The ‘Nucleobase-Cation-Symport-1’, NCS1, transporters are essential components of salvage pathways for nucleobases and related metabolites. Here, we report the 2.85 Å resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, ten of which are arranged in two inverted repeats of 5 helices. The structures of the outward-facing open and substrate-bound occluded conformations were solved showing how the outward-facing cavity closes upon binding of substrate. Comparisons with the leucine (LeuTAa) and the galactose (vSGLT) transporters reveal that the outward- and inward-facing cavities are symmetrically arranged on opposite sides of the membrane. The reciprocal opening and closing of these cavities is synchronised by the inverted repeat helices 3 and 8, providing the structural basis of the ‘alternating access’ model for membrane transport.
The hatching enzyme of zebrafish, ZHE1, was expressed, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal belonged to space group P212121 and diffracted X-rays to a resolution of 1.14 Å.
The hatching enzyme of the zebrafish, ZHE1 (29.3 kDa), is a zinc metalloprotease that catalyzes digestion of the egg envelope (chorion). ZHE1 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. Two diffraction data sets with resolution ranges 50.0–1.80 and 50.0–1.14 Å were independently collected from two crystals and were merged to give a highly complete data set over the full resolution range 50.0–1.14 Å. The space group was assigned as primitive orthorhombic P212121, with unit-cell parameters a = 32.9, b = 62.5, c = 87.4 Å. The crystal contained one ZHE1 molecule in the asymmetric unit.
ZHE1; hatching enzymes; zebrafish
Crystals of the helicase domain from a tomato mosaic virus replication protein obtained using the hanging-drop vapour-diffusion method at 285 K diffracted X-rays to 2.05 Å resolution. They belonged to the orthorhombic space group P212121, with unit-cell parameters a = 85.8, b = 128.3, c = 40.7 Å.
Tomato mosaic virus belongs to the genus Tobamovirus in the alphavirus-like superfamily of positive-strand RNA viruses. The alphavirus-like superfamily includes many plant and animal viruses of agronomical and clinical importance. These viruses encode replication-associated proteins that contain a putative superfamily 1 helicase domain. No three-dimensional structures for this domain have been determined to date. Here, the crystallization and preliminary X-ray diffraction analysis of the 130K helicase domain are reported. Diffraction data were collected and processed to 2.05 and 1.75 Å resolution from native and selenomethionine-labelled crystals, respectively. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 85.8, b = 128.3, c = 40.7 Å.
tomato mosaic virus; replicase protein; helicase domain
Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members.
A novel family GH16 β-agarase from the marine bacterium Zobellia galactanivorans was expressed, purified and crystallized. Hexagonal crystals belonging to space group P3121 diffracted to 2.2 Å resolution, whereas orthorhombic crystals belonging to space group P212121 diffracted to 1.5–Å resolution.
Marine bacteria secrete specific glycoside hydrolases such as agarases to access polysaccharides from algal cell walls as a carbon and energy source. In an attempt to identify agarases with variable degradation patterns, a novel family GH16 β-agarase from the marine bacterium Zobellia galactanivorans was expressed, purified and crystallized. The purified enzyme crystallized in two distinct forms that were grown by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. Hexagonal crystals belonging to space group P3121 diffracted to 2.2 Å resolution, whereas orthorhombic crystals belonging to space group P212121 diffracted to 1.5 Å resolution.
β-agarases; glycoside hydrolases; heterologous expression; Zobellia galactanivorans
The dihydropyrimidinase from S. meliloti CECT4114, with activity towards both hydantoin and dihydrouracil substrates, was crystallized, and diffraction data were collected to 1.85 Å resolution.
