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1.  Expression, purification, crystallization and X-ray analysis of 3-quinuclidinone reductase from Agrobacterium tumefaciens  
The purification and crystallization of 3-quinuclidinone reductase from A. tumefaciens allowed the collection of a diffraction data set to 1.72 Å resolution.
(R)-3-Quinuclidinol is a useful chiral building block for the synthesis of various pharmaceuticals and can be produced from 3-quinuclidinone by asymmetric reduction. A novel 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol with NADH as a cofactor. Recombinant AtQR was overexpressed in Escherichia coli, purified and crystallized with NADH using the sitting-drop vapour-diffusion method at 293 K. Crystals were obtained using a reservoir solution containing PEG 3350 as a precipitant. X-ray diffraction data were collected to 1.72 Å resolution on beamline BL-5A at the Photon Factory. The crystal belonged to space group P21, with unit-cell parameters a = 62.0, b = 126.4, c = 62.0 Å, β = 110.5°, and was suggested to contain four molecules in the asymmetric unit (V M = 2.08 Å3 Da−1).
doi:10.1107/S1744309112034951
PMCID: PMC3497986  PMID: 23027756
3-quinuclidinone reductase; Agrobacterium tumefaciens
2.  Expression, purification, crystallization and preliminary X-ray analysis of a novel N-substituted branched-chain l-amino-acid dioxygenase from Burkholderia ambifaria AMMD 
Diffraction data were collected to a limiting resolution of 2.4 Å from a crystal of selenomethionyl-labelled SadA, an l-amino-acid dioxygenase.
Ferrous ion- and α-ketoglutarate-dependent dioxygenase from Burkholderia ambifaria AMMD (SadA) catalyzes the C3-hydroxylation of N-substituted branched-chain l-amino acids, especially N-succinyl-l-leucine, coupled to the conversion of α-ketoglutarate to succinate and CO2. SadA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method at 293 K. Crystals of selenomethionine-substituted SadA were obtained using a reservoir solution containing PEG 3000 as the precipitant at pH 9.5 and diffracted X-rays to 2.4 Å resolution. The crystal belonged to space group P212121, with unit-cell parameters a = 49.3, b = 70.9, c = 148.2 Å. The calculated Matthews coefficient (V M = 2.1 Å3 Da−1, 41% solvent content) suggested that the crystal contains two molecules per asymmetric unit.
doi:10.1107/S1744309112031508
PMCID: PMC3433199  PMID: 22949196
C3-hydroxylation; dioxygenases; N-succinyl-l-leucine
3.  Purification, crystallization and preliminary X-ray analysis of SGR6054, a Streptomyces homologue of the mycobacterial integration host factor mIHF 
A Streptomyces homologue of the mycobacterial integration host factor mIHF was heterologously produced, purified and crystallized in the presence of a 16-mer duplex DNA by the sitting-drop vapour-diffusion method. The best crystal diffracted X-rays to 2.22 Å resolution and belonged to space group C2.
The mycobacterial integration host factor (mIHF) is a small nonspecific DNA-binding protein that is essential for the growth of Mycobacterium smegmatis. mIHF homologues are widely distributed among Actinobacteria, and a Streptomyces homologue of mIHF is involved in control of sporulation and antibiotic production in S. coelicolor A3(2). Despite their important biological functions, a structure of mIHF or its homologues has not been elucidated to date. Here, the S. griseus mIHF homologue (SGR6054) was expressed and purified from Escherichia coli and crystallized in the presence of a 16-mer duplex DNA by the sitting-drop vapour-diffusion method. The plate-shaped crystal belonged to space group C2, with unit-cell parameters a = 88.53, b = 69.35, c = 77.71 Å, β = 96.63°, and diffracted X-rays to 2.22 Å resolution.
doi:10.1107/S1744309112030631
PMCID: PMC3433204  PMID: 22949201
SGR6054; mycobacterial integration host factor; Streptomyces griseus
4.  Purification, crystallization and preliminary X-ray analysis of the DNA-binding domain of AdpA, the central transcription factor in the A-factor regulatory cascade in the filamentous bacterium Streptomyces griseus, in complex with a duplex DNA 
The DNA-binding domain of AdpA, the central transcription factor in the A-factor regulatory cascade in the filamentous bacterium S. griseus, was heterologously expressed, purified and crystallized in complex with a duplex DNA by the sitting-drop vapour-diffusion method. The best crystal diffracted X-rays to 2.8 Å resolution and belonged to space group C2221.
