Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase
Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase
复制标题
DOI：
10.1042/0264-6021:3520257
复制
发表时间：
2000-12-01
影响因子：
4.1
通讯作者：
Munro, AW
中科院分区：
文献类型：
--
作者：
Leadbeater, C;McIver, L;Munro, AW
大肠杆菌黄氧还蛋白NADP（+）氧化还原酶（FLDR）的结构将三个精氨酸（R144、R174和R184）置于nadph结合位点。通过位点定向诱变产生的突变酶，其中每个精氨酸被中性丙氨酸取代，被表征。所有突变体均表现出nadph依赖性细胞色素c还原酶活性降低(R144A, 241.6 min(-1)；R174A, 132.1 min(-1)；R184A, 305.5 min(-1)，与野生型相比，338.9 min(-1))， NADPH K-m增加（R144A, 5.3 muM; R174A, 20.2 muM; R184A, 54.4 muM，与野生型相比，3.9 muM）。与野生型（33.0 min(-1)）相比，R174A （42.3 min(-1)）和R184A （50.4 min(-1)）的NADH依赖性细胞色素c还原的k（cat）值增加，这与R174和R184通过与腺苷核糖2′-磷酸相互作用来区分NADPH/NADH的作用一致。停流研究表明，与野生型（< 5 M）相比，突变体R144A （635 mM）和R184A （2.3 mM）对NADPH的亲和力（K-d）明显降低。突变体R184A在吡啶核苷酸偏好方面表现出最大的变化，NADH/NADPH K-d比值比野生型FLDR低175倍。氢化物从NADPH转移到黄素的速率常数R174A最低(k（红色）= 8.82 s(-1)，而野生型为22.63 s(-1))， CD和荧光光谱的变化也证明了三级结构的扰动。分子模拟表明，FLDR的c端色氨酸（W248）的移动是允许NADPH的烟酰胺环接近黄素的必要条件。NADPH磷酸在模型结构中的位置与动力学数据一致，R174和R184位于腺苷核糖2'-磷酸基团附近，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 muM; R174A, 20.2 muM; R184A, 54.4 muM versus wild type, 3.9 muM). 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 muM) and R184A (2.3 mM) compared with the wild type (< 5 M). Mutant R184A displays the greatest change in pyridine nucleotide preference, with the NADH/NADPH K-d ratio > 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.