Identification and Characterization of the Terminal Enzyme of Siroheme Biosynthesis from Arabidopsis thaliana
Identification and Characterization of the Terminal Enzyme of Siroheme Biosynthesis from Arabidopsis thaliana
复制标题
拟南芥西罗血红素生物合成末端酶的鉴定和表征
DOI：
--
复制
发表时间：
2005
影响因子：
4.8
通讯作者：
M. Warren
中科院分区：
文献类型：
--
作者：
E. Raux;H. Leech;Kerry;K. J. McLean;A. Munro;P. Heathcote;S. Rigby;Alison G. Smith;M. Warren
高等植物亚硫酸盐和亚硝酸盐还原酶含有西罗血红素作为辅基。西罗血红素是由四吡咯原祖尿卟啉原 III 经甲基化、氧化和铁螯合反应三个步骤合成的。在本文中，我们报道了拟南芥西罗氢氯酸亚铁螯合酶 At-SirB。 225 个氨基酸的完整前体蛋白和较短的构建体（其中前 46 或 79 个残基已被去除）显示出与限定的大肠杆菌西罗氢氯酸亚铁螯合酶突变体互补。该蛋白质的成熟形式似乎仅由 150 个氨基酸组成，使其比之前表征的亚铁螯合酶小得多。绿色荧光蛋白标记表明它位于叶绿体中。该酶很容易在大肠杆菌中作为重组蛋白产生，并且发现分离的酶具有48.5 nmol/min/mg的比活性。值得注意的是，蛋白质纯化为棕色溶液，其紫外-可见光谱在 415 和 455 nm 处包含最大值，表明存在 Fe-S 中心。重组蛋白的 EPR 分析产生了 G 值为 2.04、1.94 和 1.90 的菱形光谱，并且具有与 2Fe-2S 中心一致的温度依赖性。氧化还原滴定表明Fe-S中心高度不稳定，表观中点还原电位约为-370 mV。这是在高等植物亚铁螯合酶中报道的第一个 Fe-S 中心。讨论了与硫和铁的代谢密切相关的酶中 Fe-S 中心的含义。
Higher plant sulfite and nitrite reductases contain siroheme as a prosthetic group. Siroheme is synthesized from the tetrapyrrole primogenitor uroporphyrinogen III in three steps involving methylation, oxidation, and ferrochelation reactions. In this paper we report on the Arabidopsis thaliana sirohydrochlorin ferrochelatase At-SirB. The complete precursor protein of 225 amino acids and shorter constructs in which the first 46 or 79 residues had been removed were shown to complement a defined Escherichia coli sirohydrochlorin ferrochelatase mutant. The mature form of the protein appeared to consist of only 150 amino acids, making it much smaller than previously characterized ferrochelatases. Green fluorescent protein tagging revealed that it is located in the chloroplast. The enzyme was easily produced in E. coli as a recombinant protein, and the isolated enzyme was found to have a specific activity of 48.5 nmol/min/mg. Significantly, the protein purified as a brown-colored solution with a UV-visible spectrum containing maxima at 415 and 455 nm, suggestive of an Fe-S center. EPR analysis of the recombinant protein produced a rhombic spectrum with G-values of 2.04, 1.94, and 1.90 and with temperature dependence consistent with a 2Fe-2S center. Redox titration demonstrated that the Fe-S center is highly unstable, with an apparent midpoint reduction potential of about -370 mV. This is the first Fe-S center to be reported in a higher plant ferrochelatase. The implications of the Fe-S center in an enzyme that is so closely associated with the metabolism of sulfur and iron are discussed.
影响因子：
4.1
作者：
Raux, E;Leech, HK;Warren, MJ
通讯作者：
Warren, MJ
影响因子：
2.9
作者：
DAILEY, HA;FINNEGAN, MG;JOHNSON, MK
通讯作者：
JOHNSON, MK
哺乳动物亚铁螯合酶，金属酶家族的新成员。
DOI：
--
发表时间：
1994
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Ferreira,GC;Franco,R;Lloyd,SG;Pereira,AS;Moura,I;Moura,JJ;Huynh,BH
通讯作者：
Huynh,BH