Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Expression, purification, crystallization and preliminary X-ray analysis of 4-hydroxy-3-methyl-2-keto-pentanoate aldolase (asHPAL) from Arthrobacter simplex strain AKU 626.

ABSTRACT: 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, ? = 122.3° and a = 116.2, b = 88.1, c = 85.0?Å, ? = 122.3°, respectively.

SUBMITTER: Guo L

PROVIDER: S-EPMC3412783 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Expression, purification, crystallization and preliminary X-ray analysis of 4-hydroxy-3-methyl-2-keto-pentanoate aldolase (asHPAL) from Arthrobacter simplex strain AKU 626.

Guo Linjun L Okai Masahiko M Mase Tomoko T Imai Fabiana Lica FL Miyakawa Takuya T Nagata Koji K Yamanaka Hiroyuki H Fujii Hidemi H Hibi Makoto M Ogawa Jun J Tanokura Masaru M

Acta crystallographica. Section F, Structural biology and crystallization communications 20120731 Pt 8

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 = 1 ...[more]

PMID: 22869132

Similar Datasets

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of 2-keto-3-deoxy-6-phosphogluconate aldolase from Zymomonas mobilis ZM4.

Project description:Zymomonas mobilis ZM4 is an organism optimized for ethanol production which uses the Entner-Doudoroff (ED) pathway for the breakdown of glucose. The key enzyme in this process is 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, which produces glyceraldehyde 3-phosphate and pyruvate. In order to provide a molecular background for the KDPG aldolase from this ethanologenic organism (zmKDPG aldolase), the ZMO0997 gene of Z. mobilis ZM4 coding for zmKDPG aldolase was cloned and expressed and the purified protein was crystallized from 25%(w/v) polyethylene glycol 3350 and 0.1 M bis-tris pH 5.5. Diffraction data were collected to 1.8 A resolution using synchrotron radiation. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 63.7, b = 83.0, c = 117.2 A. A trimeric zmKDPG aldolase molecule was present in the asymmetric unit, resulting in a crystal volume per unit protein weight of 2.40 A(3) Da(-1) and a solvent content of 48%.

| S-EPMC2852347 | biostudies-literature

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from Streptococcus suis serotype 2.

Project description:2-Keto-3-deoxy-6-phosphogluconate (KDPG) adolase from pathogenic Streptococcus suis serotype 2 was crystallized using the hanging-drop vapour-diffusion method at 291 K. X-ray diffraction data were collected to 2.8 A resolution. The crystal belonged to space group R32, with unit-cell parameters a = b = 126.4, c = 415.9 A, alpha = beta = 90, gamma = 120 degrees . Assuming the presence of six molecules in the asymmetric unit gave a V(M) value of 2.32 A(3) Da(-1) and a solvent content of 47.12%.

| S-EPMC2581687 | biostudies-literature

Expression, purification, crystallization and preliminary X-ray studies of histamine dehydrogenase from Nocardioides simplex.

Project description:Histamine dehydrogenase (HADH) from Nocardioides simplex catalyzes the oxidative deamination of histamine to produce imidazole acetaldehyde and an ammonium ion. HADH is functionally related to trimethylamine dehydrogenase (TMADH), but HADH has strict substrate specificity towards histamine. HADH is a homodimer, with each 76 kDa subunit containing two redox cofactors: a [4Fe-4S] cluster and an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-FMN. In order to understand the substrate specificity of HADH, it was sought to determine its structure by X-ray crystallography. This enzyme has been expressed recombinantly in Escherichia coli and successfully crystallized in two forms. Diffraction data were collected to 2.7 A resolution at the SSRL synchrotron with 99.7% completeness. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 101.14, b = 107.03, c = 153.35 A.

| S-EPMC2531280 | biostudies-literature

Purification, crystallization and preliminary X-ray crystallographic study of the L-fuculose-1-phosphate aldolase (FucA) from Thermus thermophilus HB8.

Project description:Fuculose phosphate aldolase catalyzes the reversible cleavage of L-fuculose-1-phosphate to dihydroxyacetone phosphate and L-lactaldehyde. The protein from Thermus thermophilus HB8 is a biological tetramer with a subunit molecular weight of 21 591 Da. Purified FucA has been crystallized using sitting-drop vapour-diffusion and microbatch techniques at 293 K. The crystals belong to space group P4, with unit-cell parameters a = b = 100.94, c = 45.87 A. The presence of a dimer of the enzyme in the asymmetric unit was estimated to give a Matthews coefficient (VM) of 2.7 A3 Da(-1) and a solvent content of 54.2%(v/v). Three-wavelength diffraction MAD data were collected to 2.3 A from zinc-containing crystals. Native diffraction data to 1.9 A resolution have been collected using synchrotron radiation at SPring-8.

| S-EPMC1978142 | biostudies-literature

Purification, crystallization and preliminary X-ray analysis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase of Streptococcus pneumoniae.

Project description:Class II 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases are potential targets for novel antibiotic development. In order to obtain a precise structural model for use in virtual screening and inhibitor design, HMG-CoA reductase of Streptococcus pneumoniae was cloned, overexpressed and purified to homogeneity using Ni-NTA affinity chromatography. Crystals were obtained using the hanging-drop vapour-diffusion method. A complete data set was collected from a single frozen crystal on a home X-ray source. The crystal diffracted to 2.3?Å resolution and belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 773.4836, b = 90.3055, c = 160.5592?Å, ? = ? = ? = 90°. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be 54.1% (V(M) = 2.68?Å(3)?Da(-1)).

| S-EPMC3001659 | biostudies-literature

Purification, crystallization and preliminary X-ray diffraction analysis of a novel keto-deoxy-D-galactarate (KDG) dehydratase from Agrobacterium tumefaciens.