Dihydropyrimidinases are involved in the reductive pathway of pyrimidine degradation, catalysing the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamoyl β-amino acids. This enzyme has often been referred to as hydantoinase owing to its industrial application in the production of optically pure amino acids starting from racemic mixtures of 5-monosubstituted hydantoins. Recombinant dihydropyrimidinase from Sinorhizobium meliloti CECT4114 (SmelDhp) has been expressed, purified and crystallized. Crystallization was performed using the counter-diffusion method with capillaries of 0.3 mm inner diameter. Crystals of SmelDhp suitable for data collection and structure determination were grown in the presence of agarose at 0.1%(w/v) in order to ensure mass transport controlled by diffusion. X-ray data were collected to a resolution of 1.85 Å. The crystal belongs to the orthorhombic space group C2221, with unit-cell parameters a = 124.89, b = 126.28, c = 196.10 Å and two molecules in the asymmetric unit. A molecular-replacement solution has been determined and refinement is in progress.
dihydropyrimidinases; pyrimidine degradation
A novel type of phosphoserine phosphatase from H. thermophilus TK-6 was heterologously expressed in E. coli, purified and crystallized by the sitting-drop vapour-diffusion method. The crystals obtained belonged to space group P212121 and diffracted X-rays to 1.5 Å resolution.
Two novel-type phosphoserine phosphatases (PSPs) with unique substrate specificity from the thermophilic and hydrogen-oxidizing bacterium Hydrogenobacter thermophilus TK-6 have previously been identified. Here, one of the PSPs (iPSP1) was heterologously expressed in Escherichia coli, purified and crystallized. Diffraction-quality crystals were obtained by the sitting-drop vapour-diffusion method using PEG 4000 as the precipitant. Two diffraction data sets with resolution ranges of 45.0–2.50 and 45.0–1.50 Å were collected from a single crystal and were merged to give a highly complete data set. The space group of the crystal was identified as primitive orthorhombic P212121, with unit-cell parameters a = 49.8, b = 73.6, c = 124.3 Å. The calculated Matthews coefficient (V
M = 2.32 Å3 Da−1) indicated that the crystal contained one iPSP1 complex per asymmetric unit.
phosphoserine phosphatases; Hydrogenobacter thermophilus TK-6
The substrate binding protein (SP_0149) of an ABC transporter from Streptococcus pneumoniae was molecularly cloned, overexpressed and purified. Diffraction quality crystals were grown using the hanging-drop vapour-diffusion technique.
A truncated (29 residues from the N-terminus) and N-terminal His-tagged form of SP_0149 from pneumococcal strain ATCC BAA-334 was overexpressed and purified to homogeneity using affinity and gel-filtration chromatography. Diffraction quality crystals were grown at 293 K using the hanging-drop vapour-diffusion technique. X-ray diffraction data were collected to 2.3 Å resolution from a single-crystal that belonged to the orthorhombic space group P212121 with the unit-cell parameters a = 54.56, b = 75.61, c = 75.52 Å. The calculated values of the Matthews coefficient assuming one molecule (with calculated molecular weight of 30 400 Da) in the crystal asymmetric unit and the corresponding solvent content were 2.56 Å3 Da−1 and 52.0%, respectively.
ABC transporter; Streptococcus pneumoniae; SP_0149; ATCC BAA-334
An exo-β-d-glucosaminidase from T. reesei (Gls93) has been crystallized by the hanging-drop vapour-diffusion method. Diffraction data have been collected using synchrotron radiation.
Chitosan is degraded to glucosamine (GlcN) by chitosanase and exo-β-d-glucosaminidase (GlcNase). GlcNase from Trichoderma reesei (Gls93) is a 93 kDa extracellular protein composed of 892 amino acids. The enzyme liberates GlcN from the nonreducing end of the chitosan chain in an exo-type manner and belongs to glycoside hydrolase family 2. For crystallographic investigations, Gls93 was overexpressed in Pichia pastoris cells. The recombinant Gls93 had two molecular forms of ∼105 kDa (Gls93-F1) and ∼100 kDa (Gls93-F2), with the difference between them being caused by N-glycosylation. Both forms were crystallized by the hanging-drop vapour-diffusion method. Crystals of Gls93-F1 belonged to the orthorhombic space group P212121, with unit-cell parameters a = 98.27, b = 98.42, c = 108.28 Å, and diffracted to 1.8 Å resolution. Crystals of Gls93-F2 belonged to the orthorhombic space group P212121, with unit-cell parameters a = 67.84, b = 81.62, c = 183.14 Å, and diffracted to 2.4 Å resolution. Both crystal forms were suitable for X-ray structure analysis at high resolution.
exo-β-d-glucosaminidases; exochitosanases; Gls93; Trichoderma reesei
A novel haemagglutinin from Jatropha curcas seeds is purified and crystallized. X-ray diffraction data collected from the rod-shaped crystals were processed in the orthorhombic space group P212121 and the crystals diffracted to 2.8 Å resolution at 103 K.
