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1.  Purification, crystallization and preliminary X-ray crystallographic analysis of rice Bowman–Birk inhibitor from Oryza sativa  
Rice Bowman–Birk inhibitor was expressed and crystallized.
Bowman–Birk inhibitors (BBIs) are cysteine-rich proteins with inhibitory activity against proteases that are widely distributed in monocot and dicot species. The expression of rice BBI from Oryza sativa is up-regulated and induced by pathogens or insects during germination of rice seeds. The rice BBI (RBTI) of molecular weight 15 kDa has been crystallized using the hanging-drop vapour-diffusion method. According to the diffraction of rice BBI crystals at a resolution of 2.07 Å, the unit cell belongs to space group P212121, with unit-cell parameters a = 74.37, b = 96.69, c = 100.36 Å. Preliminary analysis indicates four BBI molecules in an asymmetric unit, with a solvent content of 58.29%.
doi:10.1107/S1744309106014795
PMCID: PMC2243081  PMID: 16754971
Bowman–Birk inhibitors; rice
2.  Purification, crystallization and preliminary X-ray crystallographic analysis of rice lectin from Oryza sativa  
Rice lectin was crystallized and analyzed by X-ray crystallography.
Lectins with sugar-binding specificity are widely distributed in higher plants and various other species. The expression of rice lectin from Oryza sativa is up-regulated in the growing coleoptile when anaerobic stress persists. A rice lectin of molecular weight 15.2 kDa has been crystallized using the hanging-drop vapour-diffusion method. From the diffraction of the lectin crystals at 1.93 Å resolution, the unit cell belongs to space group P31, with unit-cell parameters a = 98.58, b = 98.58, c = 44.72 Å. Preliminary analysis indicates that there are two lectin molecules in an asymmetric unit with a large solvent content, 70.1%.
doi:10.1107/S1744309105040698
PMCID: PMC2150942  PMID: 16511272
lectins; rice
3.  Expression, crystallization and preliminary X-ray crystallographic analysis of XometC, a cystathionine γ-lyase-like protein from Xanthomonas oryzae pv. oryzae  
XometC, a cystathionine γ-lyase-like protein from X. oryzae pv. oryzae and an antibacterial drug-target protein against bacterial blight, was cloned, purified and crystallized. Preliminary X-ray crystallographic analysis of XometC crystals was carried out.
Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight of rice (Oryza sativa L.), one of the most devastating diseases of rice in most rice-growing countries. XometC, a cystathionine γ-lyase (CGL) like protein that is an antibacterial drug-target protein against Xoo, was cloned, expressed, purified and crystallized. CGL catalyzes the second step in the reverse-transsulfuration pathway, which is essential for the metabolic interconversion of the sulfur-containing amino acids cysteine and methionine. Crystals of two different shapes, plate-shaped and pyramid-shaped, diffracted to 2.9 and 3.2 Å resolution and belonged to the primitive orthogonal space group P212121 and the tetragonal space group P41 (or P43), with unit-cell parameters a = 73.0, b = 144.9, c = 152.3 Å and a = b = 78.2, c = 300.7 Å, respectively. For the P212121 crystals, three or four monomers exist in the asymmetric unit with a corresponding V M of 3.02 or 2.26 Å3 Da−1 and a solvent content of 59.3 or 45.7%. For the P41 (or P43) crystals, four or five monomers exist in the asymmetric unit with a corresponding V M of 2.59 or 2.09 Å3 Da−1 and a solvent content of 52.5 or 40.6%.
doi:10.1107/S1744309108021155
PMCID: PMC2494973  PMID: 18678949
bacterial blight; cystathionine γ-lyase; reverse transsulfuration pathway; Xanthomonas oryzae pv. oryzae
4.  Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of the co-chaperonin XoGroES from Xanthomonas oryzae pv. oryzae  
Bacterial blight, an infectious disease caused by X. oryzae pv. oryzae (Xoo), causes huge rice-production losses in most rice-cultivating countries. The co-chaperonin of Xoo, XoGroES, an important protein for protein folding, was cloned from Xoo, purified and crystallized for atomic resolution structure determination.
Bacterial blight (BB), a devastating disease caused by Xanthomonas oryzae pv. oryzae (Xoo), causes serious production losses of rice in Asian countries. Protein misfolding may interfere with the function of proteins in all living cells and must be prevented to avoid cellular disaster. All cells naturally contain molecular chaperones that assist the unfolded proteins in folding into the native structure. One of the well characterized chaperone complexes is GroEL–GroES. GroEL, which consists of two chambers, captures misfolded proteins and refolds them. GroES is a co-chaperonin protein that assists the GroEL protein as a lid that temporarily closes the chamber during the folding process. Xoo4289, the GroES gene from Xoo, was cloned and expressed for X-ray crystallographic study. The purified protein (XoGroES) was crystallized using the hanging-drop vapour-diffusion method and a crystal diffracted to 2.0 Å resolution. The crystal belonged to the hexagonal space group P61, with unit-cell parameters a = 64.4, c = 36.5 Å. The crystal contains a single molecule in the asymmetric unit, with a corresponding V M of 2.05 Å3 Da−1 and a solvent content of 39.9%.
doi:10.1107/S1744309110038820
PMCID: PMC3079969  PMID: 21206021
Xanthomonas oryzae pv. oryzae; bacterial blight; co-chaperonins; Xoo4289
5.  Crystallization and preliminary crystallographic studies of LipA, a secretory lipase/esterase from Xanthomonas oryzae pv. oryzae  
The crystallization and preliminary crystallographic studies of LipA, a lipase/esterase secreted by X. oryzae pv. oryzae during its infection of rice plants, are reported.
Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, a serious disease of rice. Several enzymes that are secreted through the type II secretion system of this bacterium play an important role in the plant–microbe interaction, being important for virulence and also being able to induce potent host defence responses. One of these enzymes is a secretory lipase/esterase, LipA, which shows a very weak homology to other bacterial lipases and gives a positive tributyrin plate assay. In this study, LipA was purified from the culture supernatant of an overexpressing clone of X. oryzae pv. oryzae and two types of crystals belonging to space group C2 but with two different unit-cell parameters were obtained using the hanging-drop vapour-diffusion method. Type I crystals diffract to a maximum resolution of 1.89 Å and have unit-cell parameters a = 93.1, b = 62.3, c = 66.1 Å, β = 90.8°. Type II crystals have unit-cell parameters a = 103.6, b = 54.6, c = 66.3 Å, β = 92.6° and diffract to 1.86 Å. Solvent-content analysis shows one monomer in the asymmetric unit in both the crystal forms.
doi:10.1107/S1744309107034367
PMCID: PMC2335158  PMID: 17671374
secretory lipases; Xanthomonas oryzae pv. oryzae; plant pathogens
6.  Purification, crystallization and preliminary X-ray analysis of rice BGlu1 β-glucosidase with and without 2-deoxy-2-fluoro-β-d-glucoside 
Rice BGlu1 β-glucosidase was purified from recombinant E. coli and crystallized with and without the inhibitor 2-deoxy-2-fluoro-β-d-glucose. The crystals diffracted to 2.15 and 2.75 Å, respectively.
Rice (Oryza sativa) BGlu1 β-glucosidase was expressed in Escherichia coli with N-terminal thioredoxin and hexahistidine tags and purified by immobilized metal-affinity chromatography (IMAC). After removal of the N-terminal tags, cation-exchange and S-200 gel-filtration chromatography yielded a 50 kDa BGlu1 with >95% purity. The free enzyme and a complex with 2,4-dinitrophenyl-2-­deoxy-2-fluoro-β-d-glucopyranoside inhibitor were crystallized by microbatch and hanging-drop vapour diffusion. Small tetragonal crystals of BGlu1 with and without inhibitor grew in 18%(w/v) PEG 8000 with 0.1 M sodium cacodylate pH 6.5 and 0.2 M zinc acetate. Crystals of BGlu1 with inhibitor were streak-seeded into 23%(w/v) PEG MME 5000, 0.2 M ammonium sulfate, 0.1 M MES pH 6.7 to yield larger crystals. Crystals with and without inhibitor diffracted to 2.15 and 2.75 Å resolution, respectively, and had isomorphous orthorhombic unit cells belonging to space group P212121.
doi:10.1107/S1744309106027084
PMCID: PMC2242908  PMID: 16880561
BGlu1 β-glucosidase; 2,4-dinitrophenyl-2-­deoxy-2-fluoro-β-d-glucopyranoside; rice
7.  Crystallization and preliminary X-ray diffraction study of an active-site mutant of pro-Tk-subtilisin from a hyperthermophilic archaeon 
Crystallization of and preliminary crystallographic studies on an active-site mutant of pro-Tk-subtilisin from the hyperthermophilic archaeon T. kodakaraensis were performed.
Crystallization of and preliminary crystallographic studies on an active-site mutant of pro-Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis were performed. The crystal was grown at 277 K by the sitting-drop vapour-diffusion method. Native X-ray diffraction data were collected to 2.3 Å resolution using synchrotron radiation from station BL41XU at SPring-8. The crystal belongs to the orthorhombic space group I222, with unit-cell parameters a = 92.69, b = 121.78, c = 77.53 Å. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient V M was calculated to be 2.6 Å3 Da−1 and the solvent content was 53.1%.
doi:10.1107/S1744309106030454
PMCID: PMC2242867  PMID: 16946475
pro-Tk-subtilisin; Thermococcus kodakaraensis
8.  417 Uncommon Occupational Allergy to Rice: as a Food Allergen 
Background
Rice (Oryza sativa) belongs, with other cultivated cereals, to different tribes of the Poaceae family. It is one of the most widely produced and consumed cereals in the world but hypersensitivity reactions to this grain are uncommon Most reports describe an immunologically-mediated urticaria due to contact with raw rice or reactions after the inhalation of rice fumes, whereas reports of immediate hypersensitivity reactions after ingestion of rice are scarce.
