Investigation of the Substrate Range of CYP199A4: Modification of the Partition between Hydroxylation and Desaturation Activities by Substrate and Protein Engineering
Investigation of the Substrate Range of CYP199A4: Modification of the Partition between Hydroxylation and Desaturation Activities by Substrate and Protein Engineering
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DOI：
10.1002/chem.201202776
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发表时间：
2012-12-01
影响因子：
4.3
通讯作者：
Zhou, Weihong
中科院分区：
文献类型：
--
作者：
Bell, Stephen G.;Zhou, Ruimin;Zhou, Weihong
来自沼泽红球藻HaA 2的细胞色素P450酶CYP 199 A4可以有效地使4-甲氧基苯甲酸脱甲基。它还能够氧化一系列其他相关底物。通过研究具有不同取代基和环系统的底物，我们已经能够表明羧酸酯基团和环系统的性质以及取代基对于最佳底物结合和活性都是重要的。藜芦酸，2-萘甲酸和吲哚-6-羧酸底物结合的CYP 199 A4复合物的结构揭示了底物结合模式和活性位点残基的侧链构象变化，以适应这些较大的底物。它们还为产物氧化的选择性提供了理论基础。CYP 199 A4对烷基取代苯甲酸的氧化更为复杂，去饱和反应与羟基化活性竞争。4-乙基苯甲酸结合的CYP 199 A4的结构显示，底物与4-甲氧基苯甲酸的位置相似，乙基的C β C-H键比C α更接近血红素铁（3.5与4.8埃）。当与反应中间体的相对能量相结合时，这一观察结果表明，烷基相对于血红素铁的定位在确定观察到的去饱和量时可能是关键的。通过突变CYP 199 A4（Phe 185）活性位点中的单个残基，我们能够将该酶转化为4-乙基苯甲酸去饱和酶。
The cytochrome P450 enzyme CYP199A4, from Rhodopseudomonas palustris HaA2, can efficiently demethylate 4-methoxybenzoic acid. It is also capable of oxidising a range of other related substrates. By investigating substrates with different substituents and ring systems we have been able to show that the carboxylate group and the nature of the ring system and the substituent are all important for optimal substrate binding and activity. The structures of the veratric acid, 2-naphthoic acid and indole-6-carboxylic acid substrate-bound CYP199A4 complexes reveal the substrate binding modes and the side-chain conformational changes of the active site residues to accommodate these larger substrates. They also provide a rationale for the selectivity of product oxidation. The oxidation of alkyl substituted benzoic acids by CYP199A4 is more complex, with desaturation reactions competing with hydroxylation activity. The structure of 4-ethylbenzoic acid-bound CYP199A4 revealed that the substrate is held in a similar position to 4-methoxybenzoic acid, and that the C beta C-H bonds of the ethyl group are closer to the heme iron than those of the C alpha (3.5 vs. 4.8 angstrom). This observation, when coupled to the relative energies of the reaction intermediates, indicates that the positioning of the alkyl group relative to the heme iron may be critical in determining the amount of desaturation that is observed. By mutating a single residue in the active site of CYP199A4 (Phe185) we were able to convert the enzyme into a 4-ethylbenzoic acid desaturase.