The plant Jatropha curcas (Euphorbiaceae) is an important source of biofuel from the inedible oil present in its toxic seeds. The toxicity arises from the presence of curcin, a ribosome-inactivating protein showing haemagglutination activity. In this communication, the purification, crystallization and preliminary X-ray characterization are reported of a small protein isolated from J. curcas seeds with a molecular mass of ∼10 kDa that agglutinates rabbit erythrocytes. The protein was crystallized using the hanging-drop vapour-diffusion method and also by the microbatch method in 72-well HLA plates, using PEG 8000 as the precipitant in both conditions. X-ray diffraction data collected from the rod-shaped crystals were processed in the orthorhombic space group P212121. The crystals diffracted to 2.8 Å resolution at 103 K.
Jatropha curcas; haemagglutinins
Cyanide-insensitive alternative oxidase from T. brucei brucei has been purified and crystallized for X-ray structure analysis.
Cyanide-insensitive alternative oxidase (AOX) is a mitochondrial membrane protein and a non-proton-pumping ubiquinol oxidase that catalyzes the four-electron reduction of dioxygen to water. In the African trypanosomes, trypanosome alternative oxidase (TAO) functions as a cytochrome-independent terminal oxidase that is essential for survival in the mammalian host; hence, the enzyme is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in haem-deficient Escherichia coli was purified and crystallized at 293 K by the hanging-drop vapour-diffusion method using polyethylene glycol 400 as a precipitant. X-ray diffraction data were collected at 100 K and were processed to 2.9 Å resolution with 93.1% completeness and an overall R
merge of 9.5%. The TAO crystals belonged to the orthorhombic space group I222 or I212121, with unit-cell parameters a = 63.11, b = 136.44, c = 223.06 Å. Assuming the presence of two rTAO molecules in the asymmetric unit (2 × 38 kDa), the calculated Matthews coefficient (V
M) was 3.2 Å3 Da−1, which corresponds to a solvent content of 61.0%. This is the first report of a crystal of the membrane-bound diiron proteins, which include AOXs.
alternative oxidases; membrane proteins; Trypanosoma brucei brucei; ascofuranone
Crystallization and X-ray diffraction data collection of the Fab fragment of the monoclonal antibody WO2 in the absence or presence of amyloid β peptides associated with Alzheimer’s disease are reported.
The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid β peptide (Aβ) associated with Alzheimer’s disease. This region of Aβ has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Aβ peptides Aβ1–16 and Aβ1–28 are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO4; they belonged to the orthorhombic space group P212121 and diffracted to 1.6 Å resolution. The complexes of WO2 Fab with either Aβ1–16 or Aβ1–28 were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P212121, and diffracted to 1.6 Å resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble Aβ1–42 in PEG 550 MME. This second form belonged to space group P21 and diffracted to 1.9 Å resolution.
Aβ peptides; Alzheimer’s disease; antibody Fab fragments; immunotherapy
Grass carp β2-microglobulin was expressed in E. coli, purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method with PEG 2K as precipitant. The crystals obtained belong to the orthorhombic space group P212121, with unit-cell parameters a = 38.72, b = 40.65, c = 71.12 Å.
β2-Microglobulin (β2m) is an essential subunit of MHC I molecules; it stabilizes the structure of MHC I and plays a pivotal role in coreceptor recognition. To date, structures of β2m have been solved for three different mammals: human, mouse and cattle. In order to illuminate the molecular evolutionary origin of β2m, an understanding of its structure in lower vertebrates becomes important. Here, grass carp (Ctenopharyngodon idellus) β2m (Ctid-β2m) was expressed, purified and crystallized. Diffraction data were collected to a resolution of 2.5 Å. The crystal belongs to space group P212121, with unit-cell parameters a = 38.72, b = 40.65, c = 71.12 Å. The Matthews coefficient and the solvent content were calculated to be 2.56 Å Da−1 and 52.07%, respectively, for one molecule per asymmetric unit. The structure has been solved by molecular replacement using monomeric human β2m as a model.
