Expression, Purification, and Biochemical Characterization of the Flavocytochrome P450 CYP505A30 from Myceliophthora thermophila.
Expression, Purification, and Biochemical Characterization of the Flavocytochrome P450 CYP505A30 from Myceliophthora thermophila.
复制标题
DOI：
10.1021/acsomega.7b00450
复制
发表时间：
2017-08-31
期刊：
影响因子：
4.1
通讯作者：
Munro AW
中科院分区：
文献类型：
--
作者：
Baker GJ;Girvan HM;Matthews S;McLean KJ;Golovanova M;Waltham TN;Rigby SEJ;Nelson DR;Blankley RT;Munro AW
来自嗜热真菌嗜热毁丝霉的细胞色素 P450/P450 还原酶融合酶 CYP505A30 及其血红素 (P450) 结构域在大肠杆菌中表达，并使用亲和层析、离子交换层析和尺寸排阻层析进行纯化。 CYP505A30 结合直链脂肪酸（从 ~C10 到 C20），对十三烷酸具有最高的亲和力 (KD = 2.7 μM)。还原型烟酰胺腺嘌呤二核苷酸磷酸盐是 CYP505A30 的首选还原剂（细胞色素 c 还原中，KM = 3.1 μM，而还原型烟酰胺腺嘌呤二核苷酸为 330 μM）。电子顺磁共振证实了 CYP505A30 及其血红素结构域中血红素铁的半胱氨酸硫醇盐配位。氧化还原电位测定显示，黄素腺嘌呤二核苷酸和黄素单核苷酸辅因子（E0′~-118 mV）还原具有异常正的中点电位，并且结合花生四烯酸底物的 CYP505A30 血红素结构域 FeIII/FeII 氧化还原对（约 230 mV）大幅增加。这种转换使三价铁血红素铁电位进入与还原酶黄素相同的范围。多角度激光散射分析揭示了 CYP505A30 具有二聚化能力，而血红素结构域是单体化的。这些数据表明 CYP505A30 可能作为二聚体发挥催化作用（如巨大芽孢杆菌 P450 BM3 中所述），并且 CYP505A30 血红素结构域之间的结合相互作用不是二聚体形成所必需的。 CYP505A30 催化直链脂肪酸在 ω-1 至 ω-3 位点的羟基化，在十二烷酸和十四烷酸的氧化中，对 ω-1 的羟基化优先于 ω-3 的羟基化（分别为 88 与 2% 产物和 63 与 9% 产物）。 CYP505A30 与 P450 BM3 具有重要的结构和催化相似性，但脂质底物氧化具有独特的区域选择性，具有潜在的生物技术应用。
The cytochrome P450/P450 reductase fusion enzyme CYP505A30 from the thermophilic fungus Myceliophthora thermophila and its heme (P450) domain were expressed in Escherichia coli and purified using affinity, ion exchange, and size exclusion chromatography. CYP505A30 binds straight chain fatty acids (from ∼C10 to C20), with highest affinity for tridecanoic acid (KD = 2.7 μM). Reduced nicotinamide adenine dinucleotide phosphate is the preferred reductant for CYP505A30 (KM = 3.1 μM compared to 330 μM for reduced nicotinamide adenine dinucleotide in cytochrome c reduction). Electron paramagnetic resonance confirmed cysteine thiolate coordination of heme iron in CYP505A30 and its heme domain. Redox potentiometry revealed an unusually positive midpoint potential for reduction of the flavin adenine dinucleotide and flavin mononucleotide cofactors (E0′ ∼ −118 mV), and a large increase in the CYP505A30 heme domain FeIII/FeII redox couple (ca. 230 mV) on binding arachidonic acid substrate. This switch brings the ferric heme iron potential into the same range as that of the reductase flavins. Multiangle laser light scattering analysis revealed CYP505A30’s ability to dimerize, whereas the heme domain is monomeric. These data suggest CYP505A30 may function catalytically as a dimer (as described for Bacillus megaterium P450 BM3), and that binding interactions between CYP505A30 heme domains are not required for dimer formation. CYP505A30 catalyzed hydroxylation of straight chain fatty acids at the ω-1 to ω-3 positions, with a strong preference for ω-1 over ω-3 hydroxylation in the oxidation of dodecanoic and tetradecanoic acids (88 vs 2% products and 63 vs 9% products, respectively). CYP505A30 has important structural and catalytic similarities to P450 BM3 but distinct regioselectivity of lipid substrate oxidation with potential biotechnological applications.
登录
查看更多内容
DOI：
10.1016/0005-2760(75)90089-2
发表时间：
1975-01-01
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
MIURA, Y;FULCO, AJ
通讯作者：
FULCO, AJ
影响因子：
4.8
作者：
Girvan, HM;Marshall, KR;Munro, AW
通讯作者：
Munro, AW
影响因子：
4.8
作者：
Hubbard, PA;Shen, AL;Kim, JJP
通讯作者：
Kim, JJP
影响因子：
2.9
作者：
MASSEY, V;PALMER, G
通讯作者：
PALMER, G
影响因子：
3.9
作者：
BODDUPALLI, SS;PRAMANIK, BC;PETERSON, JA
通讯作者：
PETERSON, JA