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1.  Crystallization and preliminary crystallographic analysis of dextranase from Streptococcus mutans  
S. mutans dextranase was crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 1.6 Å and belonged to space group P21.
Streptococcus mutans dextranase hydrolyzes the internal α-1,6-linkages of dextran and belongs to glycoside hydrolase family 66. An N- and C-terminal deletion mutant of S. mutans dextranase was crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 1.6 Å and belonged to space group P21, with unit-cell parameters a = 53.2, b = 89.7, c = 63.3 Å, β = 102.3°. Assuming that the asymmetric unit of the crystal contained one molecule, the Matthews coefficient was calculated to be 4.07 Å3 Da−1; assuming the presence of two molecules in the asymmetric unit it was calculated to be 2.03 Å3 Da−1.
doi:10.1107/S1744309111038425
PMCID: PMC3232134  PMID: 22139161
dextran; dextranases; glycoside hydrolase family 66; isomaltooligosaccharides; Streptococcus mutans
2.  Crystal Structure of Silkworm Bombyx mori JHBP in Complex With 2-Methyl-2,4-Pentanediol: Plasticity of JH-Binding Pocket and Ligand-Induced Conformational Change of the Second Cavity in JHBP 
PLoS ONE  2013;8(2):e56261.
Juvenile hormones (JHs) control a diversity of crucial life events in insects. In Lepidoptera which major agricultural pests belong to, JH signaling is critically controlled by a species-specific high-affinity, low molecular weight JH-binding protein (JHBP) in hemolymph, which transports JH from the site of its synthesis to target tissues. Hence, JHBP is expected to be an excellent target for the development of novel specific insect growth regulators (IGRs) and insecticides. A better understanding of the structural biology of JHBP should pave the way for the structure-based drug design of such compounds. Here, we report the crystal structure of the silkworm Bombyx mori JHBP in complex with two molecules of 2-methyl-2,4-pentanediol (MPD), one molecule (MPD1) bound in the JH-binding pocket while the other (MPD2) in a second cavity. Detailed comparison with the apo-JHBP and JHBP-JH II complex structures previously reported by us led to a number of intriguing findings. First, the JH-binding pocket changes its size in a ligand-dependent manner due to flexibility of the gate α1 helix. Second, MPD1 mimics interactions of the epoxide moiety of JH previously observed in the JHBP-JH complex, and MPD can compete with JH in binding to the JH-binding pocket. We also confirmed that methoprene, which has an MPD-like structure, inhibits the complex formation between JHBP and JH while the unepoxydated JH III (methyl farnesoate) does not. These findings may open the door to the development of novel IGRs targeted against JHBP. Third, binding of MPD to the second cavity of JHBP induces significant conformational changes accompanied with a cavity expansion. This finding, together with MPD2-JHBP interaction mechanism identified in the JHBP-MPD complex, should provide important guidance in the search for the natural ligand of the second cavity.
doi:10.1371/journal.pone.0056261
PMCID: PMC3577830  PMID: 23437107
3.  Crystallization and preliminary crystallographic analysis of the glycoside hydrolase family 115 α-­glucuronidase from Streptomyces pristinaespiralis  
α-Glucuronidase from S. pristinaespiralis was crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group R3 and diffracted to a resolution of 1.9 Å.
α-Glucuronidase from Streptomyces pristinaespiralis (SpGlcA115A) is com­posed of a single-chain peptide containing a catalytic domain belonging to glycosyl hydrolase family 115, a novel family of hemicellulolytic α-glucuronidases. The enzyme catalyzes the hydrolysis of α-linked 4-O-methylglucuronosyl and glucuronosyl residues from both polymeric xylans and oligosaccharides. SpGlcA115A was crystallized at 293 K using the sitting-drop vapour-diffusion method. The crystals belonged to space group R3 and diffracted to a resolution of 1.9 Å.
doi:10.1107/S1744309110043721
PMCID: PMC3079975  PMID: 21206027
α-glucuronidase; glycosyl hydrolase family 115; Streptomyces pristinaespiralis
4.  Crystallization and preliminary X-ray diffraction studies of a terminal oxygenase of carbazole 1,9a-­dioxygenase from Novosphingobium sp. KA1 
The terminal oxygenase component (Oxy) of carbazole 1,9a-dioxygenase (CARDO) catalyzes dihydroxylation of the aromatic ring. The Oxy of CARDO from Novosphingobium sp. KA1 was crystallized and the crystals diffracted to a resolution of 2.1 Å.
