Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase
Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase
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探究大肠杆菌黄素氧还蛋白氧化还原酶的 NADPH 结合位点
DOI：
10.1042/bj3520257
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发表时间：
2000
影响因子：
4.1
通讯作者：
A. Munro
中科院分区：
文献类型：
--
作者：
C. Leadbeater;L. McIver;D. Campopiano;S. Webster;R. Baxter;S. Kelly;N. C. Price;D. A. Lysek;M. Noble;S. Chapman;A. Munro
大肠杆菌黄色素NADP(+)氧化还原酶(FLDR)的结构将三种精氨酸(R144、R174和R184)放置在所建议的NADPH结合部位。对用中性丙氨酸取代每个精氨酸的定点突变产生的酶进行了鉴定。所有突变体的NADPH依赖细胞色素c还原酶活性降低(R144A，241.6分钟(-1)；R174A，132.1分钟(-1)；R184A，305.5分钟(-1)对野生型，338.9分钟(-1))，而NADPH的K(M)增加(R144A，5.3微米；R174A，20.2微米；R184A，54.4微米对野生型，3.9微米)。与野生型(33.0min(-1))相比，R174A(42.3min(-1))和R184A(50.4min(-1))依赖NADH的细胞色素c还原的k(CAT)值增加，这与R174和R184通过与腺苷核糖2‘-磷酸相互作用区分NADPH/NADH的作用一致。停流研究表明，突变体R144A(635微米)和R184A(2.3 mM)对NADPH的亲和力(K(D))显著低于野生型(比野生型FLDR低175倍)。R174A的氢化物从NADPH向黄素转移的速率常数最低(k(红)=8.82 S(-1)，野生型为22.63 S(-1))，CD和荧光光谱的变化也证明了这一点。分子模拟表明，FLDR的C-末端色氨酸(W248)的移动是使NADPH的烟酰胺环与黄素接近所必需的。NADPH磷酸酯在模型结构中的位置与动力学数据一致，其中R174和R184靠近腺苷核糖2‘-磷酸基团，R144可能与烟酰胺核糖5’-磷酸基团相互作用。
The structure of the Escherichia coli flavodoxin NADP(+) oxidoreductase (FLDR) places three arginines (R144, R174 and R184) in the proposed NADPH-binding site. Mutant enzymes produced by site-directed mutagenesis, in which each arginine was replaced by neutral alanine, were characterized. All mutants exhibited decreased NADPH-dependent cytochrome c reductase activity (R144A, 241.6 min(-1); R174A, 132.1 min(-1); R184A, 305.5 min(-1) versus wild type, 338.9 min(-1)) and increased K(m) for NADPH (R144A, 5.3 microM; R174A, 20.2 microM; R184A, 54.4 microM versus wild type, 3.9 microM). The k(cat) value for NADH-dependent cytochrome c reduction was increased for R174A (42.3 min(-1)) and R184A (50.4 min(-1)) compared with the wild type (33.0 min(-1)), consistent with roles for R174 and R184 in discriminating between NADPH/NADH by interaction with the adenosine ribose 2'-phosphate. Stopped-flow studies indicated that affinity (K(d)) for NADPH was markedly reduced in mutants R144A (635 microM) and R184A (2.3 mM) compared with the wild type ( 175-fold lower than for wild-type FLDR. The rate constant for hydride transfer from NADPH to flavin was lowest for R174A (k(red)=8.82 s(-1) versus 22.63 s(-1) for the wild type), which also exhibited tertiary structure perturbation, as evidenced by alterations in CD and fluorescence spectra. Molecular modelling indicated that movement of the C-terminal tryptophan (W248) of FLDR is necessary to permit close approach of the nicotinamide ring of NADPH to the flavin. The positions of NADPH phosphates in the modelled structure are consistent with the kinetic data, with R174 and R184 located close to the adenosine ribose 2'-phosphate group, and R144 likely to interact with the nicotinamide ribose 5'-phosphate group.
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影响因子：
2.9
作者：
P. Karplus;K. Walsh;J. Herriott
通讯作者：
P. Karplus;K. Walsh;J. Herriott
DOI：
10.1016/s0021-9258(18)60416-8
发表时间：
1989-07
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
R. T. Ruettinger;Long-Ping Wen;Armand J. FulcoP
通讯作者：
R. T. Ruettinger;Long-Ping Wen;Armand J. FulcoP
人肝细胞中诱导型一氧化氮合酶的分子克隆和表达
DOI：
10.1073/pnas.90.8.3491
发表时间：
1993-04-15
影响因子：
11.1
作者：
GELLER, DA;LOWENSTEIN, CJ;BILLIAR, TR
通讯作者：
BILLIAR, TR
人肝脏 NADPH-细胞色素 P-450 还原酶的结构和功能分析：人酶和 NADPH 结合位点的完整序列。
影响因子：
2.9
作者：
Haniu,M;McManus,ME;Birkett,DJ;Lee,TD;Shively,JE
通讯作者：
Shively,JE
来自大肠杆菌的黄素氧还蛋白和 NADPH-黄素氧还蛋白还原酶支持牛细胞色素 P450c17 羟化酶活性。
DOI：
--
发表时间：
1994
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Jenkins,CM;Waterman,MR
通讯作者：
Waterman,MR