Preparation, crystallization and X-ray diffraction analysis to 1.5 A resolution of rat cysteine dioxygenase, a mononuclear iron enzyme responsible for cysteine thiol oxidation.
Preparation, crystallization and X-ray diffraction analysis to 1.5 A resolution of rat cysteine dioxygenase, a mononuclear iron enzyme responsible for cysteine thiol oxidation.
复制标题
大鼠半胱氨酸双加氧酶（一种负责半胱氨酸硫醇氧化的单核铁酶）的制备、结晶和 X 射线衍射分析（分辨率为 1.5 A）。
DOI：
10.1107/s1744309105033737
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Stipanuk,MarthaH
中科院分区：
文献类型：
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作者：
Simmons,ChadR;Hao,Quan;Stipanuk,MarthaH
半胱氨酸双加氧酶（CDO; EC 1.13.11.20）是一种分子量为23 kDa的非血红素铁金属酶，负责O2氧化半胱氨酸，产生半胱氨酸亚磺酸盐。 CDO催化半胱氨酸转化为牛磺酸的第一步，以及催化半胱氨酸转化为丙酮酸加硫酸盐的第一步。重组大鼠CDO异源表达，纯化和结晶。该蛋白表达为融合蛋白，其带有多组氨酸标签以便于纯化，硫氧还蛋白标签以提高溶解度，以及因子Xa切割位点以允许去除整个N-末端，仅留下天然蛋白固有的200个氨基酸。使用多步纯化方案以实现>95%纯度的CDO。最佳的CDO晶体衍射分辨率为1.5 μ m，空间群为P43212或P41212，晶胞参数a = B = 57.55，c = 123.06 μ m，α = β = γ = 90°。 CDO与任何其他蛋白质几乎没有同源性;因此，将使用硒代甲硫酰衍生物通过从头算定相来确定酶的结构。
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 O2, 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 P43212 or P41212, 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.