Carbazole 1,9a-dioxygenase (CARDO) is the initial dioxygenase in the carbazole-degradation pathway of Novosphingobium sp. KA1. The CARDO from KA1 consists of a terminal oxygenase (Oxy), a putidaredoxin-type ferredoxin and a ferredoxin reductase. The Oxy from Novosphingobium sp. KA1 was crystallized at 277 K using the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant. Diffraction data were collected to a resolution of 2.1 Å. The crystals belonged to the monoclinic space group P21. Self-rotation function analysis suggested that the asymmetric unit contained two Oxy trimers; the Matthews coefficient and solvent content were calculated to be 5.9 Å3 Da−1 and 79.1%, respectively.
doi:10.1107/S1744309110034949
PMCID: PMC3001653  PMID: 21045300
carbazole; Novosphingobium; Rieske nonhaem iron oxygenases; sphingomonads; terminal oxygenases
5.  Structural insight into the substrate- and dioxygen-binding manner in the catalytic cycle of rieske nonheme iron oxygenase system, carbazole 1,9a-dioxygenase 
Background
Dihydroxylation of tandemly linked aromatic carbons in a cis-configuration, catalyzed by multicomponent oxygenase systems known as Rieske nonheme iron oxygenase systems (ROs), often constitute the initial step of aerobic degradation pathways for various aromatic compounds. Because such RO reactions inherently govern whether downstream degradation processes occur, novel oxygenation mechanisms involving oxygenase components of ROs (RO-Os) is of great interest. Despite substantial progress in structural and physicochemical analyses, no consensus exists on the chemical steps in the catalytic cycles of ROs. Thus, determining whether conformational changes at the active site of RO-O occur by substrate and/or oxygen binding is important. Carbazole 1,9a-dioxygenase (CARDO), a RO member consists of catalytic terminal oxygenase (CARDO-O), ferredoxin (CARDO-F), and ferredoxin reductase. We have succeeded in determining the crystal structures of oxidized CARDO-O, oxidized CARDO-F, and both oxidized and reduced forms of the CARDO-O: CARDO-F binary complex.
Results
In the present study, we determined the crystal structures of the reduced carbazole (CAR)-bound, dioxygen-bound, and both CAR- and dioxygen-bound CARDO-O: CARDO-F binary complex structures at 1.95, 1.85, and 2.00 Å resolution. These structures revealed the conformational changes that occur in the catalytic cycle. Structural comparison between complex structures in each step of the catalytic mechanism provides several implications, such as the order of substrate and dioxygen bindings, the iron-dioxygen species likely being Fe(III)-(hydro)peroxo, and the creation of room for dioxygen binding and the promotion of dioxygen binding in desirable fashion by preceding substrate binding.
Conclusions
The RO catalytic mechanism is proposed as follows: When the Rieske cluster is reduced, substrate binding induces several conformational changes (e.g., movements of the nonheme iron and the ligand residue) that create room for oxygen binding. Dioxygen bound in a side-on fashion onto nonheme iron is activated by reduction to the peroxo state [Fe(III)-(hydro)peroxo]. This state may react directly with the bound substrate, or O–O bond cleavage may occur to generate Fe(V)-oxo-hydroxo species prior to the reaction. After producing a cis-dihydrodiol, the product is released by reducing the nonheme iron. This proposed scheme describes the catalytic cycle of ROs and provides important information for a better understanding of the mechanism.
doi:10.1186/1472-6807-12-15
PMCID: PMC3423008  PMID: 22727022
6.  Crystallization and preliminary X-ray diffraction studies of a ferredoxin reductase component of carbazole 1,9a-dioxygenase from Novosphingobium sp. KA1 
The ferredoxin reductase component of carbazole 1,9a-dioxygenase (Red) is involved in electron transfer from NAD(P)H to ferredoxin. The class IIA Red from Novosphingobium sp. KA1 was crystallized and the crystal diffracted to a resolution of 1.58 Å.