Streptomyces griseus AdpA is the central transcription factor in the A-factor regulatory cascade and activates a number of genes that are required for both secondary metabolism and morphological differentiation, leading to the onset of streptomycin biosynthesis as well as aerial mycelium formation and sporulation. The DNA-binding domain of AdpA consists of two helix–turn–helix DNA-binding motifs and shows low nucleotide-sequence specificity. To reveal the molecular basis of the low nucleotide-sequence specificity, an attempt was made to obtain cocrystals of the DNA-binding domain of AdpA and several kinds of duplex DNA. The best diffracting crystal was obtained using a 14-mer duplex DNA with two-nucleotide overhangs at the 5′-ends. The crystal diffracted X-rays to 2.8 Å resolution and belonged to space group C2221, with unit-cell parameters a = 76.86, b = 100.96, c = 101.25 Å. The Matthews coefficient (V M = 3.71 Å3 Da−1) indicated that the crystal was most likely to contain one DNA-binding domain of AdpA and one duplex DNA in the asymmetric unit, with a solvent content of 66.8%.
doi:10.1107/S1744309112026899
PMCID: PMC3412780  PMID: 22869129
AdpA; Streptomyces griseus; A-factor regulatory cascade; DNA-binding domain
5.  Expression, purification, crystallization and preliminary X-ray analysis of 4-hydroxy-3-methyl-2-­keto-pentanoate aldolase (asHPAL) from Arthrobacter simplex strain AKU 626 
asHPAL, a member of the HpaI/HpcH subfamily of class II aldolases, was expressed, purified and crystallized in the absence and presence of 2-ketobutyrate as one of its substrates using the sitting-drop vapour-diffusion method. asHPAL crystals grown without and with 2-ketobutyrate diffracted to 1.60 and 1.55 Å resolution, respectively.
4-Hydroxy-3-methyl-2-keto-pentanoate aldolase (asHPAL), an enzyme used in the synthesis of (2S,3R,4S)-4-hydroxyisoleucine, was crystallized in the absence and the presence of 2-ketobutyrate as one of its substrates by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. Crystals of asHPAL grown without and with 2-ketobutyrate diffracted to 1.60 and 1.55 Å resolution and belonged to space group C2, with unit-cell parameters a = 116.8, b = 88.2, c = 85.3 Å, β = 122.3° and a = 116.2, b = 88.1, c = 85.0 Å, β = 122.3°, respectively.
doi:10.1107/S1744309112028278
PMCID: PMC3412783  PMID: 22869132
4-hydroxy-3-methyl-2-keto-pentanoate aldolase; Arthrobacter simplex strain AKU 626; aldolases
6.  Crystallization and preliminary X-ray diffraction analysis of a novel type of phosphoserine phosphatase from Hydrogenobacter thermophilus TK-6 
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.
doi:10.1107/S1744309112025213
PMCID: PMC3412771  PMID: 22869120
phosphoserine phosphatases; Hydrogenobacter thermophilus TK-6
7.  Crystallization and preliminary X-ray analysis of the haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 
The haloalkane dehalogenase DatA from A. tumefaciens C58 was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 1.70 Å resolution.
Haloalkane dehalogenases are enzymes that catalyze the hydrolytic reaction of a wide variety of haloalkyl substrates to form the corresponding alcohol and hydrogen halide products. DatA from Agrobacterium tumefaciens C58 is a haloalkane dehalogenase that has a unique pair of halide-binding residues, asparagine (Asn43) and tyrosine (Tyr109), instead of the asparagine and tryptophan that are conserved in other members of the subfamily. DatA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with a reservoir solution consisting of 0.1 M CHES pH 8.6, 1.0 M potassium sodium tartrate, 0.2 M lithium sulfate, 0.01 M barium chloride. X-ray diffraction data were collected to 1.70 Å resolution. The space group of the crystal was determined as the primitive tetragonal space group P422, with unit-cell parameters a = b = 123.7, c = 88.1 Å. The crystal contained two molecules in the asymmetric unit.
doi:10.1107/S1744309112013942
PMCID: PMC3370902  PMID: 22684062
haloalkane dehalogenases; bioremediation
8.  Expression, purification, crystallization and preliminary X-ray analysis of carbonyl reductase S1 from Candida magnoliae  
Carbonyl reductase S1 from C. magnoliae was expressed, purified and crystallized. The crystals obtained diffracted X-rays to 1.90 Å resolution and belonged to space group P6122 or P6522.
