Johnson Eachan O, Wong Luet-Lok
Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR, UK.
Catal Sci Technol. 2016 Oct 21;6(20):7549-7560. doi: 10.1039/C6CY01042C. Epub 2016 Sep 1.
Cytochrome P450 (CYP) enzymes catalyze the insertion of oxygen into carbon-hydrogen bonds and have great potential for enzymatic synthesis. Application development of class I CYPs is hampered by their dependence on two redox partners (a ferredoxin and ferredoxin reductase), slowing catalysis compared to self-sufficient CYPs such as CYP102A1 (P450BM3). Previous attempts to address this have fused all three components in several permutations and geometries, with much reduced activity compared to the native system. We report here the new approach of fusing putidaredoxin reductase (PdR) to the carboxy-terminus of CYP101A1 (P450cam) via a linker peptide and reconstituting camphor hydroxylase activity with free putidaredoxin (Pdx). Initial purification of a P450cam-PdR fusion yielded 2.0% heme incorporation. Co-expression of ferrochelatase, lengthening the linker from 5 to 20 residues, and altering culture conditions for enzyme production furnished 85% heme content. Fusion co-expression with Pdx gave a functional system with comparable camphor oxidation activity as the native system. , the fused system's steady state NADH oxidation rate was two-fold faster than that of the native system. In contrast to the native system, NADH oxidation rates for the fusion enzyme showed non-hyperbolic dependence on Pdx concentration, suggesting a role for the PdR domain; these data were consistent with a kinetic model based on two-site binding of Pdx by P450cam-PdR and inactive dimer formation of the fusion. P450cam-PdR is the first example of a class I P450 fusion that exhibits significantly more favorable behavior than that of the native system.
细胞色素P450（CYP）酶催化将氧插入碳氢键中，在酶促合成方面具有巨大潜力。I类CYP的应用开发受到其对两个氧化还原伙伴（铁氧化还原蛋白和铁氧化还原蛋白还原酶）的依赖的阻碍，与自给自足的CYP（如CYP102A1（P450BM3））相比，催化速度较慢。此前解决这一问题的尝试以多种排列和几何结构融合了所有三个组分，与天然系统相比活性大幅降低。我们在此报告一种新方法，即将恶臭假单胞菌铁氧化还原蛋白还原酶（PdR）通过连接肽融合到CYP101A1（P450cam）的羧基末端，并与游离的恶臭假单胞菌铁氧化还原蛋白（Pdx）一起重建樟脑羟化酶活性。最初纯化的P450cam-PdR融合蛋白的血红素掺入率为2.0%。共表达亚铁螯合酶、将连接肽从5个残基延长至20个残基以及改变酶生产的培养条件，使血红素含量达到85%。与Pdx共表达融合蛋白得到了一个功能系统，其樟脑氧化活性与天然系统相当。此外，融合系统的稳态NADH氧化速率比天然系统快两倍。与天然系统不同，融合酶的NADH氧化速率对Pdx浓度呈现非双曲线依赖性，表明PdR结构域发挥了作用；这些数据与基于P450cam-PdR对Pdx的双位点结合以及融合蛋白形成无活性二聚体的动力学模型一致。P450cam-PdR是I类P450融合蛋白的首个实例，其表现出比天然系统更有利的行为。