β2-microglobulin; MHC I
Thermostable recombinant L2 lipase from thermophilic Bacillus sp. L2 has been crystallized by using counter-diffusion method and diffracted to 2.7 Å resolution. The crystal belongs to the primitive orthorhombic space group P212121 with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 Å.
Purified thermostable recombinant L2 lipase from Bacillus sp. L2 was crystallized by the counter-diffusion method using 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl as precipitant. X-ray diffraction data were collected to 2.7 Å resolution using an in-house Bruker X8 PROTEUM single-crystal diffractometer system. The crystal belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 Å. The asymmetric unit contained one single molecule of protein, with a Matthews coefficient (V
M) of 2.85 Å3 Da−1 and a solvent content of 57%.
lipases; L2 lipase; Bacillus sp. L2; thermostable lipase; counter-diffusion method
The purification, crystallization and preliminary X-ray diffraction studies of vitamin D3 hydroxylase isolated from P. autotrophica are reported.
Vitamin D3 hydroxylase (Vdh) is a novel cytochrome P450 monooxygenase isolated from the actinomycete Pseudonocardia autotrophica and consisting of 403 amino-acid residues. Vdh catalyzes the activation of vitamin D3
via sequential hydroxylation reactions: these reactions involve the conversion of vitamin D3 (cholecalciferol or VD3) to 25-hydroxyvitamin D3 [25(OH)VD3] and the subsequent conversion of 25(OH)VD3 to 1α,25-dihydroxyvitamin D3 [calciferol or 1α,25(OH)2VD3]. Overexpression of recombinant Vdh was carried out using a Rhodococcus erythropolis expression system and the protein was subsequently purified and crystallized. Two different crystal forms were obtained by the hanging-drop vapour-diffusion method at 293 K using polyethylene glycol as a precipitant. The form I crystal belonged to the trigonal space group P31, with unit-cell parameters a = b = 61.7, c = 98.8 Å. There is one Vdh molecule in the asymmetric unit, with a solvent content of 47.6%. The form II crystal was grown in the presence of 25(OH)VD3 and belonged to the orthorhombic system P212121, with unit-cell parameters a = 63.4, b = 65.6 c = 102.2 Å. There is one Vdh molecule in the asymmetric unit, with a solvent content of 46.7%. Native data sets were collected to resolutions of 1.75 and 3.05 Å for form I and form II crystals, respectively, using synchrotron radiation. The structure solution was obtained by the molecular-replacement method and model refinement is in progress for the form I crystal.
cholecalciferol; CYP107; cytochrome P450 monooxygenase; hydroxylation; Pseudonocardia autotrophica; vitamin D3
Dextran glucosidase from S. mutans was crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.2 Å resolution.
Dextran glucosidase from Streptococcus mutans is an exo-hydrolase that acts on the nonreducing terminal α-1,6-glucosidic linkage of oligosaccharides and dextran with a high degree of transglucosylation. Based on amino-acid sequence similarity, this enzyme is classified into glycoside hydrolase family 13. Recombinant dextran glucosidase was purified and crystallized by the hanging-drop vapour-diffusion technique using polyethylene glycol 6000 as a precipitant. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 72.72, b = 86.47, c = 104.30 Å. A native data set was collected to 2.2 Å resolution from a single crystal.
dextran glucosidase; Streptococcus mutans; α-amylase family
A novel 15 kDa antifungal protein amaryllin has been crystallized using 30% PEG 8000 as the precipitating agent. The crystals belong to orthorhombic space group I or I212121 with cell dimensions, a = 48.6, b = 61.9 and c = 79.6–Å.
