Reconstitution of the fatty acid hydroxylase activity of cytochrome P450BM-3 utilizing its functional domains.
Reconstitution of the fatty acid hydroxylase activity of cytochrome P450BM-3 utilizing its functional domains.
复制标题
利用细胞色素 P450BM-3 的功能域重建其脂肪酸羟化酶活性。
DOI：
10.1006/abbi.1997.9895
复制
发表时间：
1997
影响因子：
3.9
通讯作者：
Peterson,JA
中科院分区：
文献类型：
--
作者：
Sevrioukova,I;Truan,G;Peterson,JA
细胞色素P450 BM-3是一种来自巨大芽孢杆菌的催化自给自足的脂肪酸单加氧酶，是一种含有血红素、FAD和FMN的多结构域蛋白。先前利用等摩尔浓度的单独的血红素（BMP）和还原酶（BMR）结构域重建完整P450 BM-3的脂肪酸单加氧酶活性的尝试是不成功的，因为快速积累的双电子还原的FMN不能将电子转移到血红素铁。本研究利用P450 BM-3的不同功能结构域的组合来重建P450 BM-3的单加氧酶活性。为此，表达并纯化了P450 BM-3的FAD/NADPH-和FMN-结合结构域以及血红素-和FMN-结合结构域的组合（BMP/FMN）。由BMP/FMN和FAD结构域或BMP、FMN和FAD结构域组成的重建系统仍然不如全酶P450 BM-3有效，但比由BMP和BMR组成的系统有效得多。新开发的重构系统的棕榈酸的最大氧化速率仍然仅为全酶活性的约5%。重构系统产生ω-1、ω-2和ω-3单羟基棕榈酸，但不产生用全酶观察到的棕榈酸羟基化的次级产物。不能完全重建全酶的最大活性以及缺乏次级产物形成的物理原因目前尚不清楚。
Cytochrome P450BM-3, a catalytically self-sufficient fatty acid monooxygenase fromBacillus megaterium,is a multidomain protein containing heme, FAD, and FMN. Previous attempts to reconstitute the fatty acid monooxygenase activity of intact P450BM-3 utilizing equimolar concentrations of the separate heme (BMP) and reductase (BMR) domains, have been unsuccessful because two-electron reduced FMN, which rapidly accumulates, is incapable of electron transfer to the heme iron. The present study of the reconstitution of the monooxygenase activity of P450BM-3 utilized combinations of the different functional domains of P450BM-3. For this purpose, the FAD/NADPH- and FMN-binding domains of P450BM-3 as well as the combination of the heme- and FMN-binding domains (BMP/FMN) have been expressed and purified. The reconstitution systems, consisting of either BMP/FMN and FAD domains or BMP, FMN, and FAD domains, were still less effective than the holoenzyme, P450BM-3, but were much more effective than a system consisting of BMP and BMR. The maximal rate of oxidation of palmitic acid by the newly developed reconstitution systems is still only approximately 5% of the activity of the holoenzyme. The reconstitution systems produced ω-1, ω-2, and ω-3 monohydroxy palmitic acid, but not the secondary products of palmitic acid hydroxylation observed with the holoenzyme. The physical cause of the inability to reconstitute fully the maximal activity of the holoenzyme as well as the lack of secondary product formation is not presently understood.
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影响因子：
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作者：
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DOI：
10.1016/0005-2760(77)90218-1
发表时间：
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期刊：
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影响因子：
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作者：
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影响因子：
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作者：
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影响因子：
2.9
作者：
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通讯作者：
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