Project description:D-galacturonic acid is the main component of pectin. It could be used to produce affordable renewable fuels, chemicals and materials through biotechnical conversion. Keto-deoxy-D-galactarate (KDG) dehydratase is an enzyme in the oxidative pathway of D-galacturonic acid in Agrobacterium tumefaciens (At). It converts 3-deoxy-2-keto-L-threo-hexarate to ?-ketoglutaric semialdehyde. At KDG dehydratase was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 169.1, b = 117.8, c = 74.3?Å, ? = 112.4° and an asymmetric unit of four monomers. X-ray diffraction data were collected to 1.9?Å resolution using synchrotron radiation. The three-dimensional structure of At KDG dehydratase will provide valuable information on the function of the enzyme and will allow it to be engineered for biorefinery-based applications.

| S-EPMC3943101 | biostudies-literature

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of fructose-1,6-bisphosphate aldolase from Escherichia coli.

Project description:Fructose-1,6-bisphosphate aldolase is one of the most important enzymes in the glycolytic pathway and catalyzes the reversible cleavage of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The full-length fbaB gene encoding fructose-1,6-bisphosphate aldolase class I (FBPA I) was cloned from Escherichia coli strain BL21. FBPA I was overexpressed in E. coli and purified. Biochemical analysis found that the optimum reaction temperature of FBPA I is 330.5?K and that the enzyme has a high temperature tolerance. Crystals of recombinant FBPA I were obtained by the sitting-drop vapour-diffusion technique in a condition consisting of 19?mg?ml(-1) FBPA I in 0.1?M Tris pH 9.0, 10%(w/v) polyethylene glycol 8000 and diffracted to 2.0?Å resolution. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 217.7, b = 114.9, c = 183.9?Å, ? = 124.6°. The asymmetric unit of these crystals may contain ten molecules, giving a Matthews coefficient of 2.48?Å(3)?Da(-1) and a solvent content of 50.5%.

| S-EPMC4188083 | biostudies-other

Purification, crystallization and preliminary X-ray diffraction of human S100A15.

Project description:Human S100A15 is a novel member of the S100 family of EF-hand calcium-binding proteins and was recently identified in psoriasis, where it is significantly upregulated in lesional skin. The protein is implicated as an effector in calcium-mediated signal transduction pathways. Although its biological function is unclear, the association of the 11.2 kDa S100A15 with psoriasis suggests that it contributes to the pathogenesis of the disease and could provide a molecular target for therapy. To provide insight into the function of S100A15, the protein was crystallized to visualize its structure and to further the understanding of how the many similar calcium-binding mediator proteins in the cell distinguish their cognate target molecules. The S100A15 protein has been cloned, expressed and purified to homogeneity and produced two crystal forms. Crystals of form I are triclinic, with unit-cell parameters a = 33.5, b = 44.3, c = 44.8 angstroms, alpha = 71.2, beta = 68.1, gamma = 67.8 degrees and an estimated two molecules in the asymmetric unit, and diffract to 1.7 angstroms resolution. Crystals of form II are monoclinic, with unit-cell parameters a = 82.1, b = 33.6, c = 52.2 angstroms, beta = 128.2 degrees and an estimated one molecule in the asymmetric unit, and diffract to 2.0 angstroms resolution. This structural analysis of the human S100A15 will further aid in the phylogenic comparison between the other members of the S100 protein family, especially the highly homologous paralog S100A7.

| S-EPMC2219979 | biostudies-literature

Purification, crystallization and preliminary crystallographic studies on 2-dehydro-3-deoxygalactarate aldolase from Leptospira interrogans.

Project description:2-Dehydro-3-deoxygalactarate (DDG) aldolase is a member of the class II aldolase family and plays an important role in the pyruvate-metabolism pathway, catalyzing the reversible aldol cleavage of DDG to pyruvate and tartronic semialdehyde. As it is a potential novel antibiotic target, it is necessary to elucidate the catalytic mechanism of DDG aldolase. To determine the crystal structure, crystals of DDG aldolase from Leptospira interrogans were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.2 A resolution using a Cu K alpha rotating-anode X-ray source. The crystal belonged to space group C2, with unit-cell parameters a = 293.5, b = 125.6, c = 87.6 A, beta = 100.9 degrees. The V(M) is calculated to be 2.4 A3 Da(-1), assuming there to be 12 protein molecules in the asymmetric unit.

| S-EPMC2225365 | biostudies-literature

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp6.

Project description:Enpp (ectonucleotide phosphodiesterase/pyrophosphatase) 6 is a membrane-bound glycoprotein that hydrolyzes choline-containing compounds such as lysophosphatidylcholine and glycerophosphorylcholine, and presumably participates in choline metabolism. The catalytic domain of mouse Enpp6 was expressed in HEK293T cells, purified using the TARGET tag/P20.1-Sepharose system and crystallized. An X-ray diffraction data set was collected to 1.8?Å resolution. The crystal belonged to space group P1, with unit-cell parameters a=63.7, b=68.8, c=69.7?Å, ?=60.6, ?=87.0, ?=68.1°. Assuming the presence of two protein molecules per asymmetric unit, the solvent content was estimated to be 49.5%.

| S-EPMC4051540 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data