A novel antifungal protein, amaryllin, has been isolated from the underground bulbs of Amaryllis belladonna, purified to homogeneity and crystallized. The protein was extracted using ammonium sulfate fractionation. The purified protein samples indicated a molecular weight of 15 kDa on SDS–PAGE. The protein showed antifungal activity against Aspergillus flavus and Fusarium oxysporum. The N-terminal sequence of the first 15 amino-acid residues was determined using Edman degradation and did not show significant sequence identity to any known protein. The protein was crystallized using the hanging-drop vapour-diffusion method with 30% PEG 8000 as precipitating agent. The crystals diffracted to 2.7 Å resolution and belonged to the orthorhombic space group I222 or I212121, with unit-cell parameters a = 48.6, b = 61.9, c = 79.6 Å. The complete sequence and structure determination of amaryllin are in progress.
antifungal activity; pathogenesis-related proteins; N-terminal sequence and structure analysis
We present the first account of the structure–function relationships of a protein of the subfamily of urea/H+ membrane transporters of fungi and plants, using Aspergillus nidulans UreA as a study model. Based on the crystal structures of the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT) and of the Nucleobase-Cation-Symport-1 benzylhydantoin transporter from Microbacterium liquefaciens (Mhp1), we constructed a three-dimensional model of UreA which, combined with site-directed and classical random mutagenesis, led to the identification of amino acids important for UreA function. Our approach allowed us to suggest roles for these residues in the binding, recognition and translocation of urea, and in the sorting of UreA to the membrane. Residues W82, Y106, A110, T133, N275, D286, Y388, Y437 and S446, located in transmembrane helixes 2, 3, 7 and 11, were found to be involved in the binding, recognition and/or translocation of urea and the sorting of UreA to the membrane. Y106, A110, T133 and Y437 seem to play a role in substrate selectivity, while S446 is necessary for proper sorting of UreA to the membrane. Other amino acids identified by random classical mutagenesis (G99, R141, A163, G168 and P639) may be important for the basic transporter's structure, its proper folding or its correct traffic to the membrane.
sodium : solute symporter-family; structure–function relationships; permease
Human S100A13 protein was cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals obtained belonged to space group P212121 and diffracted to a resolution of 1.8 Å.
S100A13 is a member of the S100 family of EF-hand-containing calcium-binding proteins and plays an important role in the secretion of fibroblast growth factor-1 and interleukin 1α, two pro-angiogenic factors released by the endoplasmic reticulum/Golgi-independent non-classical secretory pathway. Human S100A13 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals diffracted X-rays from a synchrotron-radiation source to 1.8 Å resolution and the space group was assigned as primitive orthorhombic P212121.
S100A13; EF-hand calcium-binding proteins
Crystals of native intimin and its N916Y mutant from enterohaemorrhagic E. coli O157:H7 diffracted to 2.8 and 2.6 Å resolution, respectively.
Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a primarily food-borne bacterial pathogen that is capable of causing life-threatening human infections and poses a serious challenge to public health worldwide. The bacterial outer-membrane protein intimin plays a key role in the initiation process of EHEC infection. In this study, intimin from EHEC O157:H7 (Int188) and its N916Y mutant (IntN916Y) were purified and crystals of both were obtained using the hanging-drop vapour-diffusion method at 291 K. Data were collected from Int188 and IntN916Y crystals to 2.8 and 2.6 Å resolution, respectively. The crystal of Int188 belonged to the orthorhombic space group C2, with unit-cell parameters a = 235.16, b = 44.81, c = 129.12 Å, α = γ = 90, β = 97.53°. The crystal of IntN916Y belonged to space group P212121, with unit-cell parameters a = 43.78, b = 92.49, c = 100.05 Å, α = β = γ = 90°.
EHEC O157:H7; intimin; Tir proteins
The structure of the sodium-benzylhydantoin transport protein, Mhp1, from Microbacterium liquefaciens comprises a 5-helix inverted repeat, which is widespread amongst secondary transporters. Here we report the crystal structure of an inward-facing conformation of Mhp1 at 3.8 Å resolution, complementing its previously-described structures in outward-facing and occluded states. From analyses of the three structures and molecular dynamics simulations we propose a mechanism for the transport cycle in Mhp1. Switching from the outward- to the inward- facing state, to effect the inward release of sodium and benzylhydantoin, is primarily achieved by a rigid body movement of transmembrane helices 3, 4, 8 and 9 relative to the rest of the protein. This forms the basis of an alternating access mechanism applicable to many transporters of this emerging superfamily.
Recombinant cryptochrome 3 from A. thaliana with FAD and MTHF cofactors has been crystallized using the hanging-drop vapour-diffusion technique in the orthorhombic space group P212121 and X-ray diffraction data were collected to 1.9 Å resolution.
Cryptochromes are flavoproteins which serve as blue-light receptors in plants, animals, fungi and prokaryotes and belong to the same protein family as the catalytically active DNA photolyases. Cryptochrome 3 from the plant Arabidopsis thaliana (cry3; 525 amino acids, 60.7 kDa) is a representative of the novel cryDASH subfamily of UV-A/blue-light receptors and has been expressed as a mature FAD-containing protein in Escherichia coli without the signal sequence that directs the protein into plant organelles. The purified cryptochrome was found to be complexed to methenyltetrahydrofolate as an antenna pigment. Crystals of the cryptochrome–antenna pigment complex were obtained by vapour diffusion and display orthorhombic symmetry, with unit-cell parameters a = 76.298, b = 116.782, c = 135.024 Å. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The asymmetric unit comprises a cry3 dimer, the physiological role of which remains to be elucidated.
cryptochrome 3; light receptors
The Trypanosoma cruzi dihydroorotate dehydrogenase, a key enzyme in pyrimidine de novo biosynthesis and redox homeostasis, was crystallized in complex with its first reaction product, orotate.
Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD–orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 Å resolution using synchrotron radiation (λ = 0.900 Å). X-ray diffraction data were collected at 100 K and processed to 1.9 Å resolution with 98.2% completeness and an overall R
merge of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 Å. The presence of two molecules in the asymmetric unit (2 × 34 kDa) gives a crystal volume per protein weight (V
M) of 2.2 Å3 Da−1 and a solvent content of 44%.
dihydroorotate dehydrogenase; pyrimidine biosynthesis; Trypanosoma cruzi; fumarate reductase; redox homeostasis; structure-based drug design
Two trypsins from the gastric fluid of the marine crab C. pagurus were purified and crystallized and X-ray data were collected to 0.97 and 3.2 Å resolution.
The digestive fluid of the marine crab Cancer pagurus (Decapoda, Brachyura) contains highly stable proteases which display enhanced activity in aqueous mixtures of organic solvents. Three trypsins were isolated from the gastric fluid and two of them, C.p.TryII and C.p.TryIII, were purified to homogeneity by anion-exchange chromatography and crystallized by hanging-drop vapour diffusion. Diffraction data were collected at a synchrotron to 0.97 and 3.2 Å resolution, respectively. The crystal of C.p.TryII belongs to the orthorhombic space group P212121, with unit-cell parameters a = 52.06, b = 62.00, c = 71.66 Å. Based on the Matthews coefficient, one protein molecule per asymmetric unit is suggested. In contrast, crystals of C.p.TryIII, which belong to the cubic space group P213 with unit-cell parameters a = b = c = 215.4 Å, are assumed to contain 12 molecules per asymmetric unit.
Crustacea; crabs; Cancer pagurus; gastric fluid; digestive enzymes; trypsin
YgjG is a putrescine:2-oxoglutarate aminotransferase that belongs to the class III aminotransferases. In this study, YgjG from E. coli was overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method.
Putrescine, one of the polyamines that are found in virtually all living organisms, has been implicated as an important biological material. The protein YgjG is involved in the putrescine-degradation pathway in Escherichia coli. The enzyme is a putrescine:2-oxoglutarate aminotransferase that belongs to the class III aminotransferases. In this study, YgjG from E. coli was overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected to 2.1 Å resolution using synchrotron radiation. The crystal belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 121.1, b = 129.5, c = 131.3 Å, and is estimated to contain four molecules of YgjG per asymmetric unit.
YgjG; putrescine:2-oxoglutarate aminotransferases; polyamine degradation