Prokaryotic expression of the heme- and flavin-binding domains of rat neuronal nitric oxide synthase as distinct polypeptides: identification of the heme-binding proximal thiolate ligand as cysteine-415.
Prokaryotic expression of the heme- and flavin-binding domains of rat neuronal nitric oxide synthase as distinct polypeptides: identification of the heme-binding proximal thiolate ligand as cysteine-415.
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大鼠神经元一氧化氮合酶的血红素和黄素结合域作为不同多肽的原核表达：将血红素结合近端硫醇配体鉴定为半胱氨酸-415。
DOI：
10.1021/bi00011a025
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Masters,BS
中科院分区：
文献类型：
--
作者：
McMillan,K;Masters,BS
摘要：组成型大鼠神经元一氧化氮合酶（NOS）的血红素和黄素结合域在大肠杆菌中表达为具有完整酶特性的不同多肽。使用表达载体pCW表达氨基末端血红素结合结构域（残基1-714）。表达蛋白的变性分子量为80 kDa，与兔抗NOS IgG呈免疫反应性。一氧化氮合酶血红素蛋白具有最大波长为445 nm的亚铁-一氧化碳差光谱。L-精氨酸和BH 4的光谱扰动引起了I型差异光谱，证实了这些分子在N末端NOS多肽内存在结合位点。定点诱变被施加到推定的轴向血红素配体，半胱氨酸-415，产生组氨酸突变体，这证实了近端配体的身份。使用载体pPROK-1表达具有（C1，残基715-1429）和不具有（C2，残基749-1429）氨基末端钙调蛋白结合基序的NOS黄素蛋白。C1和C2黄素蛋白的抗NOS IgG免疫反应，大小约为80 kDa。这两种纯化的黄素蛋白都具有黄素辅基的典型吸光特性，最大吸收波长在380和450 nm处，并具有NADPH依赖的电子转移到细胞色素c的能力，观察到的速率约为2-4 umol/min/mg。细菌表达的NO合酶血红素结合加氧酶和黄素蛋白氧化还原酶结构域作为分离的蛋白质与完整的酶的特定属性代表了这种复杂的酶的结构-功能研究的重要发展。
Revised Manuscript Received December 1, 1994® abstract: The heme-and flavin-binding domains of constitutive rat neuronal nitric oxide synthase (NOS) were expressed in Escherichia coli as distinct polypeptides with properties characteristic of the intact enzyme. The amino-terminal heme-binding domain (residues 1—714) was expressed using the expression vector pCW. The denatured molecular mass of the expressed protein was 80 kDa, and the protein was shown to be immunoreactive to rabbit anti-NOS IgG. The NOS hemoprotein exhibited a ferrous-carbon monoxide difference spectrum with a wavelength maximum at 445 nm. Spectral perturbation with L-arginine and BH4 elicited a type I difference spectrum, confirming thepresence of binding sites for these molecules within the N-terminal NOS polypeptide. Site-directed mutagenesis was applied to the putative axial heme ligand, cysteine-415, generating the histidine mutant, which confirmed the identity of the proximal ligand. NOS flavoproteins, with (Cl, residues 715—1429) and without (C2, residues 749—1429) an amino-terminal calmodulin-binding motif, were expressed using the vector pPROK-1. The Cl and C2 flavoproteins were immunoreactive to anti-NOS IgG and were sized at approximately 80 kDa. Both of the purified flavoproteins exhibited optical absorbance properties typical of a flavin prosthetic group, with wavelength maxima at 380 and450 nm, and were competent in NADPH-dependent electron transfer to cytochrome c, with observed rates of~ 2—4 umol/min/mg. The bacterial expression of the NO synthase heme-binding oxygenaseand flavoprotein oxidoreductase domains as isolated proteins with specific properties of the intact enzymerepresents an important development in structure—functionstudies of this complex enzyme.
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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
脑一氧化氮合酶是一种含有生物蝶呤和黄素的多功能氧化还原酶
影响因子：
3.5
作者：
B. Mayer;Mathias John;Burghard Heinzel;E. Werner;H. Wachter;G. Schultz;E. Böhme
通讯作者：
E. Böhme
影响因子：
4.1
作者：
BRILL, AS;WILLIAMS, RJP
通讯作者：
WILLIAMS, RJP
大鼠肝细胞色素 P-450d 的定点诱变：轴向配体和血红素掺入。
影响因子：
2.9
作者：
Toru Shimizu;K. Hirano;Masae Takahashi;M. Hatano;Y. Fujii‐Kuriyama
通讯作者：
Y. Fujii‐Kuriyama
影响因子：
56.9
作者：
RAVICHANDRAN, KG;BODDUPALLI, SS;DEISENHOFER, J
通讯作者：
DEISENHOFER, J