kabsch, W.; Groche, D.; Schultz, S.; Wagner, A. F. Iron center, substrate recognition and mechanism of peptide deformylase. Nat. Struct. Biol. 1998, 5, 1053−8. (11) Lucchini, G.; Bianchetti, R. Initiation of protein synthesis in isolated mitochondria and chloroplasts. Biochim. Biophys. Acta, Nucleic Acids Protein Synth. 1980, 608, 54−61. (12) Waller, J. P. The Nh2-Terminal Residues of the Proteins from Cell-Free Extracts of E. Coli. J. Mol. Biol. 1963, 7, 483−96. (13) Fry, K. T.; Lamborg, M. R. Amidohydrolase activity of Escherichia coli extracts with formylated amino acids and dipeptides as substrates. J. Mol. Biol. 1967, 28, 423−33.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b02976. Data collection and refinement statistics, sequence alignments, metal binding and coordinates, structures and comparisons, time-resolved transcriptional gene expression, and sequence alignment (PDF) Accession Codes

Atomic coordinates and structural factors for the reported crystal structures have been deposited with the Protein Data Bank under the accession codes 5E5D(apo), 5CPD(MA), 5CP0(MAS), 5CVQ(actinonin), 5CY8(FCC1), 5CY7(FCC2), 5CWY(FCC3), 5CVK(FCC4), 5CWX(FCC5), and 5CVP(FCC6).

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H.-P.-T.-N. and T.-H.H. contributed equally to this work.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +82 2-450-4090. Fax: +82 2-444-6707. 7312

DOI: 10.1021/acs.jafc.6b02976 J. Agric. Food Chem. 2016, 64, 7307−7314

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DOI: 10.1021/acs.jafc.6b02976 J. Agric. Food Chem. 2016, 64, 7307−7314