工程细胞色素 P450 酶中改变的血红素环境使单加氧酶活性转变为过氧合酶活性,ACS Catalysis

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工程细胞色素 P450 酶中改变的血红素环境使单加氧酶活性转变为过氧合酶活性
ACS Catalysis ( IF 11.3 ) Pub Date : 2022-01-12 , DOI: 10.1021/acscatal.1c05877
Matthew N. Podgorski ₁ , Joshua S. Harbort ₂ , Joel H. Z. Lee ₁ , Giang T.H. Nguyen ₃ , John B. Bruning ₄ , William A. Donald ₃ , Paul V. Bernhardt ₅ , Jeffrey R. Harmer ₂ , Stephen G. Bell ₁

Affiliation

细胞色素 P450 血红素硫醇盐单加氧酶是非常通用的酶，可将氧原子插入有机分子的非反应性 C-H 键。它们从大气中获取 O ₂并且通常通过电子转移蛋白从烟酰胺辅因子中获取电子。通过使用过氧化氢 (H ₂ O ₂）。我们证明了高度保守的苏氨酸残基（涉及双氧活化）向谷氨酸的突变会关闭 P450 酶中的单加氧酶活性并将其转化为过氧合酶。沼泽红假单胞菌CYP199A4 的苏氨酸转变为谷氨酸 (T252E) 突变体的原因HaA2 与添加底物后没有自旋态变化有关。该突变体的底物结合形式的晶体结构突出了 I-螺旋中修饰的氧结合凹槽和铁结合水配体的保留。该配体与谷氨酸残基相互作用，有利于其保留。电子顺磁共振证实突变底物结合复合物的铁血红素水配体与标准铁血红素水复合物相比具有改变的特征。对于 4-甲氧基苯甲酸到 4-羟基苯甲酸和藜芦酸到香草酸的氧化去甲基化（高达 6 倍），过氧合酶活性得到显着改善。

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An Altered Heme Environment in an Engineered Cytochrome P450 Enzyme Enables the Switch from Monooxygenase to Peroxygenase Activity

Cytochrome P450 heme-thiolate monooxygenases are exceptionally versatile enzymes which insert an oxygen atom into the unreactive C–H bonds of organic molecules. They source O₂ from the atmosphere and usually derive electrons from nicotinamide cofactors via electron transfer proteins. The requirement for an expensive nicotinamide adenine dinucleotide (phosphate) cofactor and the redox protein partners can be bypassed by driving the catalysis using hydrogen peroxide (H₂O₂). We demonstrate that the mutation of a highly conserved threonine residue, involved in dioxygen activation, to a glutamate shuts down monooxygenase activity in a P450 enzyme and converts it into a peroxygenase. The reason for this switch in the threonine to glutamate (T252E) mutant of CYP199A4 from Rhodopseudomonas palustris HaA2 was linked to the lack of a spin state change upon the addition of the substrate. The crystal structure of the substrate-bound form of this mutant highlighted a modified oxygen-binding groove in the I-helix and the retention of the iron-bound aqua ligand. This ligand interacts with the glutamate residue, which favors its retention. Electron paramagnetic resonance confirmed that the ferric heme aqua ligand of the mutant substrate-bound complex had altered characteristics compared to a standard ferric heme aqua complex. Significant improvements in peroxygenase activity were demonstrated for the oxidative demethylation of 4-methoxybenzoic acid to 4-hydroxybenzoic acid and veratric acid to vanillic acid (up to 6-fold). The detailed characterization of this engineered heme peroxygenase will facilitate the development of new methods for driving the biocatalytic generation of oxygenated organic molecules via selective C–H bond activation using heme enzymes.

更新日期：2022-02-04

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