Methods
Patient 1 (P1): A 40-year-old-man, a professional cook, presented 2 episodes of generalized urticaria minutes after rice ingestion. He tolerated the inhalation of vapours during rice-boiling, but reported icthy skin and erythema after rice handling.
Patient 2 (P2): A 30-year-old-woman, pizzeria worker for the last 10 years, complaint of sneezing and rhinorrhea after handling rice for the last 2 years, and presented diarrhea and dysphagia after rice ingestion during the last year. One week before consulting she presented eyelid angioedema, chest tightness and abdominal cramping after doing exercise right after eating rice.
None of the patients reported any additional atopic background. Skin prick tests with common inhalants and cereals extracts, Pru p 3 extract, prick-by-prick test with rice and rice flour and specific IgE determinations to rice were carried out in both patients.
Results
Skin prick tests to rice were positive in both patients (wheal diameter >3 mm). Skin prick-by-prick with rice and rice flour was also positive in patients 1 and 2. Serum specific IgE determinations against rice showed values of 0.8 kU/L and 1.48 kU/L for P1 and P2, respectively, out from a total IgE of 32.8 UI/mL and 23.7 UI/mL, respectively. SPT to common inhalants, to the rest of the cereals and to Pru p 3, showed a negative result.
Conclusions
We present 2 work-related cases of rice allergy with an unusual display and different clinical manifestations (urticaria, rhinitis and anaphylaxis) in 2 patients without atopic background and who worked handling rice and rice flour. No cross-reactivity with usual panallergens as LTP seemed to be involved.
doi:10.1097/01.WOX.0000412180.87263.db
PMCID: PMC3512861
9.  Crystallization of a nonclassical Kazal-type Carcinoscorpius rotundicauda serine protease inhibitor, CrSPI-1, complexed with subtilisin 
A recombinant serine protease inhibitor, CrSPI-1, from horseshoe crab, which inhibits subtilisin with a K i of 10−9  M, has been cloned, expressed, purified and cocrystallized with subtilisin. The crystals diffracted to 2.6 Å resolution.
Serine proteases play a major role in host–pathogen interactions. The innate immune system is known to respond to invading pathogens in a nonspecific manner. The serine protease cascade is an essential component of the innate immune system of the horseshoe crab. The serine protease inhibitor CrSPI isoform 1 (CrSPI-1), a unique nonclassical Kazal-type inhibitor of molecular weight 9.3 kDa, was identified from the hepatopancreas of the horseshoe crab Carcinoscorpius rotundicauda. It potently inhibits subtilisin and constitutes a powerful innate immune defence against invading microbes. Here, the cloning, expression, purification and cocrystallization of CrSPI-1 with subtilisin are reported. The crystals diffracted to 2.6 Å resolution and belonged to space group P21, with unit-cell parameters a = 73.8, b = 65.0, c = 111.9 Å, β = 95.4°. The Matthews coefficient (V M = 2.64 Å3 Da−1, corresponding to 53% solvent content) and analysis of the preliminary structure solution indicated the presence of one heterotrimer (1:2 ratio of CrSPI-1:subtilisin) and one free subtilisin molecule in the asymmetric unit.
doi:10.1107/S1744309109014420
PMCID: PMC2675604  PMID: 19407396
serine proteases; Kazal-type serine protease inhibitors; host–pathogen interaction; immune defence; horseshoe crab; CrSPI
10.  Preliminary crystallographic study of two cuticle-degrading proteases from the nematophagous fungi Lecanicillium psalliotae and Paecilomyces lilacinus  
Two cuticle-degrading proteases Ver112 and PL646 were purified from the nematophagous fungi L. psalliotae and P. lilacinus, respectively. The protease Ver112 and a complex between PL646 and a tetrapeptide inhibitor were crystallized. Diffraction data were collected to 1.65 and 2.2 Å resolution, respectively.
Cuticle-degrading proteases are extracellular subtilisin-like serine proteases that are secreted by entomopathogenic and nematophagous fungi. These proteases can digest the host cuticle during invasion of an insect or nematode and serve as a group of important virulence factors during the infection of nematodes by nematophagous fungi. To elucidate the mechanism of interaction between the proteases and the nematode cuticle, two cuticle-degrading proteases, Ver112 from Lecanicillium psalliotae (syn. Verticillium psalliotae) and PL646 from Paecilomyces lilacinus, were studied. The Ver112 protein and the complex between PL646 and the substrate-like tetrapeptide inhibitor methoxy­succinyl-Ala-Ala-Pro-Val-chloromethyl ketone (MSU-AAPV) were crystallized using the hanging-drop vapour-diffusion method at 289 K. The crystals were analyzed by X-ray diffraction to resolutions of 1.65 and 2.2 Å, respectively. These analyses identified that crystals of Ver112 belonged to space group P212121, with unit-cell parameters a = 43.7, b = 67.8, c = 76.3 Å, α = β = γ = 90°. In contrast, crystals of the PL646–MSU-AAPV complex belonged to space group P21, with unit-cell parameters a = 65.1, b = 62.5, c = 67.6 Å, β = 92.8°.