Carbazole 1,9a-dioxygenase (CARDO) is the initial enzyme of the carbazole-degradation pathway. The CARDO of Novosphingobium sp. KA1 consists of a terminal oxygenase, a putidaredoxin-type ferredoxin and a ferredoxin-NADH oxidoreductase (Red) and is classified as a class IIA Rieske oxygenase. Red from KA1 was crystallized at 278 K by the hanging-drop vapour-diffusion method using PEG 4000. The crystal diffracted to 1.58 Å resolution and belonged to space group P32, with unit-cell parameters a = b = 92.2, c = 78.6 Å, α = γ = 90, β = 120°. Preliminary analysis of the X-ray diffraction data revealed that the asymmetric unit contained two Red monomers. The crystal appeared to be a merohedral twin, with a twin fraction of 0.32 and twin law (−h, −k, l).
doi:10.1107/S1744309110014491
PMCID: PMC2882777  PMID: 20516607
carbazole; Rieske nonhaem iron oxygenases; ferredoxin reductases
7.  Crystallization of selenomethionyl exo-β-1,3-galactanase from the basidiomycete Phanerochaete chrysosporium  
Selenomethionyl exo-β-1,3-galactanase from P. chrysosporium K-3 produced in Pichia pastoris was crystallized. The crystals diffracted to a resolution of 1.8 Å and belonged to space group P21.
Exo-β-1,3-galactanase from Phanerochaete chrysosporium (Pc1,3Gal43A) con­sists of a glycoside hydrolase family 43 catalytic domain and a substrate-binding domain that belongs to carbohydrate-binding module family 35. It catalyzes the hydrolysis of β-1,3-galactan, which is the backbone of the arabinogalactan proteins; the C-terminal carbohydrate-binding module family 35 domain increases the local concentration of the enzyme around β-1,3-galactan by its high affinity for the substrate. To enable phase determination using the multiwavelength anomalous dispersion method, selenomethionyl Pc1,3Gal43A was crystallized at 298 K using the hanging-drop vapour-diffusion method. The presence of selenium in the crystals was confirmed from the X-ray absorption spectrum. The crystals belonged to space group P21 and diffracted to 1.8 Å resolution.
doi:10.1107/S1744309109043395
PMCID: PMC2802879  PMID: 20054127
carbohydrate-binding module family 35; exo-β-1,3-galactanases; glycoside hydrolase family 43; Phanerochaete chrysosporium; Pichia pastoris; selenomethionine
8.  Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori 
Scientific Reports  2011;1:133.
Juvenile hormone (JH) plays crucial roles in many aspects of the insect life. All the JH actions are initiated by transport of JH in the hemolymph as a complex with JH-binding protein (JHBP) to target tissues. Here, we report structural mechanism of JH delivery by JHBP based upon the crystal and solution structures of apo and JH-bound JHBP. In solution, apo-JHBP exists in equilibrium of multiple conformations with different orientations of the gate helix for the hormone-binding pocket ranging from closed to open forms. JH-binding to the gate-open form results in the fully closed JHBP-JH complex structure where the bound JH is completely buried inside the protein. JH-bound JHBP opens the gate helix to release the bound hormone likely by sensing the less polar environment at the membrane surface of target cells. This is the first report that provides structural insight into JH signaling.
doi:10.1038/srep00133
PMCID: PMC3216614  PMID: 22355650
9.  Crystallization and preliminary crystallographic analysis of poly-γ-glutamate hydrolase from bacteriophage ΦNIT1 
Poly-γ-glutamate hydrolase from bacteriophage ΦNIT1 was crystallized by the sitting-drop vapour-diffusion method and the crystals diffracted to beyond 2.4 Å resolution.