The NADPH-dependent carbonyl reductase S1 from Candida magnoliae stereoselectively catalyzes the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE), which is a chiral compound valuable as a building block for pharmaceuticals. Carbonyl reductase S1 was expressed in Escherichia coli and purified by Ni-affinity, ion-exchange and size-exclusion chromatography. Crystals of carbonyl reductase S1 were obtained by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. X-ray diffraction data were collected to 1.90 Å resolution using a synchrotron-radiation source. The crystals belonged to space group P6122 or P6522, with unit-cell parameters a = b = 77.7, c = 307.5 Å. The asymmetric unit contained two molecules of the protein, with a solvent content of 44.2%.
doi:10.1107/S1744309112011645
PMCID: PMC3374508  PMID: 22691783
stereoselectivity; short-chain dehydrogenase/reductases; carbonyl reductase S1; Candida magnoliae
9.  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
10.  Crystallization and preliminary X-ray crystallographic analysis of dioscorin from Dioscorea japonica  
Dioscorin from D. japonica was expressed, purified and crystallized using the sitting-drop vapour-diffusion method. The dioscorin crystal diffracted X-rays to 2.11 Å resolution.
Dioscorin, the major tuber storage protein in yam, has been reported to possess carbonic anhydrase, trypsin inhibitor, angiotensin-converting enzyme (ACE) inhibitor, free-radical scavenger, dehydroascorbate reductase and mono­dehydro­ascorbate reductase activities. Recent research has also found that dioscorin can enhance immune modulation via the toll-like receptor 4 (TLR-4) signal transduction pathway in RAW 264.7 cells, murine bone-marrow cells and human monocytes ex vivo. Resolving the structure of dioscorin would help in better understanding its activities and would provide clues to understanding the mechanism of its multiple functions. The full-length protein (residues 1–246) with an additional His6 tag at the N-terminus was expressed in Escherichia coli Rosetta (DE3) cells. After His-tag cleavage and purification, the protein was crystallized by the sitting-drop vapour-diffusion method at 278 K. An X-ray diffraction data set was collected to a resolution of 2.11 Å using a synchrotron X-­ray source. The crystal belonged to space group C2221, with unit-cell parameters a = 83.5, b = 156.8, c = 83.6 Å, and was estimated to contain two protein molecules per asymmetric unit.
doi:10.1107/S1744309111053723
PMCID: PMC3274401  PMID: 22297997
dioscorin; Dioscorea japonica
11.  Crystallization and preliminary X-ray analysis of crustacean hyperglycaemic hormone from the kuruma prawn Marsupenaeus japonicus in its weakly active precursor form 
Crustacean hyperglycaemic hormone from the kuruma prawn M. japonicus was crystallized by the sitting-drop vapour-diffusion method in its weakly active precursor form which has an extra glycine residue at the C-terminus. The crystals belonged to the orthorhombic space group P212121 and diffracted to 1.95 Å resolution.
Crustacean hyperglycaemic hormone (CHH) plays a pivotal role in the regulation of glucose metabolism in crustaceans. Pej-SGP-I, one of the six known CHHs in the kuruma prawn Marsupenaeus japonicus, was heterolo­gously expressed in Escherichia coli as an N-terminally His-tagged and Nus-tagged protein in its weakly active precursor form, Pej-SGP-I-Gly, which has an extra glycine residue at the C-terminus. The recombinant peptide was subjected to affinity purification, tag removal, further purification and crystallization by the sitting-drop vapour-diffusion method using NaCl as the main precipitant. The crystals diffracted to 1.95 Å resolution and the space group was assigned as primitive orthorhombic P212121, with unit-cell parameters a = 40.19, b = 53.65, c = 53.63 Å. The Matthews coefficient (V M = 1.73 Å3 Da−1) indicated that the crystal contained two Pej-SGP-I-Gly molecules per asymmetric unit, with a solvent content of 29.0%.
doi:10.1107/S1744309111040140
PMCID: PMC3232146  PMID: 22139173
Marsupenaeus japonicus; crustacean hyperglycaemic hormone
12.  Purification, crystallization and preliminary X-ray analysis of glucokinase from Streptomyces griseus in complex with glucose 
Glucokinase from S. griseus (SgGlkA) was expressed, purified and crystallized using the sitting-drop vapour-diffusion method. A crystal of SgGlkA in complex with glucose diffracted X-rays to 1.84 Å resolution.