doi:10.1107/S1744309109003595
PMCID: PMC2650454  PMID: 19255481
cuticle-degrading proteases; Ver112; PL646; Lecanicillium psalliotae; Paecilomyces lilacinus
11.  Crystallization and preliminary X-ray analysis of carnein, a serine protease from Ipomoea carnea  
The subtilisin-like serine protease carnein was isolated from the latex of the plant I. carnea, purified and crystallized by the hanging-drop vapour-diffusion method. A diffraction data set was collected to 2.0 Å resolution in-house from a single crystal at 110 K.
Carnein is an 80 kDa subtilisin-like serine protease from the latex of the plant Ipomoea carnea which displays an exceptional resistance to chemical and thermal denaturation. In order to obtain the first crystal structure of a plant subtilisin and to gain insight into the structural determinants underlying its remarkable stability, carnein was isolated from I. carnea latex, purified and crystallized by the hanging-drop vapour-diffusion method. A data set was collected to 2.0 Å resolution in-house from a single crystal at 110 K. The crystals belonged to the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 126.9, c = 84.6 Å, α = β = 90, γ = 120°. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient is 2.46 Å3 Da−1, corresponding to a solvent content of 50%. Structure determination of the enzyme is in progress.
doi:10.1107/S1744309109008288
PMCID: PMC2664766  PMID: 19342786
carnein; serine proteases; subtilisin
12.  Approach via a small retroperitoneal anterior subcostal incision in the supine position for gasless laparoendoscopic single-port radical nephrectomy: initial experience of 42 patients 
BMC Urology  2014;14:29.
Background
Gasless laparoendoscopic single-port surgery (GasLESS) for radical nephrectomy (GasLESSRN) in the flank position is a minimally invasive treatment option for patients with T1–3 renal cell carcinoma (RCC). However, RCC patients considered suitable for supine positioning rather than flank positioning for radical nephrectomy are occasionally encountered. This study evaluated the safety and feasibility of approach via a small retroperitoneal anterior subcostal incision (RASI) in the supine position for GasLESSRN (RASI-GasLESSRN) on the basis of our initial experience.
Methods
RASI-GasLESSRN was performed on 42 patients with RCC or suspected RCC from 2011–2013. The RASI, which was 6 cm long in principle, was made parallel to the tip of the rib from the lateral border of rectus abdominis muscle toward the flank in the supine position. The specimen was extracted via the RASI using a retrieval device. All procedures were performed retroperitoneally under flexible endoscopy with reusable instruments and without carbon dioxide insufflation or insertion of hands into the operative field.
Results
RASI-GasLESSRN was successfully performed in all patients without complications. The mean incision length was 6.3 cm, mean operative time was 198 minutes, and mean blood loss was 284 mL. All 42 patients were classified as Clavien grade I. The mean times to oral feeding and walking were 1.1 and 2 days, respectively. The mean number of postoperative days required for patients to be dischargeable was 3.7 days.
Conclusions
The approach via a small RASI in the supine position for GasLESSRN is a safe and feasible technique. RASI-GasLESSRN in the supine position is an alternative minimally invasive treatment option, especially for RCC patients considered suitable for supine positioning.
doi:10.1186/1471-2490-14-29
PMCID: PMC3977956  PMID: 24708621
Retroperitoneal anterior subcostal incision; Supine position; Renal cell carcinoma; Radical nephrectomy; Laparoendoscopic single-port surgery; Gasless laparoendoscopic single-port surgery
13.  Expression, crystallization and preliminary X-ray crystallographic analysis of Xoo0352, d-alanine-d-alanine ligase A, from Xanthomonas oryzae pv. oryzae  
Xoo0352, which encodes d-alanine-d-alanine ligase A (DdlA), from X. oryzae pv. oryzae was cloned, purified and crystallized. Preliminary X-ray crystallographic analysis of DdlA crystals was performed.
Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB), which is one of the most devastating diseases of rice in most rice-growing countries. d-­Alanine-d-alanine ligase A (DdlA), coded by the Xoo0352 gene, was expressed, purified and crystallized. DdlA is an enzyme that is involved in d-­alanine metabolism and the biosynthesis of an essential bacterial peptidoglycan precursor, in which it catalyzes the formation of d-alanyl-d-alanine from two d-­alanines, and is thus an attractive antibacterial drug target against Xoo. The DdlA crystals diffracted to 2.3 Å resolution and belonged to the primitive tetragonal space group P43212, with unit-cell parameters a = b = 83.0, c = 97.6 Å. There is one molecule in the asymmetric unit, with a corresponding V M of 1.88 Å3 Da−1 and a solvent content of 34.6%. The initial structure was determined by molecular replacement using d-alanine-d-alanine ligase from Staphylococcus aureus (PDB code 2i87) as a template model.
