NADPH-cytochrome P450 oxidoreductase from the mosquito Anopheles minimus: kinetic studies and the influence of Leu86 and Leu219 on cofactor binding and protein stability.
NADPH-cytochrome P450 oxidoreductase from the mosquito Anopheles minimus: kinetic studies and the influence of Leu86 and Leu219 on cofactor binding and protein stability.
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DOI：
10.1016/j.abb.2008.05.012
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发表时间：
2008-09
影响因子：
3.9
通讯作者：
Songklod Sarapusit;C. Xia;I. Misra;P. Rongnoparut;Jung‐Ja P. Kim
中科院分区：
文献类型：
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作者：
Songklod Sarapusit;C. Xia;I. Misra;P. Rongnoparut;Jung‐Ja P. Kim
缺失前55个氨基酸残基的小按蚊nadph -细胞色素c氧化还原酶在大肠杆菌中表达。纯化的酶失去FMN，导致不稳定的蛋白质和随后的聚集。为了了解这种不稳定性的基础，我们构建了L86F、L219F和P456A的单突变体和三突变体，前两个残基位于FMN结构域，第三个残基位于FAD结构域。该三突变体获得高产，化学计量学为0.97 FMN和0.55 FAD。通过向酶中添加外源FAD，克服了FAD含量的不足。野生型和三重突变体都遵循两个位点的乒乓机制，具有相似的动力学常数，因此不会发生任何全局结构变化。单突变体分析表明，脯氨酸到丙氨酸的取代对FMN结合没有影响，但亮氨酸到苯丙氨酸的取代对FMN结合和酶的最大稳定性都是必需的。
NADPH-cytochrome c oxidoreductase from the mosquito Anopheles minimus lacking the first 55 amino acid residues was expressed in Escherichia coli. The purified enzyme loses FMN, leading to an unstable protein and subsequent aggregation. To understand the basis for the instability, we constructed single and triple mutants of L86F, L219F, and P456A, with the first two residues in the FMN domain and the third in the FAD domain. The triple mutant was purified in high yield with stoichiometries of 0.97 FMN and 0.55 FAD. Deficiency in FAD content was overcome by addition of exogenous FAD to the enzyme. Both wild-type and the triple mutant follow a two-site Ping-Pong mechanism with similar kinetic constants arguing against any global structural changes. Analysis of the single mutants indicates that the proline to alanine substitution has no impact, but that both leucine to phenylalanine substitutions are essential for FMN binding and maximum stability of the enzyme.