Glucokinase catalyzes the phosphorylation of glucose using ATP to yield glucose 6-­phosphate. SgGlkA is a bacterial group III glucokinase from Streptomyces griseus that seems to play a regulatory role in carbon catabolite repression in this organism. SgGlkA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method at 293 K. A crystal of SgGlkA in complex with glucose was obtained using a reservoir solution consisting of 0.9 M sodium/potassium tartrate, 0.2 M NaCl and 0.1 M imidazole pH 8.1 and diffracted X-rays to 1.84 Å resolution. The crystal of SgGlkA in complex with glucose belonged to space group P6222 or P6422, with unit-cell parameters a = b = 109.19, c = 141.18 Å. The crystal contained one molecule in the asymmetric unit.
doi:10.1107/S1744309111022275
PMCID: PMC3151127  PMID: 21821894
GlkA; catabolite repression; Streptomyces griseus
13.  Crystallization and preliminary X-ray analysis of a putative sensor histidine kinase domain: the C-­terminal domain of HksP4 from Aquifex aeolicus VF5 
The putative sensor histidine kinase domain of the cytoplasmic protein HksP4 from the hyperthermophilic bacterium A. aeolicus VF5 was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. Crystals were obtained in the presence of ATP and AMPPNP; they were found to belong to the same space group P212121 and diffracted X-rays to 3.1 and 2.9 Å resolution, respectively.
The histidine kinase domain of the cytoplasmic protein HksP4 from the hyperthermophilic bacterium Aquifex aeolicus VF5, located in the C-terminal half of the protein, was expressed, purified and crystallized. Diffraction-quality crystals were obtained in the presence of adenosine triphosphate (ATP) or adenosine 5′-­(β,γ-imido)triphosphate (AMPPNP) by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals obtained in the presence of ATP and AMPPNP diffracted X-rays to 3.1 and 2.9 Å resolution, respectively, on BL-5A at Photon Factory (Ibaraki, Japan) and were found to belong to the same space group P212121, with unit-cell parameters a = 80.2, b = 105.5, c = 122.0 Å and a = 81.5, b = 105.5, c = 130.9 Å, respectively. Their Matthews coefficients (V M = 2.74 and 2.51 Å3 Da−1, respectively) indicated that both crystals contained four protein molecules per asymmetric unit.
doi:10.1107/S1744309111018434
PMCID: PMC3144801  PMID: 21795799
histidine kinase domains; HksP4; Aquifex aeolicus VF5
14.  Structure of flap endonuclease 1 from the hyperthermophilic archaeon Desulfuro­coccus amylolyticus  
The crystal structure of flap endonuclease 1 from the hyperthermophilic archaeon D. amylolyticus was determined at 2.00 Å resolution.
Flap endonuclease 1 (FEN1) is a key enzyme in DNA repair and DNA replication. It is a structure-specific nuclease that removes 5′-overhanging flaps and the RNA/DNA primer during maturation of the Okazaki fragment. Homologues of FEN1 exist in a wide range of bacteria, archaea and eukaryotes. In order to further understand the structural basis of the DNA recognition, binding and cleavage mechanism of FEN1, the structure of FEN1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus (DaFEN1) was determined at 2.00 Å resolution. The overall fold of DaFEN1 was similar to those of other archaeal FEN1 proteins; however, the helical clamp and the flexible loop exhibited a putative substrate-binding pocket with a unique conformation.
doi:10.1107/S1744309110053030
PMCID: PMC3034609  PMID: 21301087
FEN1; DNA repair; DNA replication
15.  Expression, purification, crystallization and preliminary X-ray analysis of the KaiC-like protein PH0187 from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 
The KaiC-like protein PH0187 from the hyperthermophilic archaeon P. horikoshii OT3 was expressed, purified and crystallized using the sitting-drop vapour-diffusion method. The crystal of PH0187 diffracted X-rays to 2.75 Å resolution.