doi:10.1107/S1744309108028820
PMCID: PMC2593706  PMID: 19052362
bacterial blight; d-alanine-d-alanine ligase; peptidoglycan biosynthesis; Xanthomonas oryzae pv. oryzae
14.  Purification, crystallization and preliminary X-ray analysis of OsAREB8 from rice, a member of the AREB/ABF family of bZIP transcription factors, in complex with its cognate DNA 
OsAREB8 from rice (O. sativa), a member of the AREB/ABF family of bZIP transcription factors, was expressed, purified and crystallized using the sitting-drop vapour-diffusion method. A crystal of OsAREB8 in complex with its cognate DNA diffracted X-rays to 3.65 Å resolution.
The AREB/ABF family of bZIP transcription factors play a key role in drought stress response and tolerance during the vegetative stage in plants. To reveal the DNA-recognition mechanism of the AREB/ABF family of proteins, the bZIP domain of OsAREB8, an AREB/ABF-family protein from Oryza sativa, was expressed in Escherichia coli, purified and crystallized with its cognate DNA. Crystals of the OsAREB8–DNA complex were obtained by the sitting-drop vapour-diffusion method at 277 K with a reservoir solution consisting of 50 mM MES pH 6.4, 29% MPD, 2 mM spermidine, 20 mM magnesium acetate and 100 mM sodium chloride. A crystal diffracted X-rays to 3.65 Å resolution and belonged to space group C222, with unit-cell parameters a = 155.1, b = 206.7, c = 38.5 Å. The crystal contained one OsAREB8–DNA complex in the asymmetric unit.
doi:10.1107/S1744309112009384
PMCID: PMC3325828  PMID: 22505428
AREB/ABF family; stress response; Oryza sativa
15.  Purification, crystallization and preliminary X-ray analysis of recombinant betaine aldehyde dehydrogenase 2 (OsBADH2), a protein involved in jasmine aroma, from Thai fragrant rice (Oryza sativa L.) 
Crystals of betaine aldehyde dehydrogenase 2 from rice (O. sativa L.) belonged to a C-centred orthorhombic space group and diffraceted X-rays to 2.6 Å resolution.
Fragrant rice (Oryza sativa L.) betaine aldehyde dehydrogenase 2 (OsBADH2) is a key enzyme in the synthesis of fragrance aroma compounds. The extremely low activity of OsBADH2 in catalyzing the oxidation of acetaldehyde is believed to be crucial for the accumulation of the volatile compound 2-acetyl-1-pyrroline (2AP) in many scented plants, including fragrant rice. Recombinant fragrant rice OsBADH2 was expressed in Escherichia coli as an N-terminal hexahistidine fusion protein, purified using Ni Sepharose affinity chromatography and crystallized using the microbatch method. Initial crystals were obtained within 24 h using 0.1 M Tris pH 8.5 with 30%(w/v) PEG 4000 and 0.2 M magnesium chloride as the precipitating agent at 291 K. Crystal quality was improved when the enzyme was cocrystallized with NAD+. Improved crystals were grown in 0.1 M HEPES pH 7.4, 24%(w/v) PEG 4000 and 0.2 M ammonium chloride and diffracted to beyond 2.95 Å resolution after being cooled in a stream of N2 immediately prior to X-ray diffraction experiments. The crystals belonged to space group C2221, with unit-cell parameters a = 66.03, b = 183.94, c = 172.28 Å. An initial molecular-replacement solution has been obtained and refinement is in progress.
doi:10.1107/S1744309111030971
PMCID: PMC3212367  PMID: 22102032
Oryza sativa L.; betaine aldehyde dehydrogenases; OsBADH2; fragrant rice
16.  Rice (Oryza) hemoglobins 
F1000Research  2014;3:253.
Hemoglobins (Hbs) corresponding to non-symbiotic (nsHb) and truncated (tHb) Hbs have been identified in rice ( Oryza). This review discusses the major findings from the current studies on rice Hbs. At the molecular level, a family of the nshb genes, consisting of hb1, hb2, hb3, hb4 and hb5, and a single copy of the thb gene exist in Oryza sativa var. indica and O. sativa var. japonica, Hb transcripts coexist in rice organs and Hb polypeptides exist in rice embryonic and vegetative organs and in the cytoplasm of differentiating cells. At the structural level, the crystal structure of rice Hb1 has been elucidated, and the structures of the other rice Hbs have been modeled. Kinetic analysis indicated that rice Hb1 and 2, and possibly rice Hb3 and 4, exhibit an extremely high affinity for O 2, whereas rice Hb5 and tHb possibly exhibit a low to moderate affinity for O 2. Based on the accumulated information on the properties of rice Hbs and data from the analysis of other plant and non-plant Hbs, it is likely that Hbs play a variety of roles in rice organs, including O 2-transport, O 2-sensing, NO-scavenging and redox-signaling. From an evolutionary perspective, an outline for the evolution of rice Hbs is available. Rice nshb and thb genes vertically evolved through different lineages, rice nsHbs evolved into clade I and clade II lineages and rice nshbs and thbs evolved under the effect of neutral selection. This review also reveals lacunae in our ability to completely understand rice Hbs. Primary lacunae are the absence of experimental information about the precise functions of rice Hbs, the properties of modeled rice Hbs and the cis-elements and trans-acting factors that regulate the expression of rice hb genes, and the partial understanding of the evolution of rice Hbs.
