Preparation, crystallization and X-ray diffraction analysis to 1.5 Å resolution of rat cysteine dioxygenase, a mononuclear iron enzyme responsible for cysteine thiol oxidation

doi:10.1107/S1744309105033737

Title: Preparation, crystallization and X-ray diffraction analysis to 1.5 Å resolution of rat cysteine dioxygenase, a mononuclear iron enzyme responsible for cysteine thiol oxidation

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Abstract

Recombinant rat cysteine dioxygenase (CDO) has been expressed, purified and crystallized and X-ray diffraction data have been collected to 1.5 Å resolution. Cysteine dioxygenase (CDO; EC 1.13.11.20) is an ∼23 kDa non-heme iron metalloenzyme that is responsible for the oxidation of cysteine by O{sub 2}, yielding cysteinesulfinate. CDO catalyzes the first step in the conversion of cysteine to taurine, as well as the first step in the catabolism of cysteine to pyruvate plus sulfate. Recombinant rat CDO was heterologously expressed, purified and crystallized. The protein was expressed as a fusion protein bearing a polyhistidine tag to facilitate purification, a thioredoxin tag to improve solubility and a factor Xa cleavage site to permit removal of the entire N-terminus, leaving only the 200 amino acids inherent to the native protein. A multi-step purification scheme was used to achieve >95% purity of CDO. The optimal CDO crystals diffracted to 1.5 Å resolution and belonged to space group P4{sub 3}2{sub 1}2 or P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 57.55, c = 123.06 Å, α = β = γ = 90°. CDO shows little homology to any other proteins; therefore, the structure of the enzyme will be determined by ab initiomore »« less

Authors:

Simmons, Chad R. ^[1]; Hao, Quan ^[2]; Stipanuk, Martha H., E-mail: mhs6@cornell.edu ^[1]

Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853-8001 (United States)
MacCHESS at the Cornell High Energy Synchrotron Source, Cornell University, Ithaca, NY 14853-8001 (United States)

Publication Date:: Tue Nov 01 00:00:00 EST 2005

OSTI Identifier:: 22356181

Resource Type:: Journal Article

Resource Relation:: Journal Name: Acta Crystallographica. Section F; Journal Volume: 61; Journal Issue: Pt 11; Other Information: PMCID: PMC1978133; PMID: 16511222; PUBLISHER-ID: gx5071; OAI: oai:pubmedcentral.nih.gov:1978133; Copyright (c) International Union of Crystallography 2005; Country of input: International Atomic Energy Agency (IAEA)

Country of Publication:: United Kingdom

Language:: English

Subject:: 75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CLEAVAGE; CONVERSION; CRYSTALS; CYSTEINE; HEME; IMPURITIES; IRON; OXIDATION; OXYGEN; RATS; RESOLUTION; SOLUBILITY; SPACE GROUPS; X-RAY DIFFRACTION

Citation Formats


                    Simmons, Chad R., Hao, Quan, and Stipanuk, Martha H., E-mail: mhs6@cornell.edu. Preparation, crystallization and X-ray diffraction analysis to 1.5 Å resolution of rat cysteine dioxygenase, a mononuclear iron enzyme responsible for cysteine thiol oxidation.  United Kingdom: N. p., 2005. 
        Web.  doi:10.1107/S1744309105033737.

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                    Simmons, Chad R., Hao, Quan, & Stipanuk, Martha H., E-mail: mhs6@cornell.edu. Preparation, crystallization and X-ray diffraction analysis to 1.5 Å resolution of rat cysteine dioxygenase, a mononuclear iron enzyme responsible for cysteine thiol oxidation.  United Kingdom.  doi:10.1107/S1744309105033737.

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                    Simmons, Chad R., Hao, Quan, and Stipanuk, Martha H., E-mail: mhs6@cornell.edu. Tue .  
        "Preparation, crystallization and X-ray diffraction analysis to 1.5 Å resolution of rat cysteine dioxygenase, a mononuclear iron enzyme responsible for cysteine thiol oxidation".  United Kingdom.  
        doi:10.1107/S1744309105033737.

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@article{osti_22356181,

  title        = {Preparation, crystallization and X-ray diffraction analysis to 1.5 Å resolution of rat cysteine dioxygenase, a mononuclear iron enzyme responsible for cysteine thiol oxidation},

  author       = {Simmons, Chad R. and Hao, Quan and Stipanuk, Martha H., E-mail: mhs6@cornell.edu},

  abstractNote = {Recombinant rat cysteine dioxygenase (CDO) has been expressed, purified and crystallized and X-ray diffraction data have been collected to 1.5 Å resolution. Cysteine dioxygenase (CDO; EC 1.13.11.20) is an ∼23 kDa non-heme iron metalloenzyme that is responsible for the oxidation of cysteine by O{sub 2}, yielding cysteinesulfinate. CDO catalyzes the first step in the conversion of cysteine to taurine, as well as the first step in the catabolism of cysteine to pyruvate plus sulfate. Recombinant rat CDO was heterologously expressed, purified and crystallized. The protein was expressed as a fusion protein bearing a polyhistidine tag to facilitate purification, a thioredoxin tag to improve solubility and a factor Xa cleavage site to permit removal of the entire N-terminus, leaving only the 200 amino acids inherent to the native protein. A multi-step purification scheme was used to achieve >95% purity of CDO. The optimal CDO crystals diffracted to 1.5 Å resolution and belonged to space group P4{sub 3}2{sub 1}2 or P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 57.55, c = 123.06 Å, α = β = γ = 90°. CDO shows little homology to any other proteins; therefore, the structure of the enzyme will be determined by ab initio phasing using a selenomethionyl derivative.},

  doi          = {10.1107/S1744309105033737},

  journal      = {Acta Crystallographica. Section F},

  number       = Pt 11,

  volume       = 61,

  place        = {United Kingdom},

  year         = {Tue Nov 01 00:00:00 EST 2005},

  month        = {Tue Nov 01 00:00:00 EST 2005}

}

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DOI: 10.1107/S1744309105033737

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