CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
J Am Chem Soc. 2023 Mar 8;145(9):5506-5511. doi: 10.1021/jacs.3c00195. Epub 2023 Feb 15.
Given prominent physicochemical similarities between HO and water, we report a new strategy for promoting the peroxygenase activity of P450 enzymes by engineering their water tunnels to facilitate HO access to the heme center buried therein. Specifically, the HO-driven activities of two native NADH-dependent P450 enzymes (CYP199A4 and CYP153A) increase significantly (by >183-fold and >15-fold, respectively). Additionally, the amount of HO required for an artificial P450 peroxygenase facilitated by a dual-functional small molecule to obtain the desired product is reduced by 95%-97.5% (with ∼95% coupling efficiency). Structural analysis suggests that mutating the residue at the bottleneck of the water tunnel may open a second pathway for HO to flow to the heme center (in addition to the natural substrate tunnel). This study highlights a promising, generalizable strategy whereby P450 monooxygenases can be modified to adopt peroxygenase activity through HO tunnel engineering, thus broadening the application scope of P450s in synthetic chemistry and synthetic biology.
鉴于 HO 和水之间显著的物理化学相似性，我们报告了一种通过工程化水通道来促进 P450 酶过氧酶活性的新策略，使 HO 能够进入埋藏在其中的血红素中心。具体来说，两种天然依赖 NADH 的 P450 酶（CYP199A4 和 CYP153A）的 HO 驱动活性显著增加（分别增加了>183 倍和>15 倍）。此外，通过双功能小分子促进的人工 P450 过氧酶所需的 HO 量减少了 95%-97.5%（耦合效率约为 95%）。结构分析表明，突变水通道瓶颈处的残基可能会为 HO 流向血红素中心开辟第二条途径（除了天然底物隧道）。这项研究强调了一种有前途的、可推广的策略，通过 HO 隧道工程可以修饰 P450 单加氧酶以采用过氧酶活性，从而拓宽 P450 在合成化学和合成生物学中的应用范围。