KaiC is the central protein in the circadian rhythm in cyanobacteria. The 28 kDa KaiC-like protein PH0187 from the hyperthermophilic archaeon Pyrococcus horikoshii was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method at 293 K. Crystals of PH0187 were obtained using a reservoir solution consisting of 1.0 M ammonium phosphate monobasic and 0.1 M sodium citrate tribasic pH 5.3 (the final pH value of the reservoir solution was 4.8) and diffracted X-rays to 2.75 Å resolution. The crystal of PH0187 belonged to space group P6322, with unit-cell parameters a = b = 239.1, c = 106.5 Å. The crystal contained four PH0187 molecules in the asymmetric unit.
doi:10.1107/S1744309110048426
PMCID: PMC3079995  PMID: 21206047
PH0187; KaiC; circadian rhythm
16.  Crystallization and preliminary X-ray analysis of 5-­keto-d-gluconate reductase from Gluconobacter suboxydans IFO12528 complexed with 5-keto-d-gluconate and NADPH 
NADPH-dependent 5-keto-d-gluconate reductase from G. suboxydans IFO12528 (5KGR) was expressed, purified and crystallized with 5-keto-d-gluconate and NADPH using the sitting-drop vapour-diffusion method. Crystals of the 5KGR–NADPH complex and of the 5KGR–NADPH–5-keto-d-gluconate complex diffracted X-rays to 1.75 and 2.26 Å resolution, respectively.
NADPH-dependent 5-keto-d-gluconate reductase from Gluconobacter suboxy­dans IFO12528 (5KGR) catalyzes oxidoreduction between 5-keto-d-gluconate and d-gluconate with high specificity. 5KGR was expressed in Escherichia coli, purified and crystallized with 5-keto-d-gluconate and NADPH using the sitting-drop vapour-diffusion method at 288 K. A crystal of the 5KGR–NADPH complex was obtained using reservoir solution containing PEG 4000 as a precipitant and diffracted X-rays to 1.75 Å resolution. The crystal of the complex belonged to space group P42212, with unit-cell parameters a = b = 128.6, c = 62.9 Å. A crystal of the 5KGR–NADPH–5-keto-d-gluconate complex was prepared by soaking the 5KGR–NADPH complex crystal in reservoir solution supplemented with 100 mM 5-keto-d-gluconate and 10 mM NADPH for 20 min and diffracted X-rays to 2.26 Å resolution. The crystal of the ternary complex belonged to space group P42212, with unit-cell parameters a = b = 128.7, c = 62.5 Å. Both crystals contained two molecules in the asymmetric unit.
doi:10.1107/S1744309110043617
PMCID: PMC2998383  PMID: 21139224
5-keto-d-gluconate reductase; Gluconobacter suboxydans IFO12528; SDR family
17.  The structure of mAG, a monomeric mutant of the green fluorescent protein Azami-Green, reveals the structural basis of its stable green emission 
The crystal structure of a monomeric mutant of Azami-Green (mAG) from G. fascicularis was determined at 2.2 Å resolution.
Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first known monomeric green-emitting fluorescent protein that is not a variant of Aequorea victoria green fluorescent protein (avGFP). These two green fluorescent proteins are only 27% identical in their amino-acid sequences. mAG is more similar in its amino-acid sequence to four fluorescent proteins: Dendra2 (a green-to-red irreversibly photoconverting fluorescent protein), Dronpa (a bright-and-dark reversibly photoswitchable fluorescent protein), KikG (a tetrameric green-emitting fluorescent protein) and Kaede (another green-to-red irreversibly photoconverting fluorescent protein). To reveal the structural basis of stable green emission by mAG, the 2.2 Å crystal structure of mAG has been determined and compared with the crystal structures of avGFP, Dronpa, Dendra2, Kaede and KikG. The structural comparison revealed that the chromophore formed by Gln62-Tyr63-Gly64 (QYG) and the fixing of the conformation of the imidazole ring of His193 by hydrogen bonds and van der Waals contacts involving His193, Arg66 and Thr69 are likely to be required for the stable green emission of mAG. The crystal structure of mAG will contribute to the design and development of new monomeric fluorescent proteins with faster maturation, brighter fluorescence, improved photostability, new colours and other preferable properties as alternatives to avGFP and its variants.
