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Isolation and purification of Thermus thermophilus HpaB by a crystallization approach.

ABSTRACT: The oxygenase HpaB is a component of the 4-hydroxyphenylacetate 3-monooxygenase enzyme that is responsible for the hydroxylation of 4-hydroxyphenylacetate. It utilizes molecular oxygen and a reduced flavin, which is provided by HpaC, the second component of the enzyme. While isolating integral membrane respiratory complexes from Thermus thermophilus, microcrystals of HpaB were formed. Further purification of the enzyme was achieved by repetitive crystallization. Subsequently, well shaped single crystals of the native enzyme that diffract to 1.82 A resolution were grown in sitting drops. They belong to the orthorhombic space group I222, with unit-cell parameters a = 91.3, b = 99.8, c = 131.7 A.

SUBMITTER: Soulimane T

PROVIDER: S-EPMC2833055 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Isolation and purification of Thermus thermophilus HpaB by a crystallization approach.

Soulimane Tewfik T O'Kane Sarah R SR Kolaj Olga O

Acta crystallographica. Section F, Structural biology and crystallization communications 20100227 Pt 3

The oxygenase HpaB is a component of the 4-hydroxyphenylacetate 3-monooxygenase enzyme that is responsible for the hydroxylation of 4-hydroxyphenylacetate. It utilizes molecular oxygen and a reduced flavin, which is provided by HpaC, the second component of the enzyme. While isolating integral membrane respiratory complexes from Thermus thermophilus, microcrystals of HpaB were formed. Further purification of the enzyme was achieved by repetitive crystallization. Subsequently, well shaped single ...[more]

PMID: 20208179

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Cloning, overexpression, purification and crystallization of malate dehydrogenase from Thermus thermophilus.

Project description:Malate dehydrogenase (MDH) has been used as a conjugate for enzyme immunoassay of a wide variety of compounds, such as drugs of abuse, drugs used in repetitive therapeutic application and hormones. In consideration of the various biotechnological applications of MDH, investigations of MDH from Thermus thermophilus were carried out to further understand the properties of this enzyme. The DNA fragment containing the open reading frame of mdh was amplified from the genomic DNA of T. thermophilus and cloned into the expression vector pET21b(+). The protein was expressed in a soluble form in Escherichia coli strain BL21(DE3). Homogeneous protein was obtained using a three-step procedure consisting of thermal treatment, Ni(2+)-chelating chromatography and size-exclusion chromatography. The purified MDH was crystallized and the crystals diffracted to a resolution of 1.80?Å on the BL13C1 beamline of the National Synchrotron Radiation Research Center (NSRRC), Taiwan. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 71.3, b = 86.1, c = 118.2?Å. The unit-cell volume of the crystal is compatible with the presence of two monomers in the asymmetric unit, with a corresponding Matthews coefficient VM of 2.52?Å(3)?Da(-1) and a solvent content of 51.2%. The crystal structure of MDH has been solved by molecular replacement and is currently under refinement.

| S-EPMC3818045 | biostudies-literature

Crystallization and preliminary X-ray analysis of the oxygenase component (HpaB) of 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8.

Project description:The 4-hydroxyphenylacetate (4HPA) 3-monooxygenase enzyme catalyzes the hydroxylation of 4HPA to 3,4-dihydroxyphenylacetate in the initial step of the degradation pathway of 4HPA. This enzyme consists of two components: an oxygenase (HpaB) and a reductase (HpaC). HpaB hydroxylates 4HPA using an oxygen molecule and a reduced flavin, which is supplied by HpaC. HpaB from Thermus thermophilus HB8 was overexpressed in Escherichia coli and crystallized. Crystals of HpaB were grown in 0.4 M 1,6-hexanediol, 0.1 M sodium acetate pH 5.0 and 25% (v/v) glycerol and diffracted X-rays to a resolution of 1.60 A. The crystals belong to the orthorhombic space group I222, with unit-cell parameters a = 91.8, b = 99.6, c = 131.1 A. The asymmetric unit volume provides space for only one subunit of the tetrameric HpaB molecule, giving a Matthews coefficient V(M) of 2.8 A3 Da(-1) and a solvent content of 55.1%. Platinum-derivatized crystals of HpaB were prepared by soaking native crystals in a solution containing 1 mM ammonium tetrachloroplatinate(II) for 1 d and diffracted X-rays to a resolution of 2.50 A. MAD data were successfully collected for structural determination using these crystals.

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Purification, crystallization and preliminary X-ray analysis of the fumarylacetoacetase family member TTHA0809 from Thermus thermophilus HB8.

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| S-EPMC2376325 | biostudies-literature

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of thymidylate kinase (TTHA1607) from Thermus thermophilus HB8.

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| S-EPMC3564610 | biostudies-literature

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of the hyperthermophilic nucleotidyltransferase TTHA1015 from Thermus thermophilus HB8.

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| S-EPMC2496871 | biostudies-literature

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