Particular Bacillus subtilis strains produce a capsular polypeptide poly-γ-­glutamate (γ-PGA) that functions as a physical barrier against bacteriophage infection. Bacteriophage ΦNIT1 can infect B. subtilis and produces a novel γ-­PGA hydrolase PghP. PghP was overexpressed, purified and crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.4 Å using a synchrotron X-ray source and were found to belong to space group P3121 or P3221.
doi:10.1107/S1744309109029881
PMCID: PMC2795599  PMID: 19724131
poly-γ-glutamic acid; bacteriophage ΦNIT1; Bacillus subtilis; hydrolases
10.  Crystallization and preliminary crystallographic analysis of β-l-arabinopyranosidase from Streptomyces avermitilis NBRC14893 
β-l-Arabinopyranosidase from S. avermitilis NBRC14893 was crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted to 1.6 Å resolution and belonged to space group P212121.
β-l-Arabinopyranosidase from Streptomyces avermitilis NBRC14893 is a monomeric protein consisting of a catalytic domain belonging to glycosyl hydrolase family 27, an unknown domain and a substrate-binding domain belonging to carbohydrate-binding module family 13. The complete enzyme (residues 45–658) has successfully been cloned and homologously expressed in the Streptomyces expression system. β-l-Arabinopyranosidase was crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted to 1.6 Å resolution and belonged to space group P212121, with unit-cell parameters a = 68.2, b = 98.9, c = 181.3 Å. The Matthews coefficient was calculated to be 2.38 Å3 Da−1.
doi:10.1107/S1744309109017230
PMCID: PMC2688429  PMID: 19478450
β-l-arabinopyranosidase; glycoside hydrolase family 27; carbohydrate-binding module family 13
11.  Crystallization and preliminary crystallographic analysis of exo-α-1,5-l-arabinofuranosidase from Streptomyces avermitilis NBRC14893 
Exo-α-1,5-l-arabinofuranosidase from S. avermitilis NBRC14893 was crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.2 Å and belonged to space group P212121.
Exo-α-1,5-l-arabinofuranosidase from Streptomyces avermitilis NBRC14893 (SaAraf43A) is composed of a single-chain peptide containing a catalytic domain belonging to glycosyl hydrolase family 43 and a substrate-binding domain belonging to carbohydrate-binding module family 42. The enzyme catalyzes the hydrolysis of an α-linked l-arabinofuranosyl residue from hemicelluloses. SaAraf43A was crystallized at 293 K using the sitting-drop vapour-diffusion method. The crystals belonged to space group P212121 and diffracted to a resolution of 2.2 Å.
doi:10.1107/S1744309108029692
PMCID: PMC2581701  PMID: 18997327
α-l-arabinofuranosidases; carbohydrate-binding modules; glycosyl hydrolase family 43; Streptomyces avermitilis
12.  Crystallization and preliminary X-ray diffraction studies of a novel ferredoxin involved in the dioxygenation of carbazole by Novosphingobium sp. KA1 
The ferredoxin component of carbazole 1,9a-dioxygenase (CARDO-F) is involved in an electron-transfer reaction. The CARDO-F from Novosphingobium sp. KA1 was crystallized under anaerobic conditions and diffracted to a resolution of 1.9 Å.
Novosphingobium sp. KA1 uses carbazole 1,9a-dioxygenase (CARDO) as the first dioxygenase in its carbazole-degradation pathway. The CARDO of KA1 contains a terminal oxygenase component and two electron-transfer components: ferredoxin and ferredoxin reductase. In contrast to the CARDO systems of other species, the ferredoxin component of KA1 is a putidaredoxin-type protein. This novel ferredoxin was crystallized at 293 K by the hanging-drop vapour-diffusion method using PEG MME 550 as the precipitant under anaerobic conditions. The crystals belong to space group C2221 and diffraction data were collected to a resolution of 1.9 Å (the diffraction limit was 1.6 Å).
doi:10.1107/S1744309108016278
PMCID: PMC2443972  PMID: 18607094
carbazole; putidaredoxin-type proteins; Rieske nonhaem iron oxygenases
13.  Structures of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels: implications for movement of the C-terminal cysteine-rich domain 
The structures of pseudechetoxin and pseudecin suggest that both proteins bind to cyclic nucleotide-gated ion channels in a manner in which the concave surface occludes the pore entrance.
Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction by retinal photoreceptors and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins belong to a cysteine-rich secretory protein (CRISP) family containing an N-terminal pathogenesis-related proteins of group 1 (PR-1) domain and a C-terminal cysteine-rich domain (CRD). PsTx and Pdc are highly homologous proteins, but their blocking affinities on CNG channels are different: PsTx blocks both the olfactory and retinal channels with ∼15–30-fold higher affinity than Pdc. To gain further insights into their structure and function, the crystal structures of PsTx, Pdc and Zn2+-bound Pdc were determined. The structures revealed that most of the amino-acid-residue differences between PsTx and Pdc are located around the concave surface formed between the PR-1 domain and the CRD, suggesting that the concave surface is functionally important for CNG-channel binding and inhibition. A structural comparison in the presence and absence of Zn2+ ion demonstrated that the concave surface can open and close owing to movement of the CRD upon Zn2+ binding. The data suggest that PsTx and Pdc occlude the pore entrance and that the dynamic motion of the concave surface facilitates interaction with the CNG channels.
doi:10.1107/S0907444908023512
PMCID: PMC2725782  PMID: 18931410
CRISPs; CNG ion-channel blockers; snake venoms; Zn2+ binding; cysteine-rich domain
14.  Crystallization and preliminary crystallographic analysis of the ferredoxin component of carbazole 1,9a-dioxygenase from Nocardioides aromaticivorans IC177 
The ferredoxin component of carbazole 1,9a-dioxygenase from N. aromaticivorans IC177 was crystallized and diffraction data were collected to 2.0 Å resolution.
Carbazole 1,9a-dioxygenase (CARDO) catalyzes the dihydroxylation of carbazole by angular position (C9a) carbon bonding to the imino nitrogen and its adjacent C1 carbon. CARDO consists of a terminal oxygenase component and two electron-transfer components: ferredoxin and ferredoxin reductase. The ferredoxin component of carbazole 1,9a-dioxygenase from Nocardioides aromaticivorans IC177 was crystallized at 293 K using the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant. The crystals, which were improved by macroseeding, diffract to 2.0 Å resolution and belong to space group P41212.
doi:10.1107/S1744309107041437
PMCID: PMC2339720  PMID: 17909288
ferredoxins; carbazole; Rieske nonhaem iron oxygenase system; Rieske-type proteins
15.  Crystallization and preliminary X-ray diffraction studies of the ferredoxin reductase component in the Rieske nonhaem iron oxygenase system carbazole 1,9a-dioxygenase 
The NAD(P)H:ferredoxin oxidoreductase in carbazole 1,9a-dioxygenase from Janthinobacterium sp. J3 was crystallized and diffraction data were collected to 2.60 Å resolution.
Carbazole 1,9a-dioxygenase (CARDO), which consists of an oxygenase component (CARDO-O) and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), catalyzes dihydroxylation at the C1 and C9a positions of carbazole. CARDO-R was crystallized at 277 K using the hanging-drop vapour-diffusion method with the precipitant PEG 8000. Two crystal types (types I and II) were obtained. The type I crystal diffracted to a maximum resolution of 2.80 Å and belonged to space group P42212, with unit-cell parameters a = b = 158.7, c = 81.4 Å. The type II crystal was obtained in drops from which type I crystals had been removed; it diffracted to 2.60 Å resolution and belonged to the same space group, with unit-cell parameters a = b = 161.8, c = 79.5 Å.
doi:10.1107/S174430910702163X
PMCID: PMC2335075  PMID: 17554172
angular dioxygenases; NAD(P)H:ferredoxin oxidoreductases; Rieske nonhaem iron oxygenase system; electron transfer; carbazole
16.  Crystallization and preliminary X-ray diffraction studies of the terminal oxygenase component of carbazole 1,9a-dioxygenase from Nocardioides aromaticivorans IC177 
The terminal oxygenase component of carbazole 1,9a-dioxygenase from N. aromaticivorans IC177 was crystallized and diffraction data were collected to 2.30 Å resolution.