doi:10.12688/f1000research.5530.1
PMCID: PMC4304225  PMID: 25653837
Evolution; function; gene expression; non-symbiotic; structure; symbiotic; truncated
17.  Rice ( Oryza) hemoglobins 
F1000Research  2014;3:253.
Hemoglobins (Hbs) corresponding to non-symbiotic (nsHb) and truncated (tHb) Hbs have been identified in rice ( Oryza). This review discusses the major findings from the current studies on rice Hbs. At the molecular level, a family of the nshb genes, consisting of hb1, hb2, hb3, hb4 and hb5, and a single copy of the thb gene exist in Oryza sativa var. indica and O. sativa var. japonica, Hb transcripts coexist in rice organs and Hb polypeptides exist in rice embryonic and vegetative organs and in the cytoplasm of differentiating cells. At the structural level, the crystal structure of rice Hb1 has been elucidated, and the structures of the other rice Hbs have been modeled. Kinetic analysis indicated that rice Hb1 and 2, and possibly rice Hb3 and 4, exhibit a very high affinity for O 2, whereas rice Hb5 and tHb possibly exhibit a low to moderate affinity for O 2. Based on the accumulated information on the properties of rice Hbs and data from the analysis of other plant and non-plant Hbs, it is likely that Hbs play a variety of roles in rice organs, including O 2-transport, O 2-sensing, NO-scavenging and redox-signaling. From an evolutionary perspective, an outline for the evolution of rice Hbs is available. Rice nshb and thb genes vertically evolved through different lineages, rice nsHbs evolved into clade I and clade II lineages and rice nshbs and thbs evolved under the effect of neutral selection. This review also reveals lacunae in our ability to completely understand rice Hbs. Primary lacunae are the absence of experimental information about the precise functions of rice Hbs, the properties of modeled rice Hbs and the cis-elements and trans-acting factors that regulate the expression of rice hb genes, and the partial understanding of the evolution of rice Hbs.
doi:10.12688/f1000research.5530.2
PMCID: PMC4304225  PMID: 25653837
Evolution; function; gene expression; non-symbiotic; structure; symbiotic; truncated
18.  Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of β-ketoacyl-ACP synthase III (FabH) from Xanthomonas oryzae pv. oryzae  
The ketoacyl-acyl carrier protein synthase III (KASIII) encoded by the fabH gene of X. oryzae pv. oryzae is responsible for elongation by two C atoms in fatty-acid synthesis. The fabH gene was cloned, the KASIII enzyme was expressed and preliminary crystallographic study was performed with the aim of understanding the reaction mechanism.
The bacterial β-ketoacyl-ACP synthase III (KASIII) encoded by the gene fabH (Xoo4209) from Xanthomonas oryzae pv. oryzae, a plant pathogen, is an important enzyme in the elongation steps of fatty-acid biosynthesis. It is expected to be one of the enzymes responsible for bacterial blight (BB), a serious disease that results in huge production losses of rice. As it represents an important target for the development of new antibacterial drugs against BB, determination of the crystal structure of the KAS III enzyme is essential in order to understand its reaction mechanism. In order to analyze the structure and function of KAS III, the fabH (Xoo4209) gene was cloned and the enzyme was expressed and purified. The KASIII crystal diffracted to 2.05 Å resolution and belonged to the orthorhombic space group P21212, with unit-cell parameters a = 69.8, b = 79.5, c = 62.3 Å. The unit-cell volume of the crystal is compatible with the presence of a single monomer in the asymmetric unit, with a corresponding Matthews coefficient V M of 2.27 Å3 Da−1 and a solvent content of 45.8%.
doi:10.1107/S1744309109009555
PMCID: PMC2675584  PMID: 19407376
bacterial blight; β-ketoacyl-ACP synthase III; fabH; Xanthomonas oryzae pv. oryzae
19.  Correlation of LNCR rasiRNAs Expression with Heterochromatin Formation during Development of the Holocentric Insect Spodoptera frugiperda 
PLoS ONE  2011;6(9):e24746.