doi:10.1107/S1744309110011127
PMCID: PMC2864674  PMID: 20445241
fluorescent proteins; β-barrel; green emission
18.  Crystallization and preliminary X-ray analysis of a monomeric mutant of Azami-Green (mAG), an Aequorea victoria green fluorescent protein-like green-emitting fluorescent protein from the stony coral Galaxea fascicularis  
A monomeric mutant of Azami-Green from G. fascicularis was expressed, purified and crystallized using the sitting-drop vapour-diffusion method. The crystal belonged to space group P1 and diffracted X-rays to 2.20 Å resolution.
Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first monomeric green-emitting fluorescent protein that is not a derivative of Aequorea victoria green fluorescent protein (avGFP). mAG and avGFP are 27% identical in amino-acid sequence. Diffraction-quality crystals of recombinant mAG were obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The mAG crystal diffracted X-rays to 2.20 Å resolution on beamline AR-NW12A at the Photon Factory (Tsukuba, Japan). The crystal belonged to space group P1, with unit-cell parameters a = 41.78, b = 51.72, c = 52.89 Å, α = 90.96, β = 103.41, γ = 101.79°. The Matthews coefficient (V M = 2.10 Å3 Da−1) indicated that the crystal contained two mAG molecules per asymmetric unit.
doi:10.1107/S1744309109045382
PMCID: PMC2802884  PMID: 20054132
Azami-Green; green-emitting fluorescent proteins; Galaxea fascicularis
19.  Expression, purification, crystallization and preliminary X-ray analysis of conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708 
Conjugated polyketone reductase C2 from C. parapsilosis IFO 0708 was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. The crystal belonged to space group P212121 and diffracted X-rays to 1.7 Å resolution.
Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708 is a member of the NADPH-dependent aldo-keto reductase (AKR) superfamily and catalyzes the stereospecific reduction of ketopantoyl lactone to d-pantoyl lactone. A diffraction-quality crystal of recombinant CPR-C2 was obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.7 Å resolution on beamline NW12A of the Photon Factory-Advanced Ring (Tsukuba, Japan). The crystal belonged to space group P212121, with unit-cell parameters a = 55.02, b = 68.30, c = 68.93 Å. The Matthews coefficient (V M = 1.76 Å3 Da−1) indicated that the crystal contained one CPR-C2 molecule per asymmetric unit.
doi:10.1107/S1744309109038238
PMCID: PMC2777045  PMID: 19923737
conjugated polyketone reductase C; Candida parapsilosis; NAD(P)H-dependent oxido­reductases
20.  Crystallization and preliminary X-ray analysis of ZHE1, a hatching enzyme from the zebrafish Danio rerio  
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.
doi:10.1107/S1744309109033016
PMCID: PMC2765890  PMID: 19851011
ZHE1; hatching enzymes; zebrafish
21.  Crystallization and preliminary X-ray analysis of flap endonuclease 1 (FEN1) from Desulfurococcus amylolyticus  
Flap endonuclease 1 from D. amylolyticus was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 2.00 Å resolution.
Flap endonuclease 1 (FEN1) is a structure-specific nuclease that removes 5′-­overhanging flaps in DNA repair and removes the RNA/DNA primer during maturation of the Okazaki fragment in lagging-strand DNA replication. FEN1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with monoammonium dihydrogen phosphate as the precipitant at pH 8.3. X-ray diffraction data were collected to 2.00 Å resolution. The space group of the crystal was determined as the primitive hexagonal space group P321, with unit-cell parameters a = b = 103.76, c = 84.58 Å. The crystal contained one molecule in the asymmetric unit.
doi:10.1107/S1744309109031248
PMCID: PMC2795602  PMID: 19724134
FEN1; DNA repair; DNA replication
22.  Crystallization and preliminary X-ray analysis of γ-­hexachlorocyclohexane dehydrochlorinase LinA from Sphingobium japonicum UT26 
Purification and crystallization of LinA allowed the collection of data to 2.25 Å resolution.