Carbazole 1,9a-dioxygenase (CARDO) catalyzes the dihydroxylation of carbazole by angular-position (C9a) carbon bonding to the imino nitrogen and its adjacent C1 carbon. CARDO consists of a terminal oxygenase component and two electron-transfer components: ferredoxin and ferredoxin reductase. The terminal oxygenase component (43.9 kDa) of carbazole 1,9a-dioxygenase from Nocardioides aromaticivorans IC177 was crystallized at 293 K using the hanging-drop vapour-diffusion method with PEG 8000 as the precipitant. The crystals diffract to 2.3 Å resolution and belong to space group C2.
doi:10.1107/S1744309106044939
PMCID: PMC2225353  PMID: 17142899
angular dioxygenases; carbazole; Rieske nonhaem iron oxygenase system; Rieske-type protein
17.  Characterization of a modular enzyme of exo-1,5-α-l-arabinofuranosidase and arabinan binding module from Streptomyces avermitilis NBRC14893 
A gene encoding an α-l-arabinofuranosidase, designated SaAraf43A, was cloned from Streptomyces avermitilis. The deduced amino acid sequence implies a modular structure consisting of an N-terminal glycoside hydrolase family 43 module and a C-terminal family 42 carbohydrate-binding module (CBM42). The recombinant enzyme showed optimal activity at pH 6.0 and 45°C and was stable over the pH range of 5.0–6.5 at 30°C. The enzyme hydrolyzed p-nitrophenol (PNP)-α-l-arabinofuranoside but did not hydrolyze PNP-α-l-arabinopyranoside, PNP-β-d-xylopyranoside, or PNP-β-d-galactopyranoside. Debranched 1,5-arabinan was hydrolyzed by the enzyme but arabinoxylan, arabinogalactan, gum arabic, and arabinan were not. Among the synthetic regioisomers of arabinofuranobiosides, only methyl 5-O-α-l-arabinofuranosyl-α-l-arabinofuranoside was hydrolyzed by the enzyme, while methyl 2-O-α-l-arabinofuranosyl-α-l-arabinofuranoside and methyl 3-O-α-l-arabinofuranosyl-α-l-arabinofuranoside were not. These data suggested that the enzyme only cleaves α-1,5-linked arabinofuranosyl linkages. The analysis of the hydrolysis product of arabinofuranopentaose suggested that the enzyme releases arabinose in exo-acting manner. These results indicate that the enzyme is definitely an exo-1,5-α-l-arabinofuranosidase. The C-terminal CBM42 did not show any affinity for arabinogalactan and debranched arabinan, although it bound arabinan and arabinoxylan, suggesting that the CBM42 bound to branched arabinofuranosyl residues. Removal of the module decreased the activity of the enzyme with regard to debranched arabinan. The CBM42 plays a role in enhancing the debranched arabinan hydrolytic action of the catalytic module in spite of its preference for binding arabinofuranosyl side chains.
doi:10.1007/s00253-008-1551-x
PMCID: PMC2518083  PMID: 18665359
α-l-Arabinofuranosidase; Glycoside hydrolase family 43; Carbohydrate binding module family 42; Arabinan binding module; Streptomyces avermitilis
18.  Overexpression, purification and crystallization of tyrosyl-tRNA synthetase from the hyperthermophilic archaeon Aeropyrum pernix K1 
Tyrosyl-tRNA synthetase from the hyperthermophilic archaeon A. pernix K1 was cloned, purified and crystallized. The crystals belonged to the tetragonal space group P43212, with unit-cell parameters a = b = 66.1, c = 196.2 Å, and diffracted to beyond 2.15 Å resolution at 100 K.