Repeat-associated small interfering RNAs (rasiRNAs) are derived from various genomic repetitive elements and ensure genomic stability by silencing endogenous transposable elements. Here we describe a novel subset of 46 rasiRNAs named LNCR rasiRNAs due to their homology with one long non-coding RNA (LNCR) of Spodoptera frugiperda. LNCR operates as the intermediate of an unclassified transposable element (TE-LNCR). TE-LNCR is a very invasive transposable element, present in high copy numbers in the S. frugiperda genome. LNCR rasiRNAs are single-stranded RNAs without a prominent nucleotide motif, which are organized in two distinct, strand-specific clusters. The expression of LNCR and LNCR rasiRNAs is developmentally regulated. Formation of heterochromatin in the genomic region where three copies of the TE-LNCR are embedded was followed by chromatin immunoprecipitation (ChIP) and we observed this chromatin undergo dynamic changes during development. In summary, increased LNCR expression in certain developmental stages is followed by the appearance of a variety of LNCR rasiRNAs which appears to correlate with subsequent accumulation of a heterochromatic histone mark and silencing of the genomic region with TE-LNCR. These results support the notion that a repeat-associated small interfering RNA pathway is linked to heterochromatin formation and/or maintenance during development to establish repression of the TE-LNCR transposable element. This study provides insights into the rasiRNA silencing pathway and its role in the formation of fluctuating heterochromatin during the development of one holocentric organism.
doi:10.1371/journal.pone.0024746
PMCID: PMC3184123  PMID: 21980354
20.  Activation of Mitogen-activated Protein Kinase (Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase) Cascade by Aldosterone 
Molecular Biology of the Cell  2002;13(9):3042-3054.
Aldosterone in some tissues increases expression of the mRNA encoding the small monomeric G protein Ki-RasA. Renal A6 epithelial cells were used to determine whether induction of Ki-ras leads to concomitant increases in the total as well as active levels of Ki-RasA and whether this then leads to subsequent activation of its effector mitogen-activated protein kinase (MAPK/extracellular signal-regulated kinase) cascade. The molecular basis and cellular consequences of this action were specifically investigated. We identified the intron 1-exon 1 region (rasI/E1) of the mouse Ki-ras gene as sufficient to reconstitute aldosterone responsiveness to a heterologous promotor. Aldosterone increased reporter gene activity containing rasI/E1 threefold. Aldosterone increased the absolute and GTP-bound levels of Ki-RasA by a similar extent, suggesting that activation resulted from mass action and not effects on GTP binding/hydrolysis rates. Aldosterone significantly increased Ki-RasA and MAPK activity as early as 15 min with activation peaking by 2 h and waning after 4 h. Inhibitors of transcription, translation, and a glucocorticoid receptor antagonist attenuated MAPK signaling. Similarly, rasI/E1-driven luciferase expression was sensitive to glucocorticoid receptor blockade. Overexpression of dominant-negative RasN17, addition of antisense Ki-rasA and inhibition of mitogen-activated protein kinase kinase also attenuated steroid-dependent increases in MAPK signaling. Thus, activation of MAPK by aldosterone is dependent, in part, on a genomic mechanism involving induction of Ki-ras transcription and subsequent activation of its downstream effectors. This genomic mechanism has a distinct time course from activation by traditional mitogens, such as serum, which affect the GTP-binding state and not absolute levels of Ras. The result of such a genomic mechanism is that peak activation of the MAPK cascade by adrenal corticosteroids is delayed but prolonged.
doi:10.1091/mbc.E02-05-0260
PMCID: PMC124141  PMID: 12221114
21.  Overexpression, purification, crystallization and preliminary X-ray cystallographic studies of a proline-specific aminopeptidase from Aneurinibacillus sp. strain AM-1 
Preliminary X-ray crystallographic study of a proline-specific aminopepitdase from Aneurinibacillus sp, strain AM-1 was carried out.
To elucidate the structure and molecular mechanism of a characteristic proline-specific aminopeptidase produced by the thermophile Aneurinibacillus sp. strain AM-1, its gene was cloned and the recombinant protein was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.8 Å resolution from the recombinant aminopeptidase crystal. The crystals belong to the orthorhombic space group P21212, with unit-cell parameters a = 93.62, b = 68.20, c = 76.84 Å. A complete data set was also obtained from crystals of SeMet-substituted aminopeptidase. Data in the resolution range 20–2.1 Å from the MAD data set from the SeMet-substituted crystal were used for phase determination.
doi:10.1107/S1744309106047543
PMCID: PMC2225360  PMID: 17142913
proline-specific aminopeptidase; Aneurinibacillus sp. strain AM-1; thermophiles
22.  Performance of pre-commercial release formulations of spinosad against five stored-product insect species on four stored commodities 
Journal of Pest Science  2011;85(3):331-339.
