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Purification, crystallization and preliminary crystallographic analysis of the catalytic core of cystathionine ?-synthase from Saccharomyces cerevisiae.

ABSTRACT: Cystathionine ?-synthase (CBS; EC 4.2.1.22) catalyzes the condensation of homocysteine and serine to form cystathionine, with the release of water. In humans, deficiency in CBS activity is the most common cause of hyperhomocysteinaemia and homocystinuria. More than 160 pathogenic mutations in the human CBS gene have been described to date. Here, the purification and preliminary crystallographic analysis of the catalytic core of CBS from Saccharomyces cerevisiae (ScCBS) is described which, in contrast to other eukaryotic CBSs, lacks the N-terminal haem-binding domain and is considered to be a useful model for investigation of the pyridoxal-5'-phosphate-mediated reactions of human CBS (hCBS). The purified protein yielded two different crystal forms belonging to space groups P41212 and P212121, with unit-cell parameters a = b = 72.390, c = 386.794?Å and a = 58.156, b = 89.988, c = 121.687?Å, respectively. Diffraction data were collected to 2.7 and 3.1?Å resolution, respectively, using synchrotron radiation. Preliminary analysis of the X-ray data suggests the presence of ScCBS homodimers in both types of crystals.

SUBMITTER: Ereno-Orbea J

PROVIDER: S-EPMC3944693 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Purification, crystallization and preliminary crystallographic analysis of the catalytic core of cystathionine β-synthase from Saccharomyces cerevisiae.

Ereño-Orbea June J Majtan Tomas T Oyenarte Iker I Kraus Jan P JP Martínez-Cruz Luis Alfonso LA

Acta crystallographica. Section F, Structural biology communications 20140219 Pt 3

Cystathionine β-synthase (CBS; EC 4.2.1.22) catalyzes the condensation of homocysteine and serine to form cystathionine, with the release of water. In humans, deficiency in CBS activity is the most common cause of hyperhomocysteinaemia and homocystinuria. More than 160 pathogenic mutations in the human CBS gene have been described to date. Here, the purification and preliminary crystallographic analysis of the catalytic core of CBS from Saccharomyces cerevisiae (ScCBS) is described which, in con ...[more]

PMID: 24598918

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Project description:BackgroundMitochondria are unique organelles that are found in most eukaryotic cells. The main role of the mitochondria is to produce ATP. The nuclear genome encoded proteins Cbs1 and Cbs2 are located at the mitochondrial inner membrane and are reported to be essential for the translation of mitochondrial cytochrome b mRNA. Genetic studies show that Cbs2 protein recognizes the 5' untranslated leader sequence of mitochondrial cytochrome b mRNA. However, due to a lack of biochemical and structural information, this biological process remains unclear. To investigate the structural characteristics of how Saccharomyces cerevisiae (S. cerevisiae) Cbs2 tethers cytochrome b mRNA to the mitochondrial inner membrane, a preliminary X-ray crystallographic study was carried out and is reported here.MethodsThe target gene from S. cerevisiae was amplified by polymerase chain reaction. The PCR fragment was digested by the NdeI and XhoI restriction endonucleases and then inserted into expression vector p28. After sequencing, the plasmid was transformed into Escherichia coli C43 competent cells. The selenomethionine derivative Cbs2 protein was overexpressed using M9 medium based on a methionine-biosynthesis inhibition method. The protein was first purified to Ni2+-nitrilotriacetate affinity chromatography and then further purified by Ion exchange chromatography and Gel-filtration chromatography. The purified Se-Cbs2 protein was concentrated to 10 mg/mL. The crystallization trials were performed using the sitting-drop vapor diffusion method at 16 °C. The complete diffraction data was processed and scaled with the HKL2000 package and programs in the CCP4 package, respectively.ResultsCbs2 from S. cerevisiae was cloned, prokaryotic expressed and purified. The analysis of the size exclusion chromatography showed that the Cbs2 protein peaked at a molecular weight of approximately 90 KDa. The crystal belonged to the space group C2, with unit-cell parameters of a = 255.11, b = 58.10, c = 76.37, and ? = 95.35°. X-ray diffraction data was collected at a resolution of 2.7 Å. The Matthews coefficient and the solvent content were estimated to be 3.22 Å 3 Da-1 and 61.82%, respectively.ConclusionsIn the present study Cbs2 from S. cerevisiae was cloned, expressed, purified, and crystallized for structural studies. The molecular weight determination results indicated that the biological assembly of Cbs2 may be a dimer.The preliminary X-ray crystallographic studies indicated the presence of two Cbs2 molecules in the asymmetric unit. This study will provide an experimental basis for exploring how Cbs2 protein mediates cytochrome b synthesis.

Dataset Information

Purification, crystallization and preliminary crystallographic analysis of the catalytic core of cystathionine ?-synthase from Saccharomyces cerevisiae.

Publications

Purification, crystallization and preliminary crystallographic analysis of the catalytic core of cystathionine β-synthase from Saccharomyces cerevisiae.

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