Hyperthermophilic archaeal tyrosyl-tRNA synthetase from Aeropyrum pernix K1 was cloned and overexpressed in Escherichia coli. The expressed protein was purified by Cibacron Blue affinity chromatography following heat treatment at 363 K. Crystals suitable for X-ray diffraction studies were obtained under optimized crystallization conditions in the presence of 1.5 M ammonium sulfate using the hanging-drop vapour-diffusion method. The crystals belonged to the tetragonal space group P43212, with unit-cell parameters a = b = 66.1, c = 196.2 Å, and diffracted to beyond 2.15 Å resolution at 100 K.
doi:10.1107/S1744309105033245
PMCID: PMC1978129  PMID: 16511219
aminoacyl-tRNA synthetase; tyrosine tRNA; Aeropyrum pernix K1
19.  Crystallization and preliminary X-ray diffraction analyses of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels 
Crystals of pseudechetoxin and pseudecin, potent peptidic inhibitors of cyclic nucleotide-gated ion channels, have been prepared and X-ray diffraction data have been collected to 2.25 and 1.90 Å resolution, respectively.
Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction of retinal and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins are structurally classified as cysteine-rich secretory proteins and exhibit structural features that are quite distinct from those of other known small peptidic channel blockers. This article describes the crystallization and preliminary X-ray diffraction analyses of these toxins. Crystals of PsTx belonged to space group P212121, with unit-cell parameters a = 60.30, b = 61.59, c = 251.69 Å, and diffraction data were collected to 2.25 Å resolution. Crystals of Pdc also belonged to space group P212121, with similar unit-cell parameters a = 60.71, b = 61.67, c = 251.22 Å, and diffraction data were collected to 1.90 Å resolution.
doi:10.1107/S1744309105020439
PMCID: PMC1952345  PMID: 16511147
CRISPs; CNG channels; channel blockers; snake venoms
20.  Crystallization and preliminary X-ray diffraction analysis of the electron-transfer complex between the terminal oxygenase component and ferredoxin in the Rieske non-haem iron oxygenase system carbazole 1,9a-dioxygenase 
The electron-transfer complex between the terminal oxygenase and ferredoxin of carbazole 1,9a-dioxygenase was crystallized and diffraction data were collected to 1.90 Å resolution.
Carbazole 1,9a-dioxygenase, which consists of an oxygenase component (CARDO-O) and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), catalyzes dihydroxylation at the C1 and C9a positions of carbazole. The electron-transport complex between CARDO-­O and CARDO-F crystallizes at 293 K using hanging-drop vapour diffusion with the precipitant PEG MME 2000 (type I crystals) or PEG 3350 (type II). Blossom-shaped crystals form from a pile of triangular plate-shaped crystals. The type I crystal diffracts to a maximum resolution of 1.90 Å and belongs to space group P21, with unit-cell parameters a = 97.1, b = 89.8, c = 104.9 Å, α = γ = 90, β = 103.8°. Diffraction data for the type I crystal gave an overall R merge of 8.0% and a completeness of 100%. Its V M value is 2.63 Å3 Da−1, indicating a solvent content of 53.2%.
doi:10.1107/S1744309105014557
PMCID: PMC1952320  PMID: 16511100
angular dioxygenases; carbazole; electron-transfer complexes; Rieske non-haem iron oxygenase systems; Rieske-type ferredoxins; Rieske-type proteins
21.  Crystallization and preliminary X-ray crystallographic studies of XynX, a family 10 xylanase from Aeromonas punctata ME-1 
XynX, a family 10 xylanase from A. punctata ME-1, was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to beyond 1.8 Å resolution.
Xylanases catalyze the hydrolysis of β-1,4-glycosidic linkages within the xylan backbone. XynX is a xylanase from Aeromonas punctata ME-1 and belongs to glycoside hydrolase family 10. While most xylanases show endo-type catalytic activities, XynX shows exo-like catalytic activities, selectively producing xylobiose from birchwood xylan. In this study, XynX was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 79.0, b = 88.6, c = 93.2 Å, and diffracted to beyond 1.8 Å resolution.
doi:10.1107/S1744309105002058
PMCID: PMC1952265  PMID: 16511010
Aeromonas punctata ME-1; glycoside hydrolase family 10; xylanases; xylobioses
22.  Crystallization and preliminary X-ray analysis of coagulation factor IX-binding protein from habu snake venom at pH 6.5 and 4.6 
Crystals of habu coagulation factor IX-binding protein have been obtained at pH 6.5 and 4.6 and characterized by X-ray diffraction.