Two liquid and one dry pre-commercial release spinosad formulations were evaluated at the labeled rate of 1 ppm against five stored-grain insect species on wheat, short-grain rice, long-grain rice, and maize. Except on maize, efficacy of spinosad was compared with a currently registered grain protectant, chlorpyrifos-methyl (3 ppm) plus deltamethrin (0.5 ppm). The 7- and 14-day mortalities of the lesser grain borer, Rhyzopertha dominica, were 99.0–100.0% on spinosad and chlorpyrifos-methyl plus deltamethrin-treated wheat, short-grain rice, and long-grain rice. Adult progeny of R. dominica after 42 days on these commodities decreased by 99.7–100.0% relative to progeny production on untreated wheat. Mortality and reduction in adult progeny of the rice weevil, Sitophilus oryzae, on the three commodities, and that of the maize weevil, Sitophilus zeamais, on maize and the red flour beetle, Tribolium castaneum, on wheat were 100.0% only with chlorpyrifos-methyl plus deltamethrin. The liquid spinosad formulations were most effective against the Indianmeal moth, Plodia interpunctella, on maize and wheat. Except for R. dominica, the effectiveness of spinosad on the other species varied with the formulation, exposure time, and commodity. Chlorpyrifos-methyl plus deltamethrin was effective against insect species on the commodities tested.
doi:10.1007/s10340-011-0395-9
PMCID: PMC3432197  PMID: 22962550
Spinosad formulations; Grain protectants; Stored-grain insects; Efficacy assessment
23.  Crystallization and initial X-ray diffraction studies of the flavoenzyme NAD(P)H:(acceptor) oxidoreductase (FerB) from the soil bacterium Paracoccus denitrificans  
The flavin-dependent enzyme FerB from P. denitrificans has been purified and both native and SeMet-substituted FerB have been crystallized. The two variants crystallized in two different crystallographic forms belonging to the monoclinic space group P21 and the orthorhombic space group P21212, respectively. X-ray diffraction data were collected to 1.75 Å resolution for both forms.
The flavin-dependent enzyme FerB from Paracoccus denitrificans reduces a broad range of compounds, including ferric complexes, chromate and most notably quinones, at the expense of the reduced nicotinamide adenine dinucleotide cofactors NADH or NADPH. Recombinant unmodified and SeMet-substituted FerB were crystallized under similar conditions by the hanging-drop vapour-diffusion method with microseeding using PEG 4000 as the precipitant. FerB crystallized in several different crystal forms, some of which diffracted to approximately 1.8 Å resolution. The crystals of native FerB belonged to space group P21, with unit-cell parameters a = 61.6, b = 110.1, c = 65.2 Å, β = 118.2° and four protein molecules in the asymmetric unit, whilst the SeMet-substituted form crystallized in space group P21212, with unit-cell parameters a = 61.2, b = 89.2, c = 71.5 Å and two protein molecules in the asymmetric unit. Structure determination by the three-wavelength MAD/MRSAD method is now in progress.
doi:10.1107/S1744309110005099
PMCID: PMC2852337  PMID: 20383015
flavoenzymes; quinone reductases; Paracoccus denitrificans
24.  Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of 3-dehydroquinate synthase, Xoo1243, from Xanthomonas oryzae pv. oryzae  
Bacterial blight is a destructive disease of rice that is caused by X. oryzae pv. oryzae (Xoo). Dehydroquinate synthase, which is the second enzyme of the shikimate pathway, was cloned from Xoo1243 (aroB), purified and crystallized in order to elucidate its three-dimensional structure.
The disease bacterial blight results in serious production losses of rice in Asian countries. The aroB gene encoding dehydroquinate synthase (DHQS), which is a potential antibiotic target, was identified from the plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae (Xoo). DHQS plays an essential role in the synthesis of aromatic compounds in the shikimate pathway. The aroB gene (Xoo1243) was cloned from Xoo and the corresponding DHQS protein was subsequently overexpressed in Escherichia coli. The purified protein was crystallized using the hanging-drop vapour-diffusion method and yielded crystals that diffracted to 2.5 Å resolution. The crystals belonged to the tetragonal space group P43212, with unit-cell parameters a = b = 118.2, c = 98.2 Å. According to a Matthews coefficient calculation, the crystal contained two molecules in the asymmetric unit, with a corresponding V M of 2.06 Å3 Da−1 and a solvent content of 40.4%.
doi:10.1107/S1744309108033575
PMCID: PMC2593707  PMID: 19052366
aroB; bacterial blight; 3-dehydroquinate synthase; shikimate pathway; Xanthomonas oryzae pv. oryzae
25.  Crystallization and preliminary X-ray crystallographic study of disproportionating enzyme from potato 
Disproportionating enzyme from potato was crystallized and preliminarily analyzed using X-ray diffraction.
Disproportionating enzyme (D-enzyme; EC 2.4.1.25) is a 59 kDa protein that belongs to the α-amylase family. D-enzyme catalyses intramolecular and intermolecular transglycosylation reactions of α-1,4 glucan. A crystal of the D-­enzyme from potato was obtained by the hanging-drop vapour-diffusion method. Preliminary X-ray data showed that the crystal diffracts to 2.0 Å resolution and belongs to space group C2221, with unit-cell parameters a = 69.7, b = 120.3, c = 174.2 Å.
doi:10.1107/S1744309104030829
PMCID: PMC1952372  PMID: 16508106
disproportionating enzyme; intramolecular and intermolecular transglycosylation reactions

Results 1-25 (1038266)