Biochemical characterization of the heteromeric Bacillus subtilis dihydroorotate dehydrogenase and its isolated subunits.
Biochemical characterization of the heteromeric Bacillus subtilis dihydroorotate dehydrogenase and its isolated subunits.
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异聚枯草芽孢杆菌二氢乳清酸脱氢酶及其分离亚基的生化特征。
DOI：
10.1006/abbi.1999.1455
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发表时间：
1999
影响因子：
3.9
通讯作者：
Switzer,RL
中科院分区：
文献类型：
--
作者：
Kahler,AE;Nielsen,FS;Switzer,RL
Bacillus subtilis dihydroorotate dehydrogenase (DHOD) consists of two subunits, PyrDI (Mr= 33,094) and PyrDII (Mr= 28,099). The two subunits were overexpressed jointly and individually and purified. PyrDI was an FMN-containing flavoprotein with an apparent native molecular mass of 85,000. Overexpressed PyrDII formed inclusion bodies and was purified by refolding and reconstitution. Refolded PyrDII bound 1 mol FAD and 1 mol [2Fe–2S] per mol PyrDII. Coexpression and purification of PyrDI and PyrDII yielded a native holoenzyme complex with an apparent native molecular mass of 114,000 that indicated a heterotetramer (PyrDI2PyrDII2). The holoenzyme possessed dihydroorotate:NAD+oxidoreductase activity and could also reduce menadione and artificial dyes. Purified PyrDI also possessed DHOD activity but could not reduce NAD+. Compared to PyrDI, the holoenzyme had a more than 20-fold smaller Kmvalue for dihydroorotate, an approximately 50-fold smaller Kivalue for orotate, and approximately 500-fold greater catalytic efficiency. Dihydroorotate:NAD+oxidoreductase activity could be recovered by mixing the purified subunits. Recovered activity showed a clear dependence on FAD reconstitution of PyrDII but not on reconstitution with FeS clusters. PyrDII had a strong preference for FAD over FMN and bound it with an estimated Kdvalue of 4.9 ± 0.8 nM. pyrDII mutants containing alanine substitutions of the cysteine ligands to the [2Fe–2S] cluster failed to complement the pyr bradytrophy of a ΔpyrDII strain, indicating a requirement for the FeS cluster in PyrDII for normal function in vivo.
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DOI：
10.1016/s0021-9258(18)61070-1
发表时间：
1987-07
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
P. Matsudaira
通讯作者：
P. Matsudaira
使用新型敏感显色剂三吡啶基-S-三嗪的简单血清铁方法。
影响因子：
9.3
作者：
D. S. Fischer;D. Price
通讯作者：
D. Price
来自乳酸乳球菌的 B 型二氢乳清酸脱氢酶由两个不同的亚基组成，由pyrDb 和pyrK 基因编码，并包含FMN、FAD 和[FeS] 氧化还原中心*
DOI：
10.1074/jbc.271.46.29359
发表时间：
1996
期刊：
The Journal of Biological Chemistry
影响因子：
--
作者：
F. Nielsen;P. Andersen;K. Jensen
通讯作者：
K. Jensen
影响因子：
3.2
作者：
FARRAND, SK;TABER, HW
通讯作者：
TABER, HW
牛肝线粒体二氢乳清酸脱氢酶的纯化和性质。
DOI：
--
发表时间：
1986
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Hines,V;Keys3rd,LD;Johnston,M
通讯作者：
Johnston,M