Coagulation factor IX-binding protein isolated from Trimeresurus flavoviridis (IX-bp) is a C-type lectin-like protein. It is an anticoagulant protein consisting of homologous subunits A and B. The subunits both contain a Ca2+-binding site with differing affinity (K d values of 14 and 130 µM at pH 7.5). These binding characteristics are pH-dependent; under acidic conditions, the affinity of the low-affinity site was reduced considerably. In order to identify which site has high affinity and also to investigate the Ca2+-releasing mechanism, IX-bp was crystallized at pH 6.5 and 4.6. The crystals at pH 6.5 and 4.6 diffracted to 1.72 and 2.29 Å resolution, respectively; the former crystals belong to the monoclinic space group P21, with unit-cell parameters a = 60.7, b = 63.5, c = 66.9 Å, β = 117.0°, while the latter belong to the monoclinic space group C2, with a = 134.1, b = 37.8, c = 55.8 Å, β = 110.4°.
doi:10.1107/S1744309104032439
PMCID: PMC1952379  PMID: 16508118
coagulation factor binding protein; calcium binding; snake venoms; pH-dependent binding
23.  Complicated water orientations in the minor groove of the B-DNA decamer d(CCATTAATGG)2 observed by neutron diffraction measurements 
Nucleic Acids Research  2005;33(9):3017-3024.
It has long been suspected that the structure and function of a DNA duplex can be strongly dependent on its degree of hydration. By neutron diffraction experiments, we have succeeded in determining most of the hydrogen (H) and deuterium (D) atomic positions in the decameric d(CCATTAATGG)2 duplex. Moreover, the D positions in 27 D2O molecules have been determined. In particular, the complex water network in the minor groove has been observed in detail. By a combined structural analysis using 2.0 Å resolution X-ray and 3.0 Å resolution neutron data, it is clear that the spine of hydration is built up, not only by a simple hexagonal hydration pattern (as reported in earlier X-ray studies), but also by many other water bridges hydrogen-bonded to the DNA strands. The complexity of the hydration pattern in the minor groove is derived from an extraordinary variety of orientations displayed by the water molecules.
doi:10.1093/nar/gki616
PMCID: PMC1140084  PMID: 15914673
24.  Crystal Structure of Bacillus subtilis α-Amylase in Complex with Acarbose 
Journal of Bacteriology  2003;185(23):6981-6984.
The crystal structure of Bacillus subtilis α-amylase, in complex with the pseudotetrasaccharide inhibitor acarbose, revealed an hexasaccharide in the active site as a result of transglycosylation. After comparison with the known structure of the catalytic-site mutant complexed with the native substrate maltopentaose, it is suggested that the present structure represents a mimic intermediate in the initial stage of the catalytic process.
doi:10.1128/JB.185.23.6981-6984.2003
PMCID: PMC262715  PMID: 14617662
25.  Characteristics of Two Forms of α-Amylases and Structural Implication 
Applied and Environmental Microbiology  1999;65(10):4652-4658.
Complete (Ba-L) and truncated (Ba-S) forms of α-amylases from Bacillus subtilis X-23 were purified, and the amino- and carboxyl-terminal amino acid sequences of Ba-L and Ba-S were determined. The amino acid sequence deduced from the nucleotide sequence of the α-amylase gene indicated that Ba-S was produced from Ba-L by truncation of the 186 amino acid residues at the carboxyl-terminal region. The results of genomic Southern analysis and Western analysis suggested that the two enzymes originated from the same α-amylase gene and that truncation of Ba-L to Ba-S occurred during the cultivation of B. subtilis X-23 cells. Although the primary structure of Ba-S was approximately 28% shorter than that of Ba-L, the two enzyme forms had the same enzymatic characteristics (molar catalytic activity, amylolytic pattern, transglycosylation ability, effect of pH on stability and activity, optimum temperature, and raw starch-binding ability), except that the thermal stability of Ba-S was higher than that of Ba-L. An analysis of the secondary structure as well as the predicted three-dimensional structure of Ba-S showed that Ba-S retained all of the necessary domains (domains A, B, and C) which were most likely to be required for functionality as α-amylase.
PMCID: PMC91620  PMID: 10508102

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