LinA from Sphingobium japonicum UT26 catalyzes two steps of dehydrochlorination from γ-hexachlorocyclohexane (γ-HCH) to 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN) via γ-pentachlorocyclohexene (γ-PCCH). LinA was crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals belonged to space group P41 or P43, with unit-cell parameters a = b = 68.9, c = 101.9 Å, and diffracted X-rays to 2.25 Å resolution. The crystal contained three molecules in the asymmetric unit.
doi:10.1107/S1744309109026645
PMCID: PMC2720343  PMID: 19652349
LinA; γ-hexachlorocyclohexane dehydrochlorinase; Sphingobium japonicum UT26
23.  Purification, crystallization and preliminary X-ray analysis of l-sorbose reductase from Gluconobacter frateurii complexed with l-sorbose or NADPH 
NADPH-dependent l-sorbose reductase from G. frateurii (SR) was expressed, purified and crystallized with l-sorbose or NADPH using the sitting-drop vapour-diffusion method. Crystals of the SR–l-sorbose complex and SR–NADPH complex diffracted X-rays to 2.38 and 1.90 Å resolution, respectively.
NADPH-dependent l-sorbose reductase (SR) from Gluconobacter frateurii was expressed in Escherichia coli, purified and crystallized with l-sorbose or NADPH using the sitting-drop vapour-diffusion method at 293 K. Crystals of the SR–l-sorbose complex and the SR–NADPH complex were obtained using reservoir solutions containing PEG 2000 or PEG 400 as precipitants and diffracted X-rays to 2.38 and 1.90 Å resolution, respectively. The crystal of the SR–l-sorbose complex belonged to space group C2221, with unit-cell parameters a = 124.2, b = 124.1, c = 60.8 Å. The crystal of the SR–NADPH complex belonged to space group P21, with unit-cell parameters a = 124.3, b = 61.0, c = 124.5 Å, β = 89.99°. The crystals contained two and eight molecules, respectively, in the asymmetric unit.
doi:10.1107/S1744309109014687
PMCID: PMC2688410  PMID: 19478431
l-sorbose reductase; Gluconobacter frateurii; SDR family
24.  Crystallization and preliminary X-ray analysis of the NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra  
The NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra was expressed, purified, and crystallized and X-ray diffraction data of this crystal were collected to 2.2 Å resolution.
(R)-3-Quinuclidinol is a useful compound that is applicable to the synthesis of various pharmaceuticals. The NADPH-dependent carbonyl reductase 3-­quinuclidinone reductase from Rhodotorula rubra catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol and is expected to be utilized in industrial production of this alcohol. 3-Quinuclidinone reductase from R. rubra was expressed in Escherichia coli and purified using Ni-affinity and ion-exchange column chromatography. Crystals of the protein were obtained by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals belonged to space group P41212, with unit-cell parameters a = b = 91.3, c = 265.4 Å, and diffracted X-rays to 2.2 Å resolution. The asymmetric unit contained four molecules of the protein and the solvent content was 48.4%.
doi:10.1107/S1744309109017588
PMCID: PMC2688433  PMID: 19478454
3-­quinuclidinone reductase; short-chain dehydrogenase/reductase family; carbonyl reductases
25.  Structure of TTHA1623, a novel metallo-β-lactamase superfamily protein from Thermus thermophilus HB8 
The crystal structures of TTHA1623 from T. thermophilus HB8 in an iron-bound and a zinc-bound form have been determined to 2.8 and 2.2 Å resolution, respectively.
TTHA1623 is a metallo-β-lactamase superfamily protein from the extremely thermophilic bacterium Thermus thermophilus HB8. Homologues of TTHA1623 exist in a wide range of bacteria and archaea and one eukaryote, Giardia lamblia, but their function remains unknown. To analyze the structural properties of TTHA1623, the crystal structures of its iron-bound and zinc-bound forms have been determined to 2.8 and 2.2 Å resolution, respectively. TTHA1623 possesses an αββα-fold similar to that of other metallo-β-lactamase superfamily proteins with glyoxalase II-type metal coordination. However, TTHA1623 exhibits a putative substrate-binding pocket with a unique shape.
doi:10.1107/S174430910901361X
PMCID: PMC2675583  PMID: 19407375
TTHA1623; Thermus thermophilus HB8; metallo-β-